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Sample records for aureus protein efb-c

  1. Electrostatic Contributions Drive the Interaction Between Staphylococcus aureus Protein Efb-C and its Complement Target C3d

    Energy Technology Data Exchange (ETDEWEB)

    Haspel, N.; Ricklin, D.; Geisbrecht, B.V.; Kavraki, L.E.; Lambris, J.D. (UPENN); (UMKC); (Rice)

    2008-11-13

    The C3-inhibitory domain of Staphylococcus aureus extracellular fibrinogen-binding protein (Efb-C) defines a novel three-helix bundle motif that regulates complement activation. Previous crystallographic studies of Efb-C bound to its cognate subdomain of human C3 (C3d) identified Arg-131 and Asn-138 of Efb-C as key residues for its activity. In order to characterize more completely the physical and chemical driving forces behind this important interaction, we employed in this study a combination of structural, biophysical, and computational methods to analyze the interaction of C3d with Efb-C and the single-point mutants R131A and N138A. Our results show that while these mutations do not drastically affect the structure of the Efb-C/C3d recognition complex, they have significant adverse effects on both the thermodynamic and kinetic profiles of the resulting complexes. We also characterized other key interactions along the Efb-C/C3d binding interface and found an intricate network of salt bridges and hydrogen bonds that anchor Efb-C to C3d, resulting in its potent complement inhibitory properties.

  2. [Protein toxins of Staphylococcus aureus].

    Science.gov (United States)

    Shamsutdinov, A F; Tiurin, Iu A

    2014-01-01

    Main scientific-research studies regarding protein bacterial toxins of the most widespread bacteria that belong to Staphylococcus spp. genus and in particular the most pathogenic species for humans--Staphylococcus aureus, are analyzed. Structural and biological properties of protein toxins that have received the name of staphylococcus pyrogenic toxins (PTSAg) are presented. Data regarding genetic regulation of secretion and synthesis of these toxins and 3 main regulatory genetic systems (agr--accessory gene regulator, xpr--extracellular protein regulator, sar--staphylococcal accessory regulator) that coordinate synthesis of the most important protein toxins and enzymes for virulence of S. aureus, are presented.

  3. Salicylic acid enhances Staphylococcus aureus extracellular adhesin protein expression.

    Science.gov (United States)

    Alvarez, Lucía P; Barbagelata, María S; Cheung, Ambrose L; Sordelli, Daniel O; Buzzola, Fernanda R

    2011-11-01

    One of the virulence factors required by Staphylococcus aureus at the early stages of infection is Eap, a secreted adhesin that binds many host proteins and is upregulated by the two-component regulatory system saeRS. The S. aureus Newman strain harbors a mutation in saeS that is thought to be responsible for the high level of Eap expression in this strain. This study was designed to ascertain whether salicylic acid (SAL) affects the expression of Eap and the internalization of S. aureus into epithelial cells. The strain Newman treated with SAL exhibited increased levels of eap transcription and protein expression. Furthermore, SAL treatment increased the eap promoter activity. SAL treatment enhanced Eap expression in the Newman and in other S. aureus strains that do not carry the mutation in saeS. Internalization of S. aureus eap and sae mutants into the MAC-T epithelial cells was significantly decreased compared with the wild-type counterparts. In conclusion, we demonstrated that a low concentration of SAL increased S. aureus Eap expression possibly due to enhancement of sae. SAL may create the conditions for S. aureus persistence in the host, not only by decreasing the capsular polysaccharide expression as shown before, but also by enhancing Eap expression.

  4. Surface proteins of Staphylococcus aureus play an important role in experimental skin infection.

    Science.gov (United States)

    Kwiecinski, Jakub; Jin, Tao; Josefsson, Elisabet

    2014-12-01

    Staphylococcus aureus is the most common cause of skin infections that range from mild diseases up to life-threatening conditions. Mechanisms of S. aureus virulence in those infections remain poorly studied. To investigate the impact of S. aureus surface proteins on skin infection, we used mouse models of skin abscess formation and skin necrosis, induced by a subcutaneous injection of bacteria. In the skin abscess model, a sortase-deficient S. aureus strain lacking all of its cell-wall anchored proteins was less virulent than its wild-type strain. Also, strains specifically lacking protein A, fibronecting binding proteins, clumping factor A or surface protein SasF were impaired in their virulence. When a model of dermonecrosis was studied, the S. aureus surface proteins could not be shown to be involved. In summary, surface proteins play an important role in virulence of S. aureus skin abscess infections, but not in formation of skin necrosis.

  5. Monoclonal Antibody Targeting Staphylococcus aureus Surface Protein A (SasA) Protect Against Staphylococcus aureus Sepsis and Peritonitis in Mice.

    Science.gov (United States)

    Yang, Yilong; Qian, Mengying; Yi, Shaoqiong; Liu, Shuling; Li, Bing; Yu, Rui; Guo, Qiang; Zhang, Xiaopeng; Yu, Changming; Li, Jianmin; Xu, Junjie; Chen, Wei

    2016-01-01

    Epidemic methicillin-resistant Staphylococcus aureus (MRSA) imposes an increasing impact on public health. Due to multi-antibiotics resistance in MRSA strains, there is an urgent need to develop novel therapeutics such as effective monoclonal antibodies (mAbs) against MRSA infections. Staphylococcus aureus surface protein A (SasA), a large surface-located protein (~240 kDa), is one of MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) and a potential target for immunotherapeutic approaches against S. aureus infections. In the present study, we analyzed the sequence of SasA with bioinformatics tools and generated a protective monoclonal antibody (2H7) targeting the conserved domain of SasA. 2H7 was shown to recognize wild-type S. aureus and promote opsonophagocytic killing of S. aureus. In both sepsis and peritoneal infection models, prophylactic administration of 2H7 improved the survival of BALB/c mice challenged by S. aureus strain USA300 and ST239 (prevalent MRSA clones in North America and Asian countries, respectively) and enhanced bacterial clearance in kidneys. Additionally, 2H7 prophylaxis prevented the formation of intraperitoneal abscess in a murine model of peritoneal infection and therapeutic administration of 2H7 showed protective efficacy in a murine sepsis model. Our results presented here provide supporting evidences that an anti-SasA mAb might be a potential component in an antibody-based immunotherapeutic treatment of MRSA infections.

  6. Non-spa-typeable clinical Staphylococcus aureus strains are naturally occurring protein A mutants

    DEFF Research Database (Denmark)

    Baum, Cathrin; Haslinger-Löffler, Bettina; Westh, Henrik;

    2009-01-01

    Staphylococcus aureus is a major human pathogen responsible for increasing the prevalence of community- and hospital-acquired infections. Protein A (SpA) is a key virulence factor of S. aureus and is highly conserved. Sequencing of the variable-number tandem-repeat region of SpA (spa typing...

  7. Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells

    NARCIS (Netherlands)

    Sinha, B; Francois, P; Que, Y A; Hussain, M; Heilmann, C; Moreillon, P; Lew, D; Krause, K H; Peters, Georg; Herrmann, M

    2000-01-01

    Staphylococcus aureus invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between S. aureus fibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin alpha(5)beta(1) (B. Sinha et al., Cell.

  8. Clumping factor A, von Willebrand factor-binding protein and von Willebrand factor anchor Staphylococcus aureus to the vessel wall.

    Science.gov (United States)

    Claes, J; Liesenborghs, L; Peetermans, M; Veloso, T R; Missiakas, D; Schneewind, O; Mancini, S; Entenza, J M; Hoylaerts, M F; Heying, R; Verhamme, P; Vanassche, T

    2017-02-09

    Essentials Staphylococcus aureus (S. aureus) binds to endothelium via von Willebrand factor (VWF). Secreted VWF-binding protein (vWbp) mediates S. aureus adhesion to VWF under shear stress. vWbp interacts with VWF and the Sortase A-dependent surface protein Clumping factor A (ClfA). VWF-vWbp-ClfA anchor S. aureus to vascular endothelium under shear stress.

  9. Serine-Aspartate Repeat Protein D Increases Staphylococcus aureus Virulence and Survival in Blood

    Science.gov (United States)

    Uchiyama, Satoshi; Valderrama, J. Andrés; Ajayi, Clement; Sollid, Johanna U. E.; van Sorge, Nina M.; Nizet, Victor; van Strijp, Jos A. G.

    2016-01-01

    ABSTRACT Staphylococcus aureus expresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and infection. Deletion of sdrD from S. aureus subsp. aureus strain NCTC8325-4 attenuated bacterial survival in human whole blood ex vivo, which was associated with increased killing by human neutrophils. Remarkably, SdrD was able to inhibit innate immune-mediated bacterial killing independently of other S. aureus proteins, since addition of recombinant SdrD protein and heterologous expression of SdrD in Lactococcus lactis promoted bacterial survival in human blood. SdrD contributes to bacterial virulence in vivo, since fewer S. aureus subsp. aureus NCTC8325-4 ΔsdrD bacteria than bacteria of the parent strain were recovered from blood and several organs using a murine intravenous infection model. Collectively, our findings reveal a new property of SdrD as an important key contributor to S. aureus survival and the ability to escape the innate immune system in blood. PMID:27795358

  10. Characterization of human and Staphylococcus aureus proteins in respiratory mucosa by in vivo- and immunoproteomics.

    Science.gov (United States)

    Schmidt, Frank; Meyer, Tanja; Sundaramoorthy, Nandakumar; Michalik, Stephan; Surmann, Kristin; Depke, Maren; Dhople, Vishnu; Gesell Salazar, Manuela; Holtappels, Gabriele; Zhang, Nan; Bröker, Barbara M; Bachert, Claus; Völker, Uwe

    2017-02-23

    Staphylococcus aureus is a Gram-positive opportunistic bacterium which can be found as a commensal in the nares of about 50% of the human population. Besides asymptomatic carriage, S. aureus has also been found to colonize nasal polyps, a subform of chronic rhinosinusitis, in 60 to 100% of cases, and even reside intracellularly in nasal polyp tissue. The aim of this study was to shed light on the behavior of S. aureus in the human airways by analyzing S. aureus-specific proteins in nasal polyp tissue from patients with chronic rhinosinusitis and to characterize the immunogenic potential of the identified (mainly secreted) proteins. As a result, in total >600 S. aureus proteins were identified by high resolution mass spectrometry or multiple reaction monitoring. Of those roughly 180 are typically localized in the membrane, surface exposed or secreted. For 115 S. aureus proteins, partially also detected in vivo by mass spectrometry, IgA- and IgG-specific antibody signals were profiled. Strong antibody signals were predominantly found for surface expose or secreted proteins.

  11. Neutrophil-generated oxidative stress and protein damage in Staphylococcus aureus.

    Science.gov (United States)

    Beavers, William N; Skaar, Eric P

    2016-08-01

    Staphylococcus aureus is a ubiquitous, versatile and dangerous pathogen. It colonizes over 30% of the human population, and is one of the leading causes of death by an infectious agent. During S. aureus colonization and invasion, leukocytes are recruited to the site of infection. To combat S. aureus, leukocytes generate an arsenal of reactive species including superoxide, hydrogen peroxide, nitric oxide and hypohalous acids that modify and inactivate cellular macromolecules, resulting in growth defects or death. When S. aureus colonization cannot be cleared by the immune system, antibiotic treatment is necessary and can be effective. Yet, this organism quickly gains resistance to each new antibiotic it encounters. Therefore, it is in the interest of human health to acquire a deeper understanding of how S. aureus evades killing by the immune system. Advances in this field will have implications for the design of future S. aureus treatments that complement and assist the host immune response. In that regard, this review focuses on how S. aureus avoids host-generated oxidative stress, and discusses the mechanisms used by S. aureus to survive oxidative damage including antioxidants, direct repair of damaged proteins, sensing oxidant stress and transcriptional changes. This review will elucidate areas for studies to identify and validate future antimicrobial targets.

  12. ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus.

    Science.gov (United States)

    Frankel, Matthew B; Wojcik, Brandon M; DeDent, Andrea C; Missiakas, Dominique M; Schneewind, Olaf

    2010-10-01

    The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion.

  13. Non-spa-typeable clinical Staphylococcus aureus strains are naturally occurring protein A mutants

    DEFF Research Database (Denmark)

    Baum, Cathrin; Haslinger-Löffler, Bettina; Westh, Henrik;

    2009-01-01

    Staphylococcus aureus is a major human pathogen responsible for increasing the prevalence of community- and hospital-acquired infections. Protein A (SpA) is a key virulence factor of S. aureus and is highly conserved. Sequencing of the variable-number tandem-repeat region of SpA (spa typing......) provides a rapid and reliable method for epidemiological studies. Rarely, non-spa-typeable S. aureus strains are encountered. The reason for this is not known. In this study, we characterized eight non-spa-typeable bacteremia isolates. Sequencing of the entire spa locus was successful for five strains...

  14. Human SAP is a novel peptidoglycan recognition protein that induces complement- independent phagocytosis of Staphylococcus aureus

    Science.gov (United States)

    An, Jang-Hyun; Kurokawa, Kenji; Jung, Dong-Jun; Kim, Min-Jung; Kim, Chan-Hee; Fujimoto, Yukari; Fukase, Koichi; Coggeshall, K. Mark; Lee, Bok Luel

    2014-01-01

    The human pathogen Staphylococcus aureus is responsible for many community-acquired and hospital-associated infections and is associated with high mortality. Concern over the emergence of multidrug-resistant strains has renewed interest in the elucidation of host mechanisms that defend against S. aureus infection. We recently demonstrated that human serum mannose-binding lectin (MBL) binds to S. aureus wall teichoic acid (WTA), a cell wall glycopolymer, a discovery that prompted further screening to identify additional serum proteins that recognize S. aureus cell wall components. In this report, we incubated human serum with 10 different S. aureus mutants and determined that serum amyloid P component (SAP) bound specifically to a WTA-deficient S. aureus ΔtagO mutant, but not to tagO-complemented, WTA-expressing cells. Biochemical characterization revealed that SAP recognizes bacterial peptidoglycan as a ligand and that WTA inhibits this interaction. Although SAP binding to peptidoglycan was not observed to induce complement activation, SAP-bound ΔtagO cells were phagocytosed by human polymorphonuclear leukocytes in an Fcγ receptor-dependent manner. These results indicate that SAP functions as a host defense factor, similar to other peptidoglycan recognition proteins and nucleotide-binding oligomerization domain (NOD)-like receptors. PMID:23966633

  15. The giant protein Ebh is a determinant of Staphylococcus aureus cell size and complement resistance.

    Science.gov (United States)

    Cheng, Alice G; Missiakas, Dominique; Schneewind, Olaf

    2014-03-01

    Staphylococcus aureus USA300, the clonal type associated with epidemic community-acquired methicillin-resistant S. aureus (MRSA) infections, displays the giant protein Ebh on its surface. Mutations that disrupt the ebh reading frame increase the volume of staphylococcal cells and alter the cross wall, a membrane-enclosed peptidoglycan synthesis and assembly compartment. S. aureus ebh variants display increased sensitivity to oxacillin (methicillin) as well as susceptibility to complement-mediated killing. Mutations in ebh are associated with reduced survival of mutant staphylococci in blood and diminished virulence in mice. We propose that Ebh, following its secretion into the cross wall, contributes to the characteristic cell growth and envelope assembly pathways of S. aureus, thereby enabling complement resistance and the pathogenesis of staphylococcal infections.

  16. Antibodies to Staphylococcus aureus Bone Sialoprotein-Binding Protein Indicate Infectious Osteomyelitis▿

    OpenAIRE

    Persson, Lena; Johansson, Christian; Rydén, Cecilia

    2009-01-01

    Discrimination of soft tissue infection from osteomyelitis in diabetic foot infections is a common clinical problem. Staphylococcus aureus isolates from patients with osteomyelitis express bone sialoprotein-binding protein (Bbp) that binds the bone matrix protein bone sialoprotein. The serological assay with Bbp discriminated cases of osteomyelitis from soft tissue infections in patients with diabetic foot ulcers.

  17. Serine aspartate repeat protein D increases Staphylococcus aureus virulence and survival in blood

    NARCIS (Netherlands)

    Askarian, Fatemeh; Uchiyama, Satoshi; Valderrama, J. Andrés; Ajayi, Clement; Sollid, Johanna U E; van Sorge, Nina M.; Nizet, Victor; van Strijp, Jos A G; Johannessen, Mona

    2017-01-01

    Staphylococcus aureus expresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and

  18. Phosphorylation of Staphylococcus aureus Protein-Tyrosine Kinase Affects the Function of Glucokinase and Biofilm Formation

    Science.gov (United States)

    Vasu, Dudipeta; Kumar, Pasupuleti Santhosh; Prasad, Uppu Venkateswara; Swarupa, Vimjam; Yeswanth, Sthanikam; Srikanth, Lokanathan; Sunitha, Manne Mudhu; Choudhary, Abhijith; Krishna Sarma, Potukuchi Venkata Gurunadha

    2017-01-01

    Background: When Staphylococcus aureus is grown in the presence of high concentration of external glucose, this sugar is phosphorylated by glucokinase (glkA) to form glucose-6-phosphate. This product subsequently enters into anabolic phase, which favors biofilm formation. The presence of ROK (repressor protein, open reading frame, sugar kinase) motif, phosphate-1 and -2 sites, and tyrosine kinase sites in glkA of S. aureus indicates that phosphorylation must regulate the glkA activity. The aim of the present study was to identify the effect of phosphorylation on the function of S. aureus glkA and biofilm formation. Methods: Pure glkA and protein-tyrosine kinase (BYK) of S. aureus ATCC 12600 were obtained by fractionating the cytosolic fractions of glkA1 and BYK-1 expressing recombinant clones through nickel metal chelate column. The pure glkA was used as a substrate for BYK, and the phosphorylation of glkA was confirmed by treating with reagent A and resolving in SDS-PAGE, as well as staining with reagent A. The kinetic parameters of glkA and phosphorylated glkA were determined spectrophotometrically, and in silico tools were used for validation. S. aureus was grown in brain heart infusion broth, which was supplemented with glucose, and then biofilm units were calculated. Results: Fourfold elevated glkA activity was observed upon the phosphorylation by BYK. Protein-protein docking analysis revealed that glkA structure docked close to the adenosine triphosphate-binding site of BYK structure corroborating the kinetic results. Further, S. aureus grown in the presence of elevated glucose concentration exhibited an increase in the rate of biofilm formation. Conclusion: The elevated function of glkA is an essential requirement for increased biofilm units in S. aureus, a key pathogenic factor that helps its survival and the progress of infection. PMID:27695030

  19. Life and Death of Proteins: A Case Study of Glucose-starved Staphylococcus aureus*

    OpenAIRE

    Michalik, Stephan; Bernhardt, Jörg; Otto, Andreas; Moche, Martin; Becher, Dörte; Meyer, Hanna; Lalk, Michael; Schurmann, Claudia; Schlüter, Rabea; Kock, Holger; Gerth, Ulf; Hecker, Michael

    2012-01-01

    The cellular amount of proteins not only depends on synthesis but also on degradation. Here, we expand the understanding of differential protein levels by complementing synthesis data with a proteome-wide, mass spectrometry-based stable isotope labeling with amino acids in cell culture analysis of protein degradation in the human pathogen Staphylococcus aureus during glucose starvation. Monitoring protein stability profiles in a wild type and an isogenic clpP protease mutant revealed that 1) ...

  20. Staphylococcus pseudintermedius expresses surface proteins that closely resemble those from Staphylococcus aureus.

    Science.gov (United States)

    Geoghegan, Joan A; Smith, Emma J; Speziale, Pietro; Foster, Timothy J

    2009-09-18

    Staphylococcus pseudintermedius is a commensal of dogs that is implicated in the pathogenesis of canine pyoderma. This study aimed to determine if S. pseudintermedius expresses surface proteins resembling those from Staphylococcus aureus and to characterise them. S. pseudintermedius strain 326 was shown to adhere strongly to purified fibrinogen, fibronectin and cytokeratin 10. It adhered to the alpha-chain of fibrinogen which, along with binding to cytokeratin 10, is the hallmark of clumping factor B of S. aureus, a surface protein that is in part responsible for colonisation of the human nares. Ligand-affinity blotting with cell-wall extracts demonstrated that S. pseudintermedius 326 expressed a cell-wall anchored fibronectin binding protein which recognised the N-terminal 29kDa fragment. The ability to bind fibronectin is an important attribute of pathogenic S. aureus and is associated with the ability of S. aureus to colonise skin of human atopic dermatitis patients. S. pseudintermedius genomic DNA was probed with labelled DNA amplified from the serine-aspartate repeat encoding region of clfA of S. aureus. This probe hybridised to a single SpeI fragment of S. pseudintermedius DNA. In the cell-wall extract of S. pseudintermedius 326, a 180kDa protein was discovered which bound to fibrinogen by ligand-affinity blotting and reacted in a Western blot with antibodies raised against the serine-aspartate repeat region of ClfA and the B-repeats of SdrD of S. aureus. It is proposed that this is an Sdr protein with B-repeats that has an A domain that binds to fibrinogen. Whether it is the same protein that binds cytokeratin 10 is not clear.

  1. Protective activity of the CnaBE3 domain conserved among Staphylococcus aureus Sdr proteins.

    Science.gov (United States)

    Becherelli, Marco; Prachi, Prachi; Viciani, Elisa; Biagini, Massimiliano; Fiaschi, Luigi; Chiarot, Emiliano; Nosari, Sarah; Brettoni, Cecilia; Marchi, Sara; Biancucci, Marco; Fontana, Maria Rita; Montagnani, Francesca; Bagnoli, Fabio; Barocchi, Michèle A; Manetti, Andrea G O

    2013-01-01

    Staphylococcus aureus is an opportunistic pathogen, commensal of the human skin and nares, but also responsible for invasive nosocomial as well as community acquired infections. Staphylococcus aureus adheres to the host tissues by means of surface adhesins, such as SdrC, SdrD, and SdrE proteins. The Sdr family of proteins together with a functional A domain, contain respectively two, three or five repeated sequences called B motifs which comprise the CnaB domains. SdrD and SdrE proteins were reported to be protective in animal models against invasive diseases or lethal challenge with human clinical S. aureus isolates. In this study we identified a 126 amino acid sequence containing a CnaB domain, conserved among the three Sdr proteins. The three fragments defined here as CnaBC2, D5 and E3 domains even though belonging to phylogenetically distinct strains, displayed high sequence similarity. Based on the sequence conservation data, we selected the CnaBE3 domain for further analysis and characterization. Polyclonal antibodies raised against the recombinant CnaBE3 domain recognized SdrE, SdrC and SdrD proteins of different S. aureus lineages. Moreover, we demonstrated that the CnaBE3 domain was expressed in vivo during S. aureus infections, and that immunization of this domain alone significantly reduces the bacterial load in mice challenged with S. aureus. Furthermore, we show that the reduction of bacteria by CnaBE3 vaccination is due to functional antibodies. Finally, we demonstrated that the region of the SdrE protein containing the CnaBE3 domain was resistant to trypsin digestion, a characteristic often associated with the presence of an isopeptide bond.

  2. A Staphylococcus aureus Proteome Overview: Shared and Specific Proteins and Protein Complexes from Representative Strains of All Three Clades.

    Science.gov (United States)

    Liang, Chunguang; Schaack, Dominik; Srivastava, Mugdha; Gupta, Shishir K; Sarukhanyan, Edita; Giese, Anne; Pagels, Martin; Romanov, Natalie; Pané-Farré, Jan; Fuchs, Stephan; Dandekar, Thomas

    2016-02-19

    Staphylococcus aureus is an important model organism and pathogen. This S. aureus proteome overview details shared and specific proteins and selected virulence-relevant protein complexes from representative strains of all three major clades. To determine the strain distribution and major clades we used a refined strain comparison combining ribosomal RNA, MLST markers, and looking at highly-conserved regions shared between strains. This analysis shows three sub-clades (A-C) for S. aureus. As calculations are complex and strain annotation is quite time consuming we compare here key representatives of each clade with each other: model strains COL, USA300, Newman, and HG001 (clade A), model strain N315 and Mu50 (clade B) and ED133 and MRSA252 (clade C). We look at these individual proteomes and compare them to a background of 64 S. aureus strains. There are overall 13,284 S. aureus proteins not part of the core proteome which are involved in different strain-specific or more general complexes requiring detailed annotation and new experimental data to be accurately delineated. By comparison of the eight representative strains, we identify strain-specific proteins (e.g., 18 in COL, 105 in N315 and 44 in Newman) that characterize each strain and analyze pathogenicity islands if they contain such strain-specific proteins. We identify strain-specific protein repertoires involved in virulence, in cell wall metabolism, and phosphorylation. Finally we compare and analyze protein complexes conserved and well-characterized among S. aureus (a total of 103 complexes), as well as predict and analyze several individual protein complexes, including structure modeling in the three clades.

  3. Phagocytosis escape by a Staphylococcus aureus protein that connects complement and coagulation proteins at the bacterial surface.

    Science.gov (United States)

    Ko, Ya-Ping; Kuipers, Annemarie; Freitag, Claudia M; Jongerius, Ilse; Medina, Eva; van Rooijen, Willemien J; Spaan, András N; van Kessel, Kok P M; Höök, Magnus; Rooijakkers, Suzan H M

    2013-01-01

    Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureus secretes the 16 kD Extracellular fibrinogen binding protein (Efb) that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a 'capsule'-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein.

  4. The role of serum proteins in Staphylococcus aureus adhesion to ethylene glycol coated surfaces.

    Science.gov (United States)

    Schuster, Swen; Yu, Wenqi; Nega, Mulugeta; Chu, Ya-Yun; Zorn, Stefan; Zhang, Fajun; Götz, Friedrich; Schreiber, Frank

    2014-11-01

    Bacterial adhesion on implants is a first step in the development of chronic foreign body associated infections. Finding strategies to minimize bacterial adhesion may contribute to minimize such infections. It is known that surfaces with oligo-ethylene-glycol (EG3OMe) or poly-ethylene-glycol (PEG2k) terminations decrease unspecific protein adsorption and bacterial adhesion. However, little is known about the influence of serum and its components on bacterial adhesion. We therefore prepared two coatings on gold surface with HS-(CH2)11EG3OMe (EG3OMe) and PEG2k-thiol and studied the role of bovine serum albumin (BSA), γ-globulins, and serum on Staphylococcus aureus adhesion. While BSA and lysozyme showed no adherence even when applied at very high concentrations (100 mg/ml), γ-globulins adsorbed already from 10 mg/ml on. The adsorption of γ-globulins was, however, significantly decreased when it was mixed with BSA in a ratio of 3:1, as it is in the serum. Pretreatment of EG3OMe and PEG2k coatings with γ-globulins or serum strongly promoted adherence of S. aureus when resuspended in buffer, suggesting that γ-globulins play a pivotal role in promoting S. aureus adhesion by its IgG binding proteins; the finding that a spa-deletion mutant, lacking the IgG binding protein A, showed decreased adherence corroborated this. Similarly, when S. aureus was pretreated with serum or γ-globulins its adherence was also significantly decreased. Our findings show that particularly γ-globulins bind to the coated surfaces thus mediating adherence of S. aureus via its protein A. As pretreatment of S. aureus with serum or γ-globulins significantly decreased adherence, treatment of patients with γ-globulins before implant surgery might lower the risk of implant-associated infections. Copyright © 2014 Elsevier GmbH. All rights reserved.

  5. Acute phase proteins in bovine milk in an experimental model of Staphylococcus aureus subclinical mastitis

    DEFF Research Database (Denmark)

    Eckersall, P D; Young, F J; Nolan, A M

    2006-01-01

    The objectives were to establish the origin of 2 acute phase proteins in milk during subclinical bovine mastitis and to characterize the relationship between those proteins in milk and blood. Haptoglobin (Hp) and mammary-associated serum amyloid A (M-SAA3) appear in milk during mastitis, whereas Hp...... and serum amyloid A increase in serum during mastitis. The concentrations of these proteins were determined in an experimental model using a field strain of Staphylococcus aureus to induce subclinical mastitis in dairy cows. The expression of mRNA coding for these proteins was assessed and the presence of M......-SAA3 in mammary tissues was determined using immunocytochemistry. Increases of M-SAA3 and Hp in milk occurred within 12 h of Staphylococcus aureus infusion, with peak concentrations occurring 3 d after infusion of the bacteria. The increase of acute phase proteins in milk (15 h) preceded the increase...

  6. YsxC, an essential protein in Staphylococcus aureus crucial for ribosome assembly/stability

    Directory of Open Access Journals (Sweden)

    García-Lara Jorge

    2009-12-01

    Full Text Available Abstract Background Bacterial growth and division requires a core set of essential proteins, several of which are still of unknown function. They are also attractive targets for the development of new antibiotics. YsxC is a member of a family of GTPases highly conserved across eubacteria with a possible ribosome associated function. Results Here, we demonstrate by the creation of a conditional lethal mutant that ysxC is apparently essential for growth in S. aureus. To begin to elucidate YsxC function, a translational fusion of YsxC to the CBP-ProteinA tag in the staphylococcal chromosome was made, enabling Tandem Affinity Purification (TAP of YsxC-interacting partners. These included the ribosomal proteins S2, S10 and L17, as well as the β' subunit of the RNA polymerase. YsxC was then shown to copurify with ribosomes as an accessory protein specifically localizing to the 50 S subunit. YsxC depletion led to a decrease in the presence of mature ribosomes, indicating a role in ribosome assembly and/or stability in S. aureus. Conclusions In this study we demonstrate that YsxC of S. aureus localizes to the ribosomes, is crucial for ribosomal stability and is apparently essential for the life of S. aureus.

  7. Improving time to optimal Staphylococcus aureus treatment using a penicillin-binding protein 2a assay.

    Science.gov (United States)

    Rao, Sonia N; Wang, Sheila K; Gonzalez Zamora, Jose; Hanson, Amy P; Polisetty, Radhika S; Singh, Kamaljit

    2016-12-01

    The penicillin-binding protein 2a (PBP2a) assay is a quick, accurate and inexpensive test for determining methicillin susceptibility in Staphylococcus aureus. A pre-post-study design was conducted using a PBP2a assay with and without the impact of an antimicrobial stewardship intervention to improve time to optimal therapy for methicillin-susceptible and methicillin-resistant S. aureus isolates. Our results demonstrate significantly improved time to optimal therapy and support the use of a PBP2a assay as part of an programme for all healthcare facilities, especially those with limited resources.

  8. Protective Role of Surfactant Protein D in Ocular Staphylococcus aureus Infection.

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    Zhiyong Zhang

    Full Text Available Staphylococcus aureus is one of the most common pathogens causing keratitis. Surfactant protein D (SP-D plays a critical role in host defense and innate immunity. In order to investigate the role of SP-D in ocular S. aureus infection, the eyes of wild-type (WT and SP-D knockout (SP-D KO C57BL/6 mice were infected with S. aureus (10(7 CFU/eye in the presence and absence of cysteine protease inhibitor(E64.Bacterial counts in the ocular surface were examined 3, 6, 12, 24 hrs after infection. Bacterial phagocytosis by neutrophils and bacterial invasion in ocular epithelial cells were evaluated quantitatively. S. aureus-induced ocular injury was determined with corneal fluorescein staining. The results demonstrated that SP-D is expressed in ocular surface epithelium and the lacrimal gland; WT mice had increased clearance of S. aureus from the ocular surface (p<0.05 and reduced ocular injury compared with SP-D KO mice. The protective effects of SP-D include increased bacterial phagocytosis by neutrophils (p<0.05 and decreased bacterial invasion into epithelial cells (p<0.05 in WT mice compared to in SP-D KO mice. In the presence of inhibitor (E64, WT mice showed enhanced bacterial clearance (p<0.05 and reduced ocular injury compared to absent E64 while SP-D KO mice did not. Collectively, we concluded that SP-D protects the ocular surface from S. aureus infection but cysteine protease impairs SP-D function in this murine model, and that cysteine protease inhibitor may be a potential therapeutic agent in S. aureus keratitis.

  9. Identification of Functional Regulatory Residues of the β-Lactam Inducible Penicillin Binding Protein in Methicillin-Resistant Staphylococcus aureus

    OpenAIRE

    Mbah, Andreas N.; Isokpehi, Raphael D

    2013-01-01

    Resistance to methicillin by Staphylococcus aureus is a persistent clinical problem worldwide. A mechanism for resistance has been proposed in which methicillin resistant Staphylococcus aureus (MRSA) isolates acquired a new protein called β -lactam inducible penicillin binding protein (PBP-2′). The PBP-2′ functions by substituting other penicillin binding proteins which have been inhibited by β -lactam antibiotics. Presently, there is no structural and regulatory information on PBP-2′ protein...

  10. Characterization of EssB, a protein required for secretion of ESAT-6 like proteins in Staphylococcus aureus

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    Chen Yi-Hsing

    2012-09-01

    Full Text Available Abstract Background Staphylococcus aureus secretes EsxA and EsxB, two small polypeptides of the WXG100 family of proteins. Genetic analyses have shown that production and secretion of EsxA and EsxB require an intact ESAT-6 Secretion System (ESS, a cluster of genes that is conserved in many Firmicutes and encompasses esxA and esxB . Here, we characterize EssB, one of the proteins encoded by the ESS cluster. EssB is highly conserved in Gram-positive bacteria and belongs to the Cluster of Orthologous Groups of protein COG4499 with no known function. Results By generating an internal deletion in essB , we demonstrate that EssB is required for secretion of EsxA. We use a polyclonal antibody to identify EssB and show that the protein fractionates with the plasma membrane of S. aureus . Yet, when produced in Escherichia coli, EssB remains mostly soluble and the purified protein assembles into a highly organized oligomer that can be visualized by electron microscopy. Production of truncated EssB variants in wild-type S. aureus confers a dominant negative phenotype on EsxA secretion. Conclusions The data presented here support the notion that EssB may oligomerize and interact with other membrane components to form the WXG100-specific translocon in S. aureus .

  11. Staphylococcus aureus infection induces protein A–mediated immune evasion in humans

    Science.gov (United States)

    Pauli, Noel T.; Kim, Hwan Keun; Falugi, Fabiana; Huang, Min; Dulac, John; Henry Dunand, Carole; Zheng, Nai-Ying; Kaur, Kaval; Andrews, Sarah F.; Huang, Yunping; DeDent, Andrea; Frank, Karen M.; Charnot-Katsikas, Angella; Schneewind, Olaf

    2014-01-01

    Staphylococcus aureus bacterial infection commonly results in chronic or recurrent disease, suggesting that humoral memory responses are hampered. Understanding how S. aureus subverts the immune response is critical for the rescue of host natural humoral immunity and vaccine development. S. aureus expresses the virulence factor Protein A (SpA) on all clinical isolates, and SpA has been shown in mice to expand and ablate variable heavy 3 (VH3) idiotype B cells. The effects of SpA during natural infection, however, have not been addressed. Acutely activated B cells, or plasmablasts (PBs), were analyzed to dissect the ongoing immune response to infection through the production of monoclonal antibodies (mAbs). The B cells that were activated by infection had a highly limited response. When screened against multiple S. aureus antigens, only high-affinity binding to SpA was observed. Consistently, PBs underwent affinity maturation, but their B cell receptors demonstrated significant bias toward the VH3 idiotype. These data suggest that the superantigenic activity of SpA leads to immunodominance, limiting host responses to other S. aureus virulence factors that would be necessary for protection and memory formation. PMID:25348152

  12. Molecular basis for the role of Staphylococcus aureus penicillin binding protein 4 in antimicrobial resistance.

    Science.gov (United States)

    Navratna, Vikas; Nadig, Savitha; Sood, Varun; Prasad, K; Arakere, Gayathri; Gopal, B

    2010-01-01

    Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the D-Ala-D-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala. Beta-lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a beta-lactamase and is not trapped as an acyl intermediate with beta-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.

  13. Evaluation of a lysostaphin-fusion protein as a dry-cow therapy for Staphylococcus aureus mastitis in dairy cattle

    Science.gov (United States)

    This study evaluated the efficacy of a lysostaphin-fusion protein (Lyso-PTD) as a dry-cow therapy for the treatment of experimentally-induced chronic, subclinical Staphylococcus aureus mastitis. Twenty-two Holstein dairy cows were experimentally infected with Staph. aureus in a single pair of diago...

  14. Silkworm apolipophorin protein inhibits hemolysin gene expression of Staphylococcus aureus via binding to cell surface lipoteichoic acids.

    Science.gov (United States)

    Omae, Yosuke; Hanada, Yuichi; Sekimizu, Kazuhisa; Kaito, Chikara

    2013-08-30

    We previously reported that a silkworm hemolymph protein, apolipophorin (ApoLp), binds to the cell surface of Staphylococcus aureus and inhibits expression of the saePQRS operon encoding a two-component system, SaeRS, and hemolysin genes. In this study, we investigated the inhibitory mechanism of ApoLp on S. aureus hemolysin gene expression. ApoLp bound to lipoteichoic acids (LTA), an S. aureus cell surface component. The addition of purified LTA to liquid medium abolished the inhibitory effect of ApoLp against S. aureus hemolysin production. In an S. aureus knockdown mutant of ltaS encoding LTA synthetase, the inhibitory effects of ApoLp on saeQ expression and hemolysin production were attenuated. Furthermore, the addition of anti-LTA monoclonal antibody to liquid medium decreased the expression of S. aureus saeQ and hemolysin genes. In S. aureus strains expressing SaeS mutant proteins with a shortened extracellular domain, ApoLp did not decrease saeQ expression. These findings suggest that ApoLp binds to LTA on the S. aureus cell surface and inhibits S. aureus hemolysin gene expression via a two-component regulatory system, SaeRS.

  15. Antimicrobial proteins from snake venoms: direct bacterial damage and activation of innate immunity against Staphylococcus aureus skin infection.

    Science.gov (United States)

    Samy, R P; Stiles, B G; Gopalakrishnakone, P; Chow, V T K

    2011-01-01

    The innate immune system is the first line of defense against microbial diseases. Antimicrobial proteins produced by snake venoms have recently attracted significant attention due to their relevance to bacterial infection and potential development into new therapeutic agents. Staphylococcus aureus is one of the major human pathogens causing a variety of infections involving pneumonia, toxic shock syndrome, and skin lesions. With the recent emergence of methicillin (MRSA) and vancomycin (VRSA) resistance, S. aureus infection is a serious clinical problem that will have a grave socio-economic impact in the near future. Although S. aureus susceptibility to innate antimicrobial peptides has been reported recently, the protective effect of snake venom phospholipase A₂ (svPLA₂) proteins on the skin from S. aureus infection has been understudied. This review details the protective function of svPLA₂s derived from venoms against skin infections caused by S. aureus. We have demonstrated in vivo that local application of svPLA₂ provides complete clearance of S. aureus within 2 weeks after treatment compared to fusidic acid ointment (FAO). In vitro experiments also demonstrate that svPLA₂ proteins have inhibitory (bacteriostatic) and killing (bactericidal) effects on S. aureus in a dose-dependant manner. The mechanism of bacterial membrane damage and perturbation was clearly evidenced by electron microscopic studies. In summary, svPLA₂s from Viperidae and Elapidae snakes are novel molecules that can activate important mechanisms of innate immunity in animals to endow them with protection against skin infection caused by S. aureus.

  16. Staphylococcus epidermidis Esp degrades specific proteins associated with Staphylococcus aureus biofilm formation and host-pathogen interaction.

    Science.gov (United States)

    Sugimoto, Shinya; Iwamoto, Takeo; Takada, Koji; Okuda, Ken-Ichi; Tajima, Akiko; Iwase, Tadayuki; Mizunoe, Yoshimitsu

    2013-04-01

    Staphylococcus aureus exhibits a strong capacity to attach to abiotic or biotic surfaces and form biofilms, which lead to chronic infections. We have recently shown that Esp, a serine protease secreted by commensal Staphylococcus epidermidis, disassembles preformed biofilms of S. aureus and inhibits its colonization. Esp was expected to degrade protein determinants of the adhesive and cohesive strength of S. aureus biofilms. The aim of this study was to elucidate the substrate specificity and target proteins of Esp and thereby determine the mechanism by which Esp disassembles S. aureus biofilms. We used a mutant Esp protein (Esp(S235A)) with defective proteolytic activity; this protein did not disassemble the biofilm formed by a clinically isolated methicillin-resistant S. aureus (MRSA) strain, thereby indicating that the proteolytic activity of Esp is essential for biofilm disassembly. Esp degraded specific proteins in the biofilm matrix and cell wall fractions, in contrast to proteinase K, which is frequently used for testing biofilm robustness and showed no preference for proteolysis. Proteomic and immunological analyses showed that Esp degrades at least 75 proteins, including 11 biofilm formation- and colonization-associated proteins, such as the extracellular adherence protein, the extracellular matrix protein-binding protein, fibronectin-binding protein A, and protein A. In addition, Esp selectively degraded several human receptor proteins of S. aureus (e.g., fibronectin, fibrinogen, and vitronectin) that are involved in its colonization or infection. These results suggest that Esp inhibits S. aureus colonization and biofilm formation by degrading specific proteins that are crucial for biofilm construction and host-pathogen interaction.

  17. Structural basis for hemoglobin capture by Staphylococcus aureus cell-surface protein, IsdH.

    Science.gov (United States)

    Krishna Kumar, Kaavya; Jacques, David A; Pishchany, Gleb; Caradoc-Davies, Tom; Spirig, Thomas; Malmirchegini, G Reza; Langley, David B; Dickson, Claire F; Mackay, Joel P; Clubb, Robert T; Skaar, Eric P; Guss, J Mitchell; Gell, David A

    2011-11-01

    Pathogens must steal iron from their hosts to establish infection. In mammals, hemoglobin (Hb) represents the largest reservoir of iron, and pathogens express Hb-binding proteins to access this source. Here, we show how one of the commonest and most significant human pathogens, Staphylococcus aureus, captures Hb as the first step of an iron-scavenging pathway. The x-ray crystal structure of Hb bound to a domain from the Isd (iron-regulated surface determinant) protein, IsdH, is the first structure of a Hb capture complex to be determined. Surface mutations in Hb that reduce binding to the Hb-receptor limit the capacity of S. aureus to utilize Hb as an iron source, suggesting that Hb sequence is a factor in host susceptibility to infection. The demonstration that pathogens make highly specific recognition complexes with Hb raises the possibility of developing inhibitors of Hb binding as antibacterial agents.

  18. Iron-regulated biofilm formation in Staphylococcus aureus Newman requires ica and the secreted protein Emp.

    Science.gov (United States)

    Johnson, Miranda; Cockayne, Alan; Morrissey, Julie A

    2008-04-01

    Staphylococcus aureus biofilm formation is induced in iron-restricted growth conditions in vitro. In this study, we showed that Emp and Eap play important roles in low-iron-induced biofilm formation of S. aureus Newman. Eap and Emp are secreted proteins which are non-covalently attached to the S. aureus cell surface and have previously been implicated in a number of aspects of S. aureus pathogenesis. We showed here that the transcription of these important virulence factors is induced by growth in low-iron medium, reflective of the in vivo environment. Our results show that iron regulation of Eap and Emp is Fur independent. However, Fur is required for full induction of eap and emp expression in low-iron conditions. In this study, we demonstrated that in addition to Fur, low-iron-induced biofilm formation requires Sae, Agr, and SarA. In iron-restricted growth conditions, Sae and Agr are essential for Emp and Eap expression and hence for biofilm formation, whereas SarA appears to have a less-significant role. We also showed that expression of the ica operon is required for biofilm formation in iron-restricted growth conditions. We demonstrated that in fact, ica is required for the expression of the important multifunctional virulence determinants eap and emp.

  19. Isolation of a membrane protein complex for type VII secretion in Staphylococcus aureus.

    Science.gov (United States)

    Aly, Khaled A; Anderson, Mark; Ohr, Ryan Jay; Missiakas, Dominique

    2017-09-05

    The ESAT6-like secretion system (ESS) of Staphylococcus aureus promotes effector protein transport across the bacterial envelope. Genes in the ESS cluster are required for S. aureus establishment of persistent abscess lesions and the modulation of immune responses during blood stream infections. The biochemical functions of most of the ESS gene products, specifically the identity of secretion machine components, are however unknown. Earlier work demonstrated that deletion of essB, which encodes a membrane protein, abolishes S. aureus ESS secretion. Loss of function mutations truncating the essB gene product cause dominant-negative phenotypes on ESS secretion, suggesting that EssB may be a central component of the secretion machinery. To test this prediction, we purified native and affinity-tagged EssB from staphylococcal membranes via dodecyl-maltoside extraction, identifying a complex assembled from five proteins, EsaA, EssA, EssB, EssD and EsxA. All five proteins are essential for secretion, as knock-out mutations in the corresponding genes abolish ESS transport. Biochemical and bacterial two-hybrid analyses revealed a direct interaction between EssB and EsaA that, by engaging a mobile machine component, EsxA, may also recruit EssA and EssD.IMPORTANCE Type VII secretion systems support the lifestyle of Gram-positive bacteria, including important human pathogens such as Bacillus anthracis, Mycobacterium tuberculosis, and Staphylococcus aureus Genes encoding SpoIIIE-FtsK-like ATPases and WXG100 secreted products are conserved features of type VII secretion pathways, however most of the genes in T7SS clusters are not conserved between different bacterial species. Here we isolate a complex of proteins from the membranes of S. aureus that appears to represent the core secretion machinery, designated ESS (ESAT-6-like secretion system). These results suggest that three membrane proteins, EsaA, EssB and EssA form a secretion complex that associates with EssC, the Spo

  20. The Staphylococcus aureus Protein IsdH Inhibits Host Hemoglobin Scavenging to Promote Heme Acquisition by the Pathogen

    DEFF Research Database (Denmark)

    Saederup, Kirstine Lindhardt; Stødkilde-Jørgensen, Kristian; Graversen, Jonas Heilskov;

    2016-01-01

    Hemolysis is a complication in septic infections with Staphylococcus aureus, which utilizes the released Hb as an iron source. S. aureus can acquire heme in vitro from hemoglobin (Hb) by a heme-sequestering mechanism that involves proteins from the S. aureus iron-regulated surface determinant (Isd....... By binding and uptake studies, we now show that the IsdH protein, which serves as an Hb receptor in the Isd system, directly interferes with the CD163-mediated clearance by binding the Hb-Hp complex and inhibiting CD163 recognition. Analysis of truncated IsdH variants including one or more of three near iron...

  1. Two-plasmid vector system for independently controlled expression of green and red fluorescent fusion proteins in Staphylococcus aureus.

    Science.gov (United States)

    Brzoska, Anthony J; Firth, Neville

    2013-05-01

    We have constructed a system for the regulated coexpression of green fluorescent protein (GFP) and red fluorescent protein (RFP) fusions in Staphylococcus aureus. It was validated by simultaneous localization of cell division proteins FtsZ and Noc and used to detect filament formation by an actin-like ParM plasmid partitioning protein in its native coccoid host.

  2. Karakterisasi Staphylococcus aureus Isolat Susu Sapi Perah Berdasar Keberadaan Protein-A pada Media Serum Soft Agar terhadap Aktivitas Fagositosis Secara In Vitro

    Directory of Open Access Journals (Sweden)

    Fajar Budi Lestari

    2017-02-01

    kompak dan 7 isolat (36,84% koloni berbentuk difus. Koloni kompak menunjukkan bakteri tersebut memiliki protein-A, koloni difus menunjukkan bakteri tersebut tidak memiliki protein-A atau memiliki protein-A tetapi tertutup oleh kapsul. Hasil uji fagositosis menunjukkan S. aureus yang memiliki protein-A lebih sedikit difagosit oleh leukosit polimorfonuklear (2,99 bakteri/sel dari pada S. aureus yang tidak memiliki protein-A, atau mempunyai protein-A tetapi tertutup oleh kapsul (3,85 bakteri/sel. Staphylococcus aureus yang memiliki protein-A lebih patogen daripada S. aureus yang tidak memiliki protein-A. Isolat S. aureus asal Jawa Tengah lebih virulen  dibandingkan isolat S. aureus asal Jawa Barat ditinjau dari sifat hemolisis, koagulase, dan  protein-A.

  3. Sequence diversity in the A domain of Staphylococcus aureus fibronectin-binding protein A

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    Speziale Pietro

    2008-05-01

    Full Text Available Abstract Background Fibronectin-binding protein A (FnBPA mediates adhesion of Staphylococcus aureus to fibronectin, fibrinogen and elastin. We previously reported that S. aureus strain P1 encodes an FnBPA protein where the fibrinogen/elastin-binding domain (A domain is substantially divergent in amino acid sequence from the archetypal FnBPA of S. aureus NCTC8325, and that these variations created differences in antigenicity. In this study strains from multilocus sequence types (MLST that spanned the genetic diversity of S.aureus were examined to determine the extent of FnBPA A domain variation within the S. aureus population and its effect on ligand binding and immuno-crossreactivity. Results Seven different isotype forms (I – VII of the FnBPA A domain were identified which were between 66 to 76% identical in amino acid sequence in any pair-wise alignment. The fnbA allelic variants in strains of different multilocus sequence type were identified by DNA hybridization using probes specific for sequences encoding the highly divergent N3 sub-domain of different isotypes. Several isotypes were not restricted to specific clones or clonal complexes but were more widely distributed. It is highly likely that certain fnbA genes have been transferred horizontally. Residues lining the putative ligand-binding trench were conserved, which is consistent with the ability of each A domain isotype to bind immobilized fibrinogen and elastin by the dock-latch-lock mechanism. Variant amino acid residues were mapped on a three-dimensional model of the FnBPA A domain and were predicted to be surface-exposed. Polyclonal antibodies raised against the recombinant isotype I A domain bound that protein with a 4 – 7 fold higher apparent affinity compared to the A domains of isotypes II – VII, while some monoclonal antibodies generated against the isotype I A domain showed reduced or no binding to the other isotypes. Conclusion The FnBPA A domain occurs in at least 7

  4. Rotating wall vessel exposure alters protein secretion and global gene expression in Staphylococcus aureus

    Science.gov (United States)

    Rosado, Helena; O'Neill, Alex J.; Blake, Katy L.; Walther, Meik; Long, Paul F.; Hinds, Jason; Taylor, Peter W.

    2012-04-01

    Staphylococcus aureus is routinely recovered from air and surface samples taken aboard the International Space Station (ISS) and poses a health threat to crew. As bacteria respond to the low shear forces engendered by continuous rotation conditions in a Rotating Wall Vessel (RWV) and the reduced gravitational field of near-Earth flight by altering gene expression, we examined the effect of low-shear RWV growth on protein secretion and gene expression by three S. aureus isolates. When cultured under 1 g, the total amount of protein secreted by these strains varied up to fourfold; under continuous rotation conditions, protein secretion by all three strains was significantly reduced. Concentrations of individual proteins were differentially reduced and no evidence was found for increased lysis. These data suggest that growth under continuous rotation conditions reduces synthesis or secretion of proteins. A limited number of changes in gene expression under continuous rotation conditions were noted: in all isolates vraX, a gene encoding a polypeptide associated with cell wall stress, was down-regulated. A vraX deletion mutant of S. aureus SH1000 was constructed: no differences were found between SH1000 and ΔvraX with respect to colony phenotype, viability, protein export, antibiotic susceptibility, vancomycin kill kinetics, susceptibility to cold or heat and gene modulation. An ab initio protein-ligand docking simulation suggests a major binding site for β-lactam drugs such as imipenem. If such changes to the bacterial phenotype occur during spaceflight, they will compromise the capacity of staphylococci to cause systemic infection and to circumvent antibacterial chemotherapy.

  5. Role of biofilm-associated protein bap in the pathogenesis of bovine Staphylococcus aureus.

    Science.gov (United States)

    Cucarella, Carme; Tormo, M Angeles; Ubeda, Carles; Trotonda, M Pilar; Monzón, Marta; Peris, Critòfol; Amorena, Beatriz; Lasa, Iñigo; Penadés, José R

    2004-04-01

    Staphylococcus aureus is a common cause of intramammary infections, which frequently become chronic, associated with the ability of the bacteria to produce biofilm. Here, we report a relationship between the ability to produce chronic bovine mastitis and biofilm formation. We have classified bovine mastitis S. aureus isolates into three groups based on the presence of particular genetic elements required for biofilm formation: group 1 (ica(+) bap(+)), group 2 (ica(+), bap negative), and group 3 (ica negative, bap negative). Overall, animals naturally infected with group 1 and 2 isolates had a lower milk somatic cell count than those infected with isolates of group 3. In addition, Bap-positive isolates were significantly more able to colonize and persist in the bovine mammary gland in vivo and were less susceptible to antibiotic treatments when forming biofilms in vitro. Analysis of the structural bap gene revealed the existence of alternate forms of expression of the Bap protein in S. aureus isolates obtained under field conditions throughout the animal's life. The presence of anti-Bap antibodies in serum samples taken from animals with confirmed S. aureus infections indicated the production of Bap during infection. Furthermore, disruption of the ica operon in a bap-positive strain had no effect on in vitro biofilm formation, a finding which strongly suggested that Bap could compensate for the deficiency of the PIA/PNAG product (a biofilm matrix polysaccharide). Altogether, these results demonstrate that, in the bovine intramammary gland, the presence of Bap may facilitate a biofilm formation connected with the persistence of S. aureus.

  6. Molecular typing of nosocomial Staphylococcus aureus strains associated to biofilm based on the coagulase and protein A gene polymorphisms

    Science.gov (United States)

    Salehzadeh, Ali; Zamani, Hojjatolah; Langeroudi, Maedeh Keshtkar; Mirzaie, Amir

    2016-01-01

    Objective(s): Staphylococcus aureus is an important bacterial pathogen responsible for a variety numbers of nosocomial and community acquired infections. Biofilm formation is regarded as an important factor in the establishment of S. aureus infection. The contribution of the genetic background of S. aureus to biofilm formation is poorly understood. The aim of the present work was to genotype S. aureus strains associated to biofilm based on the coagulase and protein A genes and to evaluate the association between the genetic background and the biofilm forming ability of clinical S. aureus isolates. Materials and Methods: A total number of 100 S. aureus were isolated from nosocomial infections and biofilm formation capability was investigated using phenotypic assay and molecular detection of biofilm associated genes. The strains were genotyped based on coagulase (coa) and protein A (spa) gene polymorphisms using restriction fragments length polymorphism-polymerase chain reaction (RFLP-PCR). Results: RFLP-PCR of coa gene generated two types and three subtypes. Amplification of spa gene resulted in two banding patterns and their restriction digestion generated three subtypes. The combined coa and spa RFLP patterns generated nine genotypes (G1-G9). The genotypes G4 and G1 were the most prevalent (32.1% and 24.3%, respectively). Conclusion: High clonal diversity of S. aureus strains able to produce biofilm was observed. Biofilm formation correlates with the spa and coa clonal lineage in our population and testing for multiple gene polymorphisms could be employed for local epidemiologic purposes. PMID:28096965

  7. Molecular typing of nosocomial Staphylococcus aureus strains associated to biofilm based on the coagulase and protein A gene polymorphisms

    Directory of Open Access Journals (Sweden)

    Ali Salehzadeh

    2016-12-01

    Full Text Available Objective(s: Staphylococcus aureus is an important bacterial pathogen responsible for a variety numbers of nosocomial and community acquired infections. Biofilm formation is regarded as an important factor in the establishment of S. aureus infection. The contribution of the genetic background of S. aureus to biofilm formation is poorly understood. The aim of the present work was to genotype S. aureus strains associated to biofilm based on the coagulase and protein A genes and to evaluate the association between the genetic background and the biofilm forming ability of clinical S. aureus isolates. Materials and Methods: A total number of 100 S. aureus were isolated from nosocomial infections and biofilm formation capability was investigated using phenotypic assay and molecular detection of biofilm associated genes. The strains were genotyped based on coagulase (coa and protein A (spa gene polymorphisms using restriction fragments length polymorphism-polymerase chain reaction (RFLP-PCR. Results: RFLP-PCR of coa gene generated two types and three subtypes. Amplification of spa gene resulted in two banding patterns and their restriction digestion generated three subtypes. The combined coa and spa RFLP patterns generated nine genotypes (G1-G9. The genotypes G4 and G1 were the most prevalent (32.1% and 24.3%, respectively. Conclusion: High clonal diversity of S. aureus strains able to produce biofilm was observed. Biofilm formation correlates with the spa and coa clonal lineage in our population and testing for multiple gene polymorphisms could be employed for local epidemiologic purposes.

  8. Antibacterial effect of silver nanoparticles along with protein synthesis-inhibiting antibiotics on Staphylococcus aureus isolated from cattle mastitis

    Directory of Open Access Journals (Sweden)

    Malahat Ahmadi

    2014-01-01

    Full Text Available Introduction: Staphylococcus aureus is an opportunistic pathogen in dairy ruminants which is also found in healthy carriage and can be a major cause of mastitis. Various mastitis control programs have been used to combat the problem but have not always been efficient. In most countries, antibiotic resistance is extremely common. Silver nanoparticles have shown antimicrobial activity against S. aureus. In the present study the effect of silver nanoparticles on S. aureus isolated from cattle mastitis along with antibiotics of operative on protein bacterial synthesis investigated. Materials and methods: Three hundred eleven milk samples were collected from the cow farms. Each milk sample was cultured on mannitol salt agar and was incubated. A total of 72 isolates of S. aureus were isolated from the bovine mastitis milk samples. S. aureus DNA extracted by DNA purification kit according to the manufacturer protocol. 58 isolates were confirmed as S. aureus by biochemical tests as well as nuc gene detection. MIC and MBC determined for silver nanoparticles with antibiotics on 50 isolates. Results: The resistance of S. aureus isolates against erythromycin, gentamicin, streptomycin and doxycycline were 100, 22, 100 and 8%, respectively. 8 of all isolates were sensitive to 25 µg/ml concentration of silver nanoparticles. The 92% growth of the samples were inhibited at concentrations between 50-100 µg/ml. Discussion and conclusion: The present study suggests that antibiotics which can inhibit protein synthesis have significant synergistic effect along with silver nanoparticles.

  9. Changes in Holstein cow milk and serum proteins during intramammary infection with three different strains of Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Robert Claude

    2011-09-01

    Full Text Available Abstract Background Staphylococcus aureus is one of the most prevalent pathogens to cause mastitis in dairy cattle. Intramammary infection of dairy cows with S. aureus is often subclinical, due to the pathogen's ability to evade the innate defense mechanisms, but this can lead to chronic infection. A sub-population of S. aureus, known as small colony variant (SCV, displays atypical phenotypic characteristics, causes persistent infections, and is more resistant to antibiotics than parent strains. Therefore, it was hypothesized that the host immune response will be different for SCV than its parental or typical strains of S. aureus. In this study, the local and systemic immune protein responses to intramammary infection with three strains of S. aureus, including a naturally occurring bovine SCV strain (SCV Heba3231, were characterized. Serum and casein-depleted milk cytokine levels (interleukin-8, interferon-γ, and transforming growth factor-β1, as well as serum haptoglobin concentrations were monitored over time after intramammary infection with each of the three S. aureus strains. Furthermore, comparative proteomics was used to evaluate milk proteome profiles during acute and chronic phases of S. aureus intramammary infection. Results Serum IL-8, IFN-γ, and TGF-β1 responses differed in dairy cows challenged with different strains of S. aureus. Changes in overall serum haptoglobin concentrations were observed for each S. aureus challenge group, but there were no significant differences observed between groups. In casein-depleted milk, strain-specific differences in the host IFN-γ response were observed, but inducible IL-8 and TGF-β1 concentrations were not different between groups. Proteomic analysis of the milk following intramammary infection revealed unique host protein expression profiles that were dependent on the infecting strain as well as phase of infection. Notably, the protein, component-3 of the proteose peptone (CPP3, was

  10. Phase-variable expression of the biofilm-associated protein (Bap) in Staphylococcus aureus.

    Science.gov (United States)

    Tormo, M Angeles; Ubeda, Carles; Martí, Miguel; Maiques, Elisa; Cucarella, Carme; Valle, Jaione; Foster, Timothy J; Lasa, Iñigo; Penadés, José R

    2007-06-01

    A process of phase variation is described that affects the expression of Bap (biofilm-associated protein) in Staphylococcus aureus. Upon subculture of the Bap-positive S. aureus strain V329 on Congo red agar, spontaneous smooth biofilm-negative colonies appeared at a low frequency (5 x 10(-4)). Northern blot analysis of these variants with a bap-specific gene probe showed that transcription of the bap gene did not occur. However, DNA typing, Southern blot hybridization and DNA sequencing did not show any differences between the parent V329 strain and the biofilm-negative variants. The biofilm-negative phenotype reverted to wild-type at a similar frequency upon subculture of Bap-negative variants in liquid media. Experimental infection of ovine mammary glands with Bap-negative variants showed that phase variation occurred in vivo, because Bap-expressing, biofilm-positive revertants were isolated from infected mammary glands. The absence of Bap correlated with increased adherence to fibrinogen and fibronectin. It is possible that S. aureus can detach from a biofilm by switching to a Bap-negative state.

  11. Effective interaction studies for inhibition of DNA ligase protein from Staphylococcus aureus.

    Science.gov (United States)

    Vijayalakshmi, Periyasamy; Daisy, Pitchai

    2015-02-01

    Staphylococcus aureus has been recognized as an important human pathogen for more than 100 years. It is among the most important causative agent of human infections in the twenty-first century. DNA ligase is the main protein responsible for the replication of S. aureus. In order to control the replication mechanism, DNA ligase is a successive drug target, hence we have chosen this protein for this study. We performed virtual screening using ZINC database for identification of potent inhibitor against DNA ligase. Based on the scoring methods, we have selected best five compounds from the ZINC database. In order to improve the accuracy, selected compounds were subjected into Quantum Polarized Ligand Docking (QPLD) docking, for which the results showed high docking score, compared to glide docking score. QPLD is more accurate as it includes charges in the scoring function, which was not available in the glide docking. Binding energy calculation results also indicated that selected compounds have good binding capacity with the target protein. In addition, these compounds on screening have good absorption, distribution, metabolism, excretion and toxicity property. In this study, we identified few compounds that particularly work against DNA ligase protein, having better interaction phenomenon and it would help further the experimental analysis.

  12. The Sbi protein is a multifunctional immune evasion factor of Staphylococcus aureus.

    Science.gov (United States)

    Smith, Emma Jane; Visai, Livia; Kerrigan, Steven W; Speziale, Pietro; Foster, Timothy J

    2011-09-01

    The second immunoglobulin-binding protein (Sbi) of Staphylococcus aureus has two N-terminal domains that bind the Fc region of IgG in a fashion similar to that of protein A and two domains that can bind to the complement protein C3 and promote its futile consumption in the fluid phase. It has been proposed that Sbi helps bacteria to avoid innate immune defenses. By comparing a mutant defective in Sbi with mutants defective in protein A, clumping factor A, iron-regulated surface determinant H, and capsular polysaccharide, it was shown that Sbi is indeed an immune evasion factor that promotes bacterial survival in whole human blood and the avoidance of neutrophil-mediated opsonophagocytosis. Sbi is present in the culture supernatant and is also associated with the cell envelope. S. aureus strains that expressed truncates of Sbi lacking N-terminal domains D1 and D2 (D1D2) or D3 and D4 (D3D4) or a C-terminal truncate that was no longer retained in the cell envelope were analyzed. Both the secreted and envelope-associated forms of Sbi contributed to immune evasion. The IgG-binding domains contributed only when Sbi was attached to the cell, while only the secreted C3-binding domains were biologically active.

  13. Purification and activity testing of the full-length YycFGHI proteins of Staphylococcus aureus.

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    Michael Türck

    Full Text Available BACKGROUND: The YycFG two-component regulatory system (TCS of Staphylococcus aureus represents the only essential TCS that is almost ubiquitously distributed in gram-positive bacteria with a low G+C-content. YycG (WalK/VicK is a sensor histidine-kinase and YycF (WalR/VicR is the cognate response regulator. Both proteins play an important role in the biosynthesis of the cell envelope and mutations in these proteins have been involved in development of vancomycin and daptomycin resistance. METHODOLOGY/PRINCIPAL FINDINGS: Here we present high yield expression and purification of the full-length YycG and YycF proteins as well as of the auxiliary proteins YycH and YycI of Staphylococcus aureus. Activity tests of the YycG kinase and a mutated version, that harbours an Y306N exchange in its cytoplasmic PAS domain, in a detergent-micelle-model and a phosholipid-liposome-model showed kinase activity (autophosphorylation and phosphoryl group transfer to YycF only in the presence of elevated concentrations of alkali salts. A direct comparison of the activity of the kinases in the liposome-model indicated a higher activity of the mutated YycG kinase. Further experiments indicated that YycG responds to fluidity changes in its microenvironment. CONCLUSIONS/SIGNIFICANCE: The combination of high yield expression, purification and activity testing of membrane and membrane-associated proteins provides an excellent experimental basis for further protein-protein interaction studies and for identification of all signals received by the YycFGHI system.

  14. Life and death of proteins: a case study of glucose-starved Staphylococcus aureus.

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    Michalik, Stephan; Bernhardt, Jörg; Otto, Andreas; Moche, Martin; Becher, Dörte; Meyer, Hanna; Lalk, Michael; Schurmann, Claudia; Schlüter, Rabea; Kock, Holger; Gerth, Ulf; Hecker, Michael

    2012-09-01

    The cellular amount of proteins not only depends on synthesis but also on degradation. Here, we expand the understanding of differential protein levels by complementing synthesis data with a proteome-wide, mass spectrometry-based stable isotope labeling with amino acids in cell culture analysis of protein degradation in the human pathogen Staphylococcus aureus during glucose starvation. Monitoring protein stability profiles in a wild type and an isogenic clpP protease mutant revealed that 1) proteolysis mainly affected proteins with vegetative functions, anabolic and selected catabolic enzymes, whereas the expression of TCA cycle and gluconeogenesis enzymes increased; 2) most proteins were prone to aggregation in the clpP mutant; 3) the absence of ClpP correlated with protein denaturation and oxidative stress responses, deregulation of virulence factors and a CodY repression. We suggest that degradation of redundant, inactive proteins disintegrated from functional complexes and thereby amenable to proteolytic attack is a fundamental cellular process in all organisms to regain nutrients and guarantee protein homeostasis.

  15. Impact of Staphylococcus aureus protein A (spa) genetic typing in cases of prosthetic shunt graft infections.

    Science.gov (United States)

    Konstantiniuk, P; Grisold, A; Schramayer, G; Santler, S C; Koter, S; Cohnert, T

    In January 2014 an internal audit was performed at the department of surgery, division of vascular surgery of the Medical University Graz, Austria, to assess the short and long-term outcomes of prosthetic shunt graft implantations performed between December 1998 and December 2013. A 10.8 % explantation rate due to graft infection was detected. The majority of the cases were associated with Staphylococcus aureus. The aim of this study was to clarify whether this constitutes a nosocomial problem. Between December 1998 and December 2014 a total of 490 prosthetic shunt grafts were implanted. After exclusion of 54 cases, 436 shunts remained for statistical analysis. Genetic analysis (spa typing) was acquired from three new cases with involvement of S. aureus in 2014. The impact of several factors (e.g. sex, institute for dialysis, age, type of prosthesis, implantation surgeon and position of shunt) on the shunt graft infection rate was statistically analyzed. Of the prostheses 14.0 % (61 out of 436) had to be explanted of which 12.4 % (54/436) were due to infection. In 77.8 % (42/54) bacteria were found in blood and/or wound cultures. Staphylococcus aureus was present in 76.2 % (32/42) of the cases with detected bacteria and in all cases was sensitive to methicillin. The infection rate was not significantly dependent on any of the investigated factors; however, the factor "institute for dialysis" had a remarkable p‑value of 0.060 with the infection rate ranging from 8.5 % to 18.2 % depending on the institution. Three different S. aureus protein A (spa) types were found: t015, t359, t6265. The detection of three different spa types means that these patients had different sources of S. aureus so that a nosocomial problem is very unlikely. Genetic typing of spa is a suitable technique for distinguishing between nosocomial and community acquired sources of prosthetic shunt graft infections.

  16. Polymorphisms in Fibronectin Binding Protein A of Staphylococcus Aureus are Associated with Infection of Cardiovascular Devices

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    Lower, Steven; Lamlertthon, Supaporn; Casillas-Ituarte, Nadia; Lins, Roberto D.; Yongsunthon, Ruchirej; Taylor, Eric S.; DiBartola, Alex; Edmondson, Catherine; McIntyre, Lauren M.; Reller, L. Barth; Que, Yok-Ai; Ros, Robert; Lower, Brian; Fowler, Vance

    2011-11-08

    Medical implants, like cardiovascular devices, improve the quality of life for countless individuals but may become infected with bacteria like Staphylococcus aureus. Such infections take the form of a bio-film, a structured community of bacterial cells adherent to the surface of a solid substrate. Every bio-film begins with an attractive force or bond between bacterium and substratum. We used atomic force microscopy to probe experimentally forces between a fibronectin-coated surface (i.e., proxy for an implanted cardiac device) and fibronectin-binding receptors on the surface of individual living bacteria from each of 80 clinical isolates of S. aureus. These isolates originated from humans with infected cardiac devices (CDI; n = 26), uninfected cardiac devices (n = 20), and the anterior nares of asymptomatic subjects (n = 34). CDI isolates exhibited a distinct bindingforce signature and had speci!c single amino acid polymorphisms in fibronectin-binding protein A corresponding to E652D, H782Q, and K786N. In silico molecular dynamics simulations demonstrate that residues D652, Q782, and N786 in fibronectin-binding protein A form extra hydrogen bonds with fibronectin, complementing the higher binding force and energy measured by atomic force microscopy for the CDI isolates. This study is significant, because it links pathogenic bacteria biofilms from the length scale of bonds acting across a nanometer-scale space to the clinical presentation of disease at the human dimension.

  17. The Plasmin-Sensitive Protein Pls in Methicillin-Resistant Staphylococcus aureus (MRSA) Is a Glycoprotein

    Science.gov (United States)

    Pohlentz, Gottfried; Xia, Guoqing; Hussain, Muzaffar; Foster, Simon; Peters, Georg

    2017-01-01

    Most bacterial glycoproteins identified to date are virulence factors of pathogenic bacteria, i.e. adhesins and invasins. However, the impact of protein glycosylation on the major human pathogen Staphylococcus aureus remains incompletely understood. To study protein glycosylation in staphylococci, we analyzed lysostaphin lysates of methicillin-resistant Staphylococcus aureus (MRSA) strains by SDS-PAGE and subsequent periodic acid-Schiff’s staining. We detected four (>300, ∼250, ∼165, and ∼120 kDa) and two (>300 and ∼175 kDa) glycosylated surface proteins with strain COL and strain 1061, respectively. The ∼250, ∼165, and ∼175 kDa proteins were identified as plasmin-sensitive protein (Pls) by mass spectrometry. Previously, Pls has been demonstrated to be a virulence factor in a mouse septic arthritis model. The pls gene is encoded by the staphylococcal cassette chromosome (SCC)mec type I in MRSA that also encodes the methicillin resistance-conferring mecA and further genes. In a search for glycosyltransferases, we identified two open reading frames encoded downstream of pls on the SCCmec element, which we termed gtfC and gtfD. Expression and deletion analysis revealed that both gtfC and gtfD mediate glycosylation of Pls. Additionally, the recently reported glycosyltransferases SdgA and SdgB are involved in Pls glycosylation. Glycosylation occurs at serine residues in the Pls SD-repeat region and modifying carbohydrates are N-acetylhexosaminyl residues. Functional characterization revealed that Pls can confer increased biofilm formation, which seems to involve two distinct mechanisms. The first mechanism depends on glycosylation of the SD-repeat region by GtfC/GtfD and probably also involves eDNA, while the second seems to be independent of glycosylation as well as eDNA and may involve the centrally located G5 domains. Other previously known Pls properties are not related to the sugar modifications. In conclusion, Pls is a glycoprotein and Pls glycosyl

  18. Effect of gamma irradiation on the expressed proteins in the foodborne pathogen Staphylococcus aureus

    Science.gov (United States)

    Trudeau, Karine; Dang Vu, Khanh; Shareck, François; Lacroix, Monique

    2012-08-01

    A capillary electrophoresis method with UV detection was developed to analyze protein composition of the foodborne pathogen Staphylococcus aureus. Bacterial samples containing 109 CFU/ml, obtained after two cycles of incubations of 24 h, were gamma irradiated at different doses of 1.2, 3.5 and 2.9 kGy to respectively create damage cells, to kill cells and to provoke viable but non cultivable cells (VBNC). It was observed that an irradiation at a sensitive dose of 1.2 kGy caused a significantly increase in the protein with molecular weight (MW) of 17.7 kDa (from 0.61% to 1.2%). This treatment also caused decreases in the expressed proteins with the MWs of 16.3 kDa (from 6.2% to 5.3%) and of 23.4 kDa (from 4.0% to 2.30%). Irradiation at a VBCN dose of 2.9 kGy caused increases in expressed proteins with the MWs of 17.7 kDa (from 0.61% to 3.43%), 18.7 kDa (from 1.04% to 4.30%), 19.5 kDa (from 0.71% to 2.30%), 21.1 kDa (from 1.20% to 3.80%). Moreover, this treatment (2.9 kGy) also caused significantly decreases (P≤0.05) in the expressed proteins with the MW of 30.7 kDa (from 8.6% to 5.15%), 36.3 kDa (from 3.1% to 2.7%) and 40.5 kDa (from 11.3% to 8.5%). Finally, for the irradiation at a lethal dose of 3.5 kGy, it can be found that the expressed proteins with the MW of 17.7 kDa, 18.7 kDa and 19.5 kDa were increased less than that of expressed proteins at the VCNC dose (2.9 kGy) and these might be the very important proteins which are responsible for the survival of the S. aureus. Further, there were also the decreases in expressed proteins with the MW of 30.7 kDa, 36.3 kDa and 75.1 kDa at this dose of treatment (3.5 kGy) which can be expected that these proteins are seriously affected at high dose of γ-irradiation treatment.

  19. Gold nanoprobe functionalized with specific fusion protein selection from phage display and its application in rapid, selective and sensitive colorimetric biosensing of Staphylococcus aureus.

    Science.gov (United States)

    Liu, Pei; Han, Lei; Wang, Fei; Petrenko, Valery A; Liu, Aihua

    2016-08-15

    Staphylococcus aureus (S. aureus) is one of the most ubiquitous pathogens in public healthcare worldwide. It holds great insterest in establishing robust analytical method for S. aureus. Herein, we report a S. aureus-specific recognition element, isolated from phage monoclone GQTTLTTS, which was selected from f8/8 landscape phage library against S. aureus in a high-throughput way. By functionalizing cysteamine (CS)-stabilized gold nanoparticles (CS-AuNPs) with S. aureus-specific pVIII fusion protein (fusion-pVIII), a bifunctional nanoprobe (CS-AuNPs@fusion-pVIII) for S. aureus was developed. In this strategy, the CS-AuNPs@fusion-pVIII could be induced to aggregate quickly in the presence of target S. aureus, resulting in a rapid colorimetric response of gold nanoparticles. More importantly, the as-designed probe exhibited excellent selectivity over other bacteria. Thus, the CS-AuNPs@fusion-pVIII could be used as the indicator of target S. aureus. This assay can detect as low as 19CFUmL(-1)S. aureus within 30min. Further, this approach can be applicable to detect S. aureus in real water samples. Due to its sensitivity, specificity and rapidness, this proposed method is promising for on-site testing of S. aureus without using any costly instruments.

  20. Staphylococcus aureus proteins Sbi and Efb recruit human plasmin to degrade complement C3 and C3b.

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    Tina K Koch

    Full Text Available Upon host infection, the human pathogenic microbe Staphylococcus aureus (S. aureus immediately faces innate immune reactions such as the activated complement system. Here, a novel innate immune evasion strategy of S. aureus is described. The staphylococcal proteins surface immunoglobulin-binding protein (Sbi and extracellular fibrinogen-binding protein (Efb bind C3/C3b simultaneously with plasminogen. Bound plasminogen is converted by bacterial activator staphylokinase or by host-specific urokinase-type plasminogen activator to plasmin, which in turn leads to degradation of complement C3 and C3b. Efb and to a lesser extend Sbi enhance plasmin cleavage of C3/C3b, an effect which is explained by a conformational change in C3/C3b induced by Sbi and Efb. Furthermore, bound plasmin also degrades C3a, which exerts anaphylatoxic and antimicrobial activities. Thus, S. aureus Sbi and Efb comprise platforms to recruit plasmin(ogen together with C3 and its activation product C3b for efficient degradation of these complement components in the local microbial environment and to protect S. aureus from host innate immune reactions.

  1. A Staphylococcus aureus TIR domain protein virulence factor blocks TLR2-mediated NF-κB signaling.

    Science.gov (United States)

    Askarian, Fatemeh; van Sorge, Nina M; Sangvik, Maria; Beasley, Federico C; Henriksen, Jørn R; Sollid, Johanna U E; van Strijp, Jos A G; Nizet, Victor; Johannessen, Mona

    2014-01-01

    Signaling through Toll-like receptors (TLRs), crucial molecules in the induction of host defense responses, requires adaptor proteins that contain a Toll/interleukin-1 receptor (TIR) domain. The pathogen Staphylococcus aureus produces several innate immune-evasion molecules that interfere with the host's innate immune response. A database search analysis suggested the presence of a gene encoding a homologue of the human TIR domain in S. aureus MSSA476 which was named staphylococcal TIR domain protein (TirS). Ectopic expression of TirS in human embryonic kidney, macrophage and keratinocyte cell lines interfered with signaling through TLR2, including MyD88 and TIRAP, NF-κB and/or mitogen-activated protein kinase pathways. Moreover, the presence of TirS reduced the levels of cytokines MCP-1 and G-CSF secreted in response to S. aureus. The effects on NF-κB pathway were confirmed using S. aureus MSSA476 wild type, an isogenic mutant MSSA476ΔtirS, and complemented MSSA476ΔtirS +pTirS in a Transwell system where bacteria and host cells were physically separated. Finally, in a systematic mouse infection model, TirS promoted bacterial accumulation in several organs 4 days postinfection. The results of this study reveal a new S. aureus virulence factor that can interfere with PAMP-induced innate immune signaling in vitro and bacterial survival in vivo.

  2. Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins.

    Science.gov (United States)

    Lone, Abdul G; Atci, Erhan; Renslow, Ryan; Beyenal, Haluk; Noh, Susan; Fransson, Boel; Abu-Lail, Nehal; Park, Jeong-Jin; Gang, David R; Call, Douglas R

    2015-08-01

    A partial-thickness epidermal explant model was colonized with green fluorescent protein (GFP)-expressing Staphylococcus aureus, and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. The oxygen concentration in explants was quantified using microelectrodes. The relative effective diffusivity and porosity of the epidermis were determined using magnetic resonance imaging, while hydrogen peroxide (H2O2) concentration in explant media was measured by using microelectrodes. Secreted proteins were identified and quantified using elevated-energy mass spectrometry (MS(E)). S. aureus biofilm grows predominantly in lipid-rich areas around hair follicles and associated skin folds. Dissolved oxygen was selectively depleted (2- to 3-fold) in these locations, but the relative effective diffusivity and porosity did not change between colonized and control epidermis. Histological analysis revealed keratinocyte damage across all the layers of colonized epidermis after 4 days of culture. The colonized explants released significantly (P < 0.01) more antioxidant proteins of both epidermal and S. aureus origin, consistent with elevated H2O2 concentrations found in the media from the colonized explants (P< 0.001). Caspase-14 was also elevated significantly in the media from the colonized explants. While H2O2 induces primary keratinocyte differentiation, caspase-14 is required for terminal keratinocyte differentiation and desquamation. These results are consistent with a localized biological impact from S. aureus in response to colonization of the skin surface.

  3. Prevalence of Staphylococcus aureus protein A (spa) mutants in the community and hospitals in Oxfordshire.

    Science.gov (United States)

    Votintseva, Antonina A; Fung, Rowena; Miller, Ruth R; Knox, Kyle; Godwin, Heather; Wyllie, David H; Bowden, Rory; Crook, Derrick W; Walker, A Sarah

    2014-03-12

    Staphylococcal protein A (spa) is an important virulence factor which enables Staphylococcus aureus to evade host immune responses. Genotypes known as "spa-types", based on highly variable Xr region sequences of the spa-gene, are frequently used to classify strains. A weakness of current spa-typing primers is that rearrangements in the IgG-binding region of the gene cause 1-2% of strains to be designated as "non-typeable". We developed an improved primer which enabled sequencing of all strains, containing any type of genetic rearrangement, in a large study among community carriers and hospital inpatients in Oxfordshire, UK (6110 isolates). We identified eight novel spa-gene variants, plus one previously described. Three of these rearrangements would be designated "non-typeable" using current spa-typing methods; they occurred in 1.8% (72/3905) asymptomatically carried and 0.6% (14/2205) inpatient S. aureus strains. Some individuals were simultaneously colonized by both formerly non-typeable and typeable strains; previously such patients would have been identified as carrying only currently typeable strains, underestimating mixed carriage prevalence and diversity. Formerly non-typeable strains were found in more spa-types associated with multilocus sequence type ST398 (35%), common among livestock, compared to other groups with any non-typeable strains (1-4%), suggesting particular spa-types may have been under-represented in previous human studies. This improved method allows us to spa-type previously non-typeable strains with rearrangements in the spa-gene and to resolve cases of mixed colonization with deletions in one or more strains, thus accounting for hidden diversity of S. aureus in both community and hospital environments.

  4. Crystal structure of the capsular polysaccharide synthesizing protein CapE of Staphylococcus aureus.

    Science.gov (United States)

    Miyafusa, Takamitsu; Caaveiro, Jose M M; Tanaka, Yoshikazu; Tanner, Martin E; Tsumoto, Kouhei

    2013-06-11

    Enzymes synthesizing the bacterial CP (capsular polysaccharide) are attractive antimicrobial targets. However, we lack critical information about the structure and mechanism of many of them. In an effort to reduce that gap, we have determined three different crystal structures of the enzyme CapE of the human pathogen Staphylococcus aureus. The structure reveals that CapE is a member of the SDR (short-chain dehydrogenase/reductase) super-family of proteins. CapE assembles in a hexameric complex stabilized by three major contact surfaces between protein subunits. Turnover of substrate and/or coenzyme induces major conformational changes at the contact interface between protein subunits, and a displacement of the substrate-binding domain with respect to the Rossmann domain. A novel dynamic element that we called the latch is essential for remodelling of the protein-protein interface. Structural and primary sequence alignment identifies a group of SDR proteins involved in polysaccharide synthesis that share the two salient features of CapE: the mobile loop (latch) and a distinctive catalytic site (MxxxK). The relevance of these structural elements was evaluated by site-directed mutagenesis.

  5. Identification of putative drug targets in Vancomycin-resistant Staphylococcus aureus (VRSA) using computer aided protein data analysis.

    Science.gov (United States)

    Hasan, Md Anayet; Khan, Md Arif; Sharmin, Tahmina; Hasan Mazumder, Md Habibul; Chowdhury, Afrin Sultana

    2016-01-01

    Vancomycin-resistant Staphylococcus aureus (VRSA) is a Gram-positive, facultative aerobic bacterium which is evolved from the extensive exposure of Vancomycin to Methicillin resistant S. aureus (MRSA) that had become the most common cause of hospital and community-acquired infections. Due to the emergence of different antibiotic resistance strains, there is an exigency to develop novel drug targets to address the provocation of multidrug-resistant bacteria. In this study, in-silico genome subtraction methodology was used to design potential and pathogen specific drug targets against VRSA. Our study divulged 1987 proteins from the proteome of 34,549 proteins, which have no homologues in human genome after sequential analysis through CD-HIT and BLASTp. The high stringency analysis of the remaining proteins against database of essential genes (DEG) resulted in 169 proteins which are essential for S. aureus. Metabolic pathway analysis of human host and pathogen by KAAS at the KEGG server sorted out 19 proteins involved in unique metabolic pathways. 26 human non-homologous membrane-bound essential proteins including 4 which were also involved in unique metabolic pathway were deduced through PSORTb, CELLO v.2.5, ngLOC. Functional classification of uncharacterized proteins through SVMprot derived 7 human non-homologous membrane-bound hypothetical essential proteins. Study of potential drug target against Drug Bank revealed pbpA-penicillin-binding protein 1 and hypothetical protein MQW_01796 as the best drug target candidate. 2D structure was predicted by PRED-TMBB, 3D structure and functional analysis was also performed. Protein-protein interaction network of potential drug target proteins was analyzed by using STRING. The identified drug targets are expected to have great potential for designing novel drugs against VRSA infections and further screening of the compounds against these new targets may result in the discovery of novel therapeutic compounds that can be

  6. Proteomic and transcriptomic profiling of Staphylococcus aureus surface LPXTG-proteins: correlation with agr genotypes and adherence phenotypes.

    Science.gov (United States)

    Ythier, Mathilde; Resch, Grégory; Waridel, Patrice; Panchaud, Alexandre; Gfeller, Aurélie; Majcherczyk, Paul; Quadroni, Manfredo; Moreillon, Philippe

    2012-11-01

    Staphylococcus aureus infections involve numerous adhesins and toxins, which expression depends on complex regulatory networks. Adhesins include a family of surface proteins covalently attached to the peptidoglycan via a conserved LPXTG motif. Here we determined the protein and mRNA expression of LPXTG-proteins of S. aureus Newman in time-course experiments, and their relation to fibrinogen adherence in vitro. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa), and fibrinogen-binding protein A (ClfA), as well as during growth in iron-rich or iron-poor media. Surface proteins were recovered by trypsin-shaving of live bacteria. Released peptides were analyzed by liquid chromatography coupled to tandem mass-spectrometry. To unambiguously identify peptides unique to LPXTG-proteins, the analytical conditions were refined using a reference library of S. aureus LPXTG-proteins heterogeneously expressed in surrogate Lactococcus lactis. Transcriptomes were determined by microarrays. Sixteen of the 18 LPXTG-proteins present in S. aureus Newman were detected by proteomics. Nine LPXTG-proteins showed a bell-shape agr-like expression that was abrogated in agr-negative mutants including Spa, fibronectin-binding protein A (FnBPA), ClfA, iron-binding IsdA, and IsdB, immunomodulator SasH, functionally uncharacterized SasD, biofilm-related SasG and methicillin resistance-related FmtB. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr- mutant, whereas all other LPXTG-proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in fibrinogen-adherence tests during late growth (24 h), whereas it remained poorly detected by proteomics. On the other hand, iron-regulated IsdA-B-C increased their protein expression by >10-times in iron-poor conditions. Thus, proteomic, transcriptomic, and adherence

  7. MreC and MreD Proteins Are Not Required for Growth of Staphylococcus aureus.

    Science.gov (United States)

    Tavares, Andreia C; Fernandes, Pedro B; Carballido-López, Rut; Pinho, Mariana G

    2015-01-01

    The transmembrane proteins MreC and MreD are present in a wide variety of bacteria and are thought to be involved in cell shape determination. Together with the actin homologue MreB and other morphological elements, they play an essential role in the synthesis of the lateral cell wall in rod-shaped bacteria. In ovococcus, which lack MreB homologues, mreCD are also essential and have been implicated in peripheral cell wall synthesis. In this work we addressed the possible roles of MreC and MreD in the spherical pathogen Staphylococcus aureus. We show that MreC and MreD are not essential for cell viability and do not seem to affect cell morphology, cell volume or cell cycle control. MreC and MreD localize preferentially to the division septa, but do not appear to influence peptidoglycan composition, nor the susceptibility to different antibiotics and to oxidative and osmotic stress agents. Our results suggest that the function of MreCD in S. aureus is not critical for cell division and cell shape determination.

  8. Staphylococcus aureus surface protein SdrE binds complement regulator factor H as an immune evasion tactic.

    Directory of Open Access Journals (Sweden)

    Julia A Sharp

    Full Text Available Similar to other highly successful invasive bacterial pathogens, Staphylococcus aureus recruits the complement regulatory protein factor H (fH to its surface to inhibit the alternative pathway of complement. Here, we report the identification of the surface-associated protein SdrE as a fH-binding protein using purified fH overlay of S. aureus fractionated cell wall proteins and fH cross-linking to S. aureus followed by mass spectrometry. Studies using recombinant SdrE revealed that rSdrE bound significant fH whether from serum or as a purified form, in both a time- and dose-dependent manner. Furthermore, rSdrE-bound fH exhibited cofactor functionality for factor I (fI-mediated cleavage of C3b to iC3b which correlated positively with increasing amounts of fH. Expression of SdrE on the surface of the surrogate bacterium Lactococcus lactis enhanced recruitment of fH which resulted in increased iC3b generation. Moreover, surface expression of SdrE led to a reduction in C3-fragment deposition, less C5a generation, and reduced killing by polymorphonuclear cells. Thus, we report the first identification of a S. aureus protein associated with the staphylococcal surface that binds factor H as an immune evasion mechanism.

  9. Immunization routes in cattle impact the levels and neutralizing capacity of antibodies induced against S. aureus immune evasion proteins.

    Science.gov (United States)

    Boerhout, Eveline; Vrieling, Manouk; Benedictus, Lindert; Daemen, Ineke; Ravesloot, Lars; Rutten, Victor; Nuijten, Piet; van Strijp, Jos; Koets, Ad; Eisenberg, Susanne

    2015-09-28

    Vaccines against S. aureus bovine mastitis are scarce and show limited protection only. All currently available vaccines are applied via the parenteral (usually intramuscular) route. It is unknown, however, whether this route is the most suitable to specifically increase intramammary immunity to combat S. aureus at the site of infection. Hence, in the present study, immunization via mucosal (intranasal; IN), intramuscular (triangle of the neck; IM), intramammary (IMM) and subcutaneous (suspensory ligament; SC) routes were analyzed for their effects on the quantity of the antibody responses in serum and milk as well as the neutralizing capacity of the antibodies within serum. The experimental vaccine comprised the recombinant S. aureus immune evasion proteins extracellular fibrinogen-binding protein (Efb) and the leukotoxin subunit LukM in an oil-in-water adjuvant combined with a hydrogel and alginate. The highest titer increases for both Efb and LukM specific IgG1 and IgG2 antibody levels in serum and milk were observed following SC/SC immunizations. Furthermore, the harmful effects of Efb and leukotoxin LukMF' on host-defense were neutralized by serum antibodies in a route-dependent manner. SC/SC immunization resulted in a significant increase in the neutralizing capacity of serum antibodies towards Efb and LukMF', shown by increased phagocytosis of S. aureus and increased viability of bovine leukocytes. Therefore, a SC immunization route should be considered when aiming to optimize humoral immunity against S. aureus mastitis in cattle.

  10. Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins

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    Lone , Abdul G.; Atci, Erhan; Renslow, Ryan S.; Beyenal, Haluk; Noh, S.; Fransson, B.; Abu-Lail, Nehal; Park, Jeong-Jin; Gang, David R.; Call, Douglas R.

    2015-08-31

    A partial-thickness epidermal explant model was colonized with GFP-expressing S. aureus and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. Oxygen concentration in explants was quantified using microelectrodes. The relative effective diffusivity and porosity of the epidermis were determined using magnetic resonance imaging, while hydrogen peroxide (H2O2) concentration in explant media was measured by using microelectrodes. Secreted proteins were identified and quantified using MSE mass spectrometry. We found that S. aureus biofilm grows predominantly in sebum-rich areas around hair follicles and associated skin folds. Dissolved oxygen was selectively depleted (2-3 fold) in these locations, but the relative effective diffusivity and porosity did not change between colonized and control epidermis. Histological analysis revealed keratinocyte damage across all the layers of colonized epidermis after four days of culture. The colonized explants released significantly (P< 0.01) more anti-oxidant proteins of both epidermal and S. aureus origin, consistent with elevated H2O2 concentration found in the media from the colonized explants (P< 0.001). Caspase-14 was also elevated significantly in media from infected explants. While H2O2 induces primary keratinocyte differentiation, caspase-14 is required for terminal keratinocyte differentiation and desquamation. These results are consistent with a localized biological impact from S. aureus in response to colonization of the skin surface.

  11. Undetectable bacterial resistance to phage lytic proteins from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88

    Science.gov (United States)

    The increase in antibiotic resistance world-wide revitalized the interest in the use of phage lysins to combat pathogenic bacteria. In this work, we tested for the emergence of resistant Staphylococcus aureus to any of three phage lytic proteins constructs. The investigated cell wall lytic enzymes w...

  12. Differential localization of LTA synthesis proteins and their interaction with the cell division machinery in Staphylococcus aureus.

    Science.gov (United States)

    Reichmann, Nathalie T; Piçarra Cassona, Carolina; Monteiro, João M; Bottomley, Amy L; Corrigan, Rebecca M; Foster, Simon J; Pinho, Mariana G; Gründling, Angelika

    2014-04-01

    Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria. In Staphylococcus aureus it consists of a polyglycerolphosphate-chain that is retained within the membrane via a glycolipid. Using an immunofluorescence approach, we show here that the LTA polymer is not surface exposed in S. aureus, as it can only be detected after digestion of the peptidoglycan layer. S. aureus mutants lacking LTA are enlarged and show aberrant positioning of septa, suggesting a link between LTA synthesis and the cell division process. Using a bacterial two-hybrid approach, we show that the three key LTA synthesis proteins, YpfP and LtaA, involved in glycolipid production, and LtaS, required for LTA backbone synthesis, interact with one another. All three proteins also interacted with numerous cell division and peptidoglycan synthesis proteins, suggesting the formation of a multi-enzyme complex and providing further evidence for the co-ordination of these processes. When assessed by fluorescence microscopy, YpfP and LtaA fluorescent protein fusions localized to the membrane while the LtaS enzyme accumulated at the cell division site. These data support a model whereby LTA backbone synthesis proceeds in S. aureus at the division site in co-ordination with cell division, while glycolipid synthesis takes place throughout the membrane. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  13. Staphylococcus aureus-fibronectin interactions with and without fibronectin-binding proteins and their role in adhesion and desorption

    NARCIS (Netherlands)

    Xu, Chun; Boks, Niels P; de Vries, Jacob; Kaper, Harm; Norde, Willem; Busscher, Hendrik; van der Mei, Henderina

    2008-01-01

    Adhesion and residence-time-dependent desorption of two Staphylococcus aureus strains with and without fibronectin (Fn) binding proteins (FnBPs) on Fn-coated glass were compared under flow conditions. To obtain a better understanding of the role of Fn-FnBP binding, the adsorption enthalpies of Fn wi

  14. Staphylococcus aureus-Fibronectin Interactions with and without Fibronectin-Binding Proteins and Their Role in Adhesion and Desorption

    NARCIS (Netherlands)

    Xu, C.P.; Boks, N.P.; Vries, de J.; Kaper, H.J.; Norde, W.; Busscher, H.J.; Mei, van der H.C.

    2008-01-01

    Adhesion and residence-time-dependent desorption of two Staphylococcus aureus strains with and without fibronectin (Fn) binding proteins (FnBPs) on Fn-coated glass were compared under flow conditions. To obtain a better understanding of the role of Fn-FnBP binding, the adsorption enthalpies of Fn wi

  15. Bap, a biofilm matrix protein of Staphylococcus aureus prevents cellular internalization through binding to GP96 host receptor.

    Science.gov (United States)

    Valle, Jaione; Latasa, Cristina; Gil, Carmen; Toledo-Arana, Alejandro; Solano, Cristina; Penadés, José R; Lasa, Iñigo

    2012-01-01

    The biofilm matrix, composed of exopolysaccharides, proteins, nucleic acids and lipids, plays a well-known role as a defence structure, protecting bacteria from the host immune system and antimicrobial therapy. However, little is known about its responsibility in the interaction of biofilm cells with host tissues. Staphylococcus aureus, a leading cause of biofilm-associated chronic infections, is able to develop a biofilm built on a proteinaceous Bap-mediated matrix. Here, we used the Bap protein as a model to investigate the role that components of the biofilm matrix play in the interaction of S. aureus with host cells. The results show that Bap promotes the adhesion but prevents the entry of S. aureus into epithelial cells. A broad analysis of potential interaction partners for Bap using ligand overlayer immunoblotting, immunoprecipitation with purified Bap and pull down with intact bacteria, identified a direct binding between Bap and Gp96/GRP94/Hsp90 protein. The interaction of Bap with Gp96 provokes a significant reduction in the capacity of S. aureus to invade epithelial cells by interfering with the fibronectin binding protein invasion pathway. Consistent with these results, Bap deficient bacteria displayed an enhanced capacity to invade mammary gland epithelial cells in a lactating mice mastitis model. Our observations begin to elucidate the mechanisms by which components of the biofilm matrix can facilitate the colonization of host tissues and the establishment of persistent infections.

  16. Construction of a New Fusion Protein Vector Associated to Fibronectin Binding Protein A and Clumping Factor A derived from Staphylococcus aureus NCTC8325

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    Jamshid Faghri

    2009-03-01

    Full Text Available Objective(s Staphylococcus aureus is a leading cause of many nosocomial and community acquired infections. According to many reports, antibiotic therapy can not guarantee the eradication of S. aureus infections. Thus designing an adhesin based vaccine could restrain the S. aureus infections. This study designed for construction of a new fusion protein vaccine against S. aureus infections based on adhesin molecules fibronectin binding protein A (FnBPA and clumping factor A (ClfA. Materials and Methods Bioinformatic experiments were performed using Oligo analyzer and DNAMAN softwares. The fragments corresponding to fnbA binding domain and a C-terminal fragment from clfA were amplified from S. aureus NCTC8325 genomic DNA. Purified PCR products and the vector, pET15b, were digested with NcoI and BamHI. The digested PCR products were hybridized together and then ligated to digested vector. Finally incomplete construct was assembled by Taq DNA polymerase. To quick confirmation of cloning procedure the new construct designated pfnbA-clfA was digested with NcoI and BamHI. To further verification, the product was sent for sequencing. Results The data based on bioinformatic analysis showed no homology between fusion protein and human proteins. Digestion of new vector with NcoI and BamHI confirmed the ligation of fusion protein sequence into pET15b. Sequencing results verified the integrity of target sequences. Conclusion This study is the first effort to construct a new fusion protein vector based on S. aureus adhesins using a new design. This project is being continued to study the expression and biological activity of the fusion protein in a cell culture model.

  17. The Staphylococcus aureus Membrane Protein SA2056 Interacts with Peptidoglycan Synthesis Enzymes

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    Brigitte Berger-Bächi

    2013-01-01

    Full Text Available The yet uncharacterized membrane protein SA2056 belongs to the ubiquitous RND (Resistance-Nodulation-cell Division family of transmembrane efflux transporters. The sa2056 gene is located downstream of femX, the gene encoding the essential, non-ribosomal peptidyl-transferase adding the first glycine in the staphylococcal cell wall pentaglycine interpeptide. Due to its proximity to and weak co-transcription with femX, we assumed that sa2056 may somehow be involved in peptidoglycan synthesis. Specific antibodies against SA2056 showed that this protein is expressed during growth and present in the membrane fraction of cell preparations. Using a bacterial two hybrid system, SA2056 was shown to interact (i with itself, (ii with FemB, which adds glycines 4 and 5 to the peptidoglycan interpeptide and (iii with the essential penicillin binding proteins, PBP1 and PBP2, required for cell division and incorporation of the peptidoglycan into the cell wall. Unexpectedly, deletion of sa2056 led to no phenotype regarding growth, antibiotic resistances or cell morphology; nor did sa2056 deletion in combination with femB inactivation alter b-lactam and lysostaphin sensitivity and resistance, respectively, pointing to possible redundancy in the cell wall synthesis pathway. These results suggest an accessory role of SA2056 in S. aureus peptidoglycan synthesis, broadening the range of biological functions of RND proteins.

  18. Protein A is released into the Staphylococcus aureus culture supernatant with an unprocessed sorting signal.

    Science.gov (United States)

    O'Halloran, Dara P; Wynne, Kieran; Geoghegan, Joan A

    2015-04-01

    The immunoglobulin binding protein A (SpA) of Staphylococcus aureus is synthesized as a precursor with a C-terminal sorting signal. The sortase A enzyme mediates covalent attachment to peptidoglycan so that SpA is displayed on the surface of the bacterium. Protein A is also found in the extracellular medium, but the processes involved in its release are not fully understood. Here, we show that a portion of SpA is released into the supernatant with an intact sorting signal, indicating that it has not been processed by sortase A. Release of SpA was reduced when the native sorting signal of SpA was replaced with the corresponding region of another sortase-anchored protein (SdrE). Similarly, a reporter protein fused to the sorting signal of SpA was released to a greater extent than the same polypeptide fused to the SdrE sorting signal. Released SpA protected bacteria from killing in human blood, indicating that it contributes to immune evasion.

  19. Zinc-dependent mechanical properties of Staphylococcus aureus biofilm-forming surface protein SasG.

    Science.gov (United States)

    Formosa-Dague, Cécile; Speziale, Pietro; Foster, Timothy J; Geoghegan, Joan A; Dufrêne, Yves F

    2016-01-12

    Staphylococcus aureus surface protein SasG promotes cell-cell adhesion during the accumulation phase of biofilm formation, but the molecular basis of this interaction remains poorly understood. Here, we unravel the mechanical properties of SasG on the surface of living bacteria, that is, in its native cellular environment. Nanoscale multiparametric imaging of living bacteria reveals that Zn(2+) strongly increases cell wall rigidity and activates the adhesive function of SasG. Single-cell force measurements show that SasG mediates cell-cell adhesion via specific Zn(2+)-dependent homophilic bonds between β-sheet-rich G5-E domains on neighboring cells. The force required to unfold individual domains is remarkably strong, up to ∼500 pN, thus explaining how SasG can withstand physiological shear forces. We also observe that SasG forms homophilic bonds with the structurally related accumulation-associated protein of Staphylococcus epidermidis, suggesting the possibility of multispecies biofilms during host colonization and infection. Collectively, our findings support a model in which zinc plays a dual role in activating cell-cell adhesion: adsorption of zinc ions to the bacterial cell surface increases cell wall cohesion and favors the projection of elongated SasG proteins away from the cell surface, thereby enabling zinc-dependent homophilic bonds between opposing cells. This work demonstrates an unexpected relationship between mechanics and adhesion in a staphylococcal surface protein, which may represent a general mechanism among bacterial pathogens for activating cell association.

  20. Naturally occurring IgG antibody levels to the Staphylococcus aureus protein IsdB in humans.

    Science.gov (United States)

    Zorman, Julie K; Esser, Mark; Raedler, Michael; Kreiswirth, Barry N; Ala'Aldeen, Dlawer A A; Kartsonis, Nicholas; Smugar, Steven S; Anderson, Annaliesa S; McNeely, Tessie; Arduino, Jean Marie

    2013-09-01

    Staphylococcus aureus is a well-recognized, clinically important cause of nosocomial infections, and as such, a vaccine to prevent S. aureus infections would be an important achievement. A Phase IIB/III study of V710, a vaccine containing iron-regulated surface determinant B (IsdB), demonstrated significant sero-conversion rates in cardiovascular surgery patients following a single pre-surgery immunization. However, the vaccine was not efficacious in preventing bacteremia or deep sternal wound infection post-surgery, thus raising the possibility that IsdB might not be available for immune recognition during infection. The purpose of the work described herein was to evaluate and quantify the naturally occurring anti-IsdB levels at baseline and over time during infection, to understand whether IsdB is expressed during a S. aureus infection in hospitalized non-vaccinated patients. We evaluated baseline and follow-up titers in 3 populations: (1) healthy subjects, (2) hospitalized patients with non-S. aureus infections, and (3) hospitalized patients with S. aureus infections. Baseline anti-IsdB levels generally overlapped between the 3 groups, but were highly variable within each group. In healthy subjects, baseline and follow-up levels were highly correlated (Spearman's rho = 0.93), and the geometric mean fold-rise (GMFR) in anti-IsdB levels between study entry and last value was 0.9-fold (95% confidence interval (CI): 0.8 to 1.0 ; p = 0.09), showing no trend over time. The convalescent GMFR in anti-IsdB levels from baseline was 1.7-fold (95% CI: 1.3 to 2.2, p = 0.0008) during S. aureus infection, significantly different from the 1.0-fold GMFR (95% CI: 0.9-1.2, p = 0.60) in non-S. aureus infection, p = 0.005. Additionally, S. aureus isolates (51) obtained from the hospitalized patient group expressed the IsdB protein in vitro. Collectively, these data suggest that IsdB expression levels rise substantially following infection with S. aureus, but not with other pathogens

  1. Combining in vitro protein detection and in vivo antibody detection identifies potential vaccine targets against Staphylococcus aureus during osteomyelitis.

    Science.gov (United States)

    den Reijer, P Martijn; Sandker, Marjan; Snijders, Susan V; Tavakol, Mehri; Hendrickx, Antoni P A; van Wamel, Willem J B

    2017-02-01

    Currently, little is known about the in vivo human immune response against Staphylococcus aureus during a biofilm-associated infection, such as osteomyelitis, and how this relates to protein production in biofilms in vitro. Therefore, we characterized IgG responses in 10 patients with chronic osteomyelitis against 50 proteins of S. aureus, analyzed the presence of these proteins in biofilms of the infecting isolates on polystyrene (PS) and human bone in vitro, and explored the relation between in vivo and in vitro data. IgG levels against 15 different proteins were significantly increased in patients compared to healthy controls. Using a novel competitive Luminex-based assay, eight of these proteins [alpha toxin, Staphylococcus aureus formyl peptide receptor-like 1 inhibitor (FlipR), glucosaminidase, iron-responsive surface determinants A and H, the putative ABC transporter SACOL0688, staphylococcal complement inhibitor (SCIN), and serine-aspartate repeat-containing protein E (SdrE)] were also detected in a majority of the infecting isolates during biofilm formation in vitro. However, 4 other proteins were detected in only a minority of isolates in vitro while, vice versa, 7 proteins were detected in multiple isolates in vitro but not associated with significantly increased IgG levels in patients. Detection of proteins was largely confirmed using a transcriptomic approach. Our data provide further insights into potential therapeutic targets, such as for vaccination, to reduce S. aureus virulence and biofilm formation. At the same time, our data suggest that either in vitro or immunological in vivo data alone should be interpreted cautiously and that combined studies are necessary to identify potential targets.

  2. Phospholipase C-related catalytically inactive protein participates in the autophagic elimination of Staphylococcus aureus infecting mouse embryonic fibroblasts.

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    Kae Harada-Hada

    Full Text Available Autophagy is an intrinsic host defense system that recognizes and eliminates invading bacterial pathogens. We have identified microtubule-associated protein 1 light chain 3 (LC3, a hallmark of autophagy, as a binding partner of phospholipase C-related catalytically inactive protein (PRIP that was originally identified as an inositol trisphosphate-binding protein. Here, we investigated the involvement of PRIP in the autophagic elimination of Staphylococcus aureus in infected mouse embryonic fibroblasts (MEFs. We observed significantly more LC3-positive autophagosome-like vacuoles enclosing an increased number of S. aureus cells in PRIP-deficient MEFs than control MEFs, 3 h and 4.5 h post infection, suggesting that S. aureus proliferates in LC3-positive autophagosome-like vacuoles in PRIP-deficient MEFs. We performed autophagic flux analysis using an mRFP-GFP-tagged LC3 plasmid and found that autophagosome maturation is significantly inhibited in PRIP-deficient MEFs. Furthermore, acidification of autophagosomes was significantly inhibited in PRIP-deficient MEFs compared to the wild-type MEFs, as determined by LysoTracker staining and time-lapse image analysis performed using mRFP-GFP-tagged LC3. Taken together, our data show that PRIP is required for the fusion of S. aureus-containing autophagosome-like vacuoles with lysosomes, indicating that PRIP is a novel modulator in the regulation of the innate immune system in non-professional phagocytic host cells.

  3. Atomic force microscopy recognition of protein A on Staphylococcus aureus cell surfaces by labelling with IgG–Au conjugates

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    Elena B. Tatlybaeva

    2013-11-01

    Full Text Available The labelling of functional molecules on the surface of bacterial cells is one way to recognize the bacteria. In this work, we have developed a method for the selective labelling of protein A on the cell surfaces of Staphylococcus aureus by using nanosized immunogold conjugates as cell-surface markers for atomic force microscopy (AFM. The use of 30-nm size Au nanoparticles conjugated with immunoglobulin G (IgG allowed the visualization, localization and distribution of protein A–IgG complexes on the surface of S. aureus. The selectivity of the labelling method was confirmed in mixtures of S. aureus with Bacillus licheniformis cells, which differed by size and shape and had no IgG receptors on the surface. A preferential binding of the IgG–Au conjugates to S. aureus was obtained. Thus, this novel approach allows the identification of protein A and other IgG receptor-bearing bacteria, which is useful for AFM indication of pathogenic microorganisms in poly-component associations.

  4. Rapid Exchange of Bound ADP on the Staphylococcus aureus Replication Initiation Protein DnaA*

    OpenAIRE

    2009-01-01

    In Escherichia coli, regulatory inactivation of the replication initiator DnaA occurs after initiation as a result of hydrolysis of bound ATP to ADP, but it has been unknown how DnaA is controlled to coordinate cell growth and chromosomal replication in Gram-positive bacteria such as Staphylococcus aureus. This study examined the roles of ATP binding and its hydrolysis in the regulation of the S. aureus DnaA activity. In vitro, S. aureus DnaA melted S. aureus oriC in the presence of ATP but n...

  5. Mechanical Strength and Inhibition of the Staphylococcus aureus Collagen-Binding Protein Cna

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    Philippe Herman-Bausier

    2016-10-01

    Full Text Available The bacterial pathogen Staphylococcus aureus expresses a variety of cell surface adhesion proteins that bind to host extracellular matrix proteins. Among these, the collagen (Cn-binding protein Cna plays important roles in bacterium-host adherence and in immune evasion. While it is well established that the A region of Cna mediates ligand binding, whether the repetitive B region has a dedicated function is not known. Here, we report the direct measurement of the mechanical strength of Cna-Cn bonds on living bacteria, and we quantify the antiadhesion activity of monoclonal antibodies (MAbs targeting this interaction. We demonstrate that the strength of Cna-Cn bonds in vivo is very strong (~1.2 nN, consistent with the high-affinity “collagen hug” mechanism. The B region is required for strong ligand binding and has been found to function as a spring capable of sustaining high forces. This previously undescribed mechanical response of the B region is of biological significance as it provides a means to project the A region away from the bacterial surface and to maintain bacterial adhesion under conditions of high forces. We further quantified the antiadhesion activity of MAbs raised against the A region of Cna directly on living bacteria without the need for labeling or purification. Some MAbs are more efficient in blocking single-cell adhesion, suggesting that they act as competitive inhibitors that bind Cna residues directly involved in ligand binding. This report highlights the role of protein mechanics in activating the function of staphylococcal adhesion proteins and emphasizes the potential of antibodies to prevent staphylococcal adhesion and biofilm formation.

  6. Mechanical Strength and Inhibition of the Staphylococcus aureus Collagen-Binding Protein Cna

    Science.gov (United States)

    Herman-Bausier, Philippe; Valotteau, Claire; Pietrocola, Giampiero; Rindi, Simonetta; Alsteens, David; Foster, Timothy J.

    2016-01-01

    ABSTRACT The bacterial pathogen Staphylococcus aureus expresses a variety of cell surface adhesion proteins that bind to host extracellular matrix proteins. Among these, the collagen (Cn)-binding protein Cna plays important roles in bacterium-host adherence and in immune evasion. While it is well established that the A region of Cna mediates ligand binding, whether the repetitive B region has a dedicated function is not known. Here, we report the direct measurement of the mechanical strength of Cna-Cn bonds on living bacteria, and we quantify the antiadhesion activity of monoclonal antibodies (MAbs) targeting this interaction. We demonstrate that the strength of Cna-Cn bonds in vivo is very strong (~1.2 nN), consistent with the high-affinity “collagen hug” mechanism. The B region is required for strong ligand binding and has been found to function as a spring capable of sustaining high forces. This previously undescribed mechanical response of the B region is of biological significance as it provides a means to project the A region away from the bacterial surface and to maintain bacterial adhesion under conditions of high forces. We further quantified the antiadhesion activity of MAbs raised against the A region of Cna directly on living bacteria without the need for labeling or purification. Some MAbs are more efficient in blocking single-cell adhesion, suggesting that they act as competitive inhibitors that bind Cna residues directly involved in ligand binding. This report highlights the role of protein mechanics in activating the function of staphylococcal adhesion proteins and emphasizes the potential of antibodies to prevent staphylococcal adhesion and biofilm formation. PMID:27795393

  7. Utilizing rabbit immunoglobulin G (IgG) protein for mark-capture studies on the desert subterranean termite, Heterotermes aureus (Snyder)

    Science.gov (United States)

    A series of mark-capture dispersal studies were conducted to investigate the feasibility of marking the southwestern desert subterranean termite, Heterotermes aureus (Snyder) with rabbit immunoglobulin G (IgG) protein. In turn, short-range dispersal patterns of H. aureus were measured across a 20-m ...

  8. Molecular Detection of Methicillin-Resistant Staphylococcus aureus by Non-Protein Coding RNA-Mediated Monoplex Polymerase Chain Reaction

    Science.gov (United States)

    Soo Yean, Cheryl Yeap; Selva Raju, Kishanraj; Xavier, Rathinam; Subramaniam, Sreeramanan; Gopinath, Subash C. B.; Chinni, Suresh V.

    2016-01-01

    Non-protein coding RNA (npcRNA) is a functional RNA molecule that is not translated into a protein. Bacterial npcRNAs are structurally diversified molecules, typically 50–200 nucleotides in length. They play a crucial physiological role in cellular networking, including stress responses, replication and bacterial virulence. In this study, by using an identified npcRNA gene (Sau-02) in Methicillin-resistant Staphylococcus aureus (MRSA), we identified the Gram-positive bacteria S. aureus. A Sau-02-mediated monoplex Polymerase Chain Reaction (PCR) assay was designed that displayed high sensitivity and specificity. Fourteen different bacteria and 18 S. aureus strains were tested, and the results showed that the Sau-02 gene is specific to S. aureus. The detection limit was tested against genomic DNA from MRSA and was found to be ~10 genome copies. Further, the detection was extended to whole-cell MRSA detection, and we reached the detection limit with two bacteria. The monoplex PCR assay demonstrated in this study is a novel detection method that can replicate other npcRNA-mediated detection assays. PMID:27367909

  9. Cloning, purification and preliminary crystallographic analysis of a putative DNA-binding membrane protein, YmfM, from Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ling; Sedelnikova, Svetlana E.; Baker, Patrick J.; Rice, David W., E-mail: d.rice@sheffield.ac.uk [Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN (United Kingdom)

    2008-07-01

    Truncation by the removal of the C-terminal hydrophobic transmembrane anchor has enabled the overexpression of a soluble domain of S. aureus YmfM in Escherichia coli, which has then been purified and subsequently crystallized. The Staphylococcus aureus protein YmfM contains a helix–turn–helix motif and is thought to be a putative DNA-binding protein which is associated with the membrane through a C-terminal hydrophobic transmembrane anchor. Truncation of the protein by the removal of this C-terminal hydrophobic segment has enabled the overexpression of a soluble domain of S. aureus YmfM (ΔYmfM) in Escherichia coli, which has been purified and subsequently crystallized. Crystals of ΔYmfM diffract to beyond 1.0 Å resolution and belong to one of the pair of enantiomorphic tetragonal space groups P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 45.5, c = 72.9 Å and one molecule in the asymmetric unit. The crystals of ΔYmfM have an unusually low V{sub M} of 1.6 Å{sup 3} Da{sup −1}, which is one of the lowest values observed for any protein to date. A full structure determination is under way in order to provide insights into the function of this protein.

  10. Staphylococcus aureus immunodominant surface antigen B is a cell-surface associated nucleic acid binding protein

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    Cerca Nuno

    2009-03-01

    Full Text Available Abstract Background Staphylococcus aureus immunodominant surface antigen B (IsaB elicits an immune response during septicemia and is generally classified as a virulence factor, but its biological function remains completely undefined. In an attempt to identify staphylococcal RNA-binding proteins, we designed an RNA Affinity Chromatography assay and subsequently isolated IsaB. Results Western analysis indicated that IsaB was both secreted and cell-surface associated. Gel Shift analysis confirmed the RNA binding activity but revealed that IsaB bound to any nucleic acid without sequence specificity. IsaB exhibited the highest affinity for double-stranded DNA followed by single-stranded DNA and RNA. Because extracellular DNA has been shown to play a role in biofilm formation, we investigated the biofilm-forming capacity of an isogenic isaB deletion mutant but we found that IsaB did not contribute to biofilm formation under any conditions tested. Conclusion IsaB is an extracellular nucleic acid binding protein, with little to no sequence specificity, but its role in virulence remains unclear.

  11. Differential profiles of gastrointestinal proteins interacting with peptidoglycans from Lactobacillus plantarum and Staphylococcus aureus.

    Science.gov (United States)

    Baik, Jung Eun; Jang, Young-Oh; Kang, Seok-Seong; Cho, Kun; Yun, Cheol-Heui; Han, Seung Hyun

    2015-05-01

    Peptidoglycan (PGN) is a major cell wall component of Gram-positive bacteria that contributes to the regulation of host immunity in the gastrointestinal tract (GIT). Although Gram-positive bacteria contain structurally distinct PGNs that are considered to differently interact with the GIT, PGN-binding proteins (PGN-BPs) in the GIT have been poorly understood. In the present study, we purified PGNs from Lactobacillus plantarum and Staphylococcus aureus (named as Lp.PGN and Sa.PGN, respectively) and identified Lp.PGN-BPs and Sa.PGN-BPs in the lysate of mouse GIT. Lp.PGN activated nucleotide-binding oligomerization domain (NOD) 1 and NOD2, whereas Sa.PGN activated NOD2, but not NOD1, implying that both PGNs retained the biological activity and were differently recognized by the host cells. PGN-BPs were isolated by precipitation with Lp.PGN or Sa.PGN and identified using LTQ-Orbitrap hybrid Fourier transform mass spectrometry. Three independent experiments demonstrated that 18 Lp.PGN-BPs and 6 Sa.PGN-BPs were reproducibly obtained with statistical significance (Pproteins which are related to cytoskeleton, microbial adhesion, and mucosal integrity. Lp.PGN selectively bound to proteins related to gene expression, chaperone, and antimicrobial function. However, Sa.PGN preferentially interacted with proteins involved in adherence and invasion of pathogens. Collectively, these results suggest that bacterial PGNs interact with the proteins regulating mucosal homeostasis and immunity in the gut and PGNs of commensals and pathogens might be also differentially recognized in the GIT.

  12. Dual roles of F123 in protein homodimerization and inhibitor binding to biotin protein ligase from Staphylococcus aureus.

    Science.gov (United States)

    Soares da Costa, Tatiana P; Yap, Min Y; Perugini, Matthew A; Wallace, John C; Abell, Andrew D; Wilce, Matthew C J; Polyak, Steven W; Booker, Grant W

    2014-01-01

    Protein biotinylation is catalysed by biotin protein ligase (BPL). The most characterized BPL is from Escherichia coli where it functions as both a biotin ligase and a homodimeric transcriptional repressor. Here we investigated another bifunctional BPL from the clinically important Staphylococcus aureus (SaBPL). Unliganded SaBPL (apo) exists in a dimer-monomer equilibrium at low micromolar concentrations - a stark contrast to E. coli BPL (EcBPL) that is monomeric under the same conditions. EMSA and SAXS analysis demonstrated that dimeric apo SaBPL adopted a conformation that was competent to bind DNA and necessary for it to function as a transcription factor. The SaBPL dimer-monomer dissociation constant was 5.8-fold tighter when binding the inhibitor biotin acetylene, but unchanged with biotin. F123, located in the dimer interface, was critical for homodimerization. Inhibition studies together with surface plasmon resonance analyses revealed a strong correlation between inhibitor potency and slow dissociation kinetics. A 24-fold difference in Ki values for these two enzymes was explained by differences in enzyme:inhibitor dissociation rates. Substitution of F123 in SaBPL and its equivalent in EcBPL altered both inhibitor potency and dissociation. Hence, F123 in SaBPL has novel roles in both protein dimerization and ligand-binding that have not been reported in EcBPL.

  13. The adhesive and immunomodulating properties of the multifunctional Staphylococcus aureus protein Eap

    NARCIS (Netherlands)

    Harraghy, Niamh; Hussain, Muzaffar; Haggar, Axana; Chavakis, Triantafyllos; Sinha, Bhanu; Herrmann, Mathias; Flock, Jan-Ingmar

    2003-01-01

    Adherence of Staphylococcus aureus to the host tissue is an important step in the initiation of pathogenesis. At least 10 adhesins produced by S. aureus have been described and it is becoming clear that the expression of these adhesins and their interactions with eukaryotic cells involve complex pro

  14. The Spl Serine Proteases Modulate Staphylococcus aureus Protein Production and Virulence in a Rabbit Model of Pneumonia

    Science.gov (United States)

    Salgado-Pabon, Wilmara; Meyerholz, David K.; White, Mark J.; Schlievert, Patrick M.

    2016-01-01

    ABSTRACT The Spl proteases are a group of six serine proteases that are encoded on the νSaβ pathogenicity island and are unique to Staphylococcus aureus. Despite their interesting biochemistry, their biological substrates and functions in virulence have been difficult to elucidate. We found that an spl operon mutant of the community-associated methicillin-resistant S. aureus USA300 strain LAC induced localized lung damage in a rabbit model of pneumonia, characterized by bronchopneumonia observed histologically. Disease in the mutant-infected rabbits was restricted in distribution compared to that in wild-type USA300-infected rabbits. We also found that SplA is able to cleave the mucin 16 glycoprotein from the surface of the CalU-3 lung cell line, suggesting a possible mechanism for wild-type USA300 spreading pneumonia to both lungs. Investigation of the secreted and surface proteomes of wild-type USA300 and the spl mutant revealed multiple alterations in metabolic proteins and virulence factors. This study demonstrates that the Spls modulate S. aureus physiology and virulence, identifies a human target of SplA, and suggests potential S. aureus targets of the Spl proteases. IMPORTANCE Staphylococcus aureus is a versatile human pathogen that produces an array of virulence factors, including several proteases. Of these, six proteases called the Spls are the least characterized. Previous evidence suggests that the Spls are expressed during human infection; however, their function is unknown. Our study shows that the Spls are required for S. aureus to cause disseminated lung damage during pneumonia. Further, we present the first example of a human protein cut by an Spl protease. Although the Spls were predicted not to cut staphylococcal proteins, we also show that an spl mutant has altered abundance of both secreted and surface-associated proteins. This work provides novel insight into the function of Spls during infection and their potential ability to degrade

  15. The structure of SAV1646 from Staphylococcus aureus belonging to a new `ribosome-associated' subfamily of bacterial proteins.

    Science.gov (United States)

    Chirgadze, Yuri N; Clarke, Teresa E; Romanov, Vladimir; Kisselman, Gera; Wu-Brown, Jean; Soloveychik, Maria; Chan, Tiffany S Y; Gordon, Roni D; Battaile, Kevin P; Pai, Emil F; Chirgadze, Nickolay Y

    2015-02-01

    The crystal structure of the SAV1646 protein from the pathogenic microorganism Staphylococcus aureus has been determined at 1.7 Å resolution. The 106-amino-acid protein forms a two-layer sandwich with α/β topology. The protein molecules associate as dimers in the crystal and in solution, with the monomers related by a pseudo-twofold rotation axis. A sequence-homology search identified the protein as a member of a new subfamily of yet uncharacterized bacterial `ribosome-associated' proteins with at least 13 members to date. A detailed analysis of the crystal protein structure along with the genomic structure of the operon containing the sav1646 gene allowed a tentative functional model of this protein to be proposed. The SAV1646 dimer is assumed to form a complex with ribosomal proteins L21 and L27 which could help to complete the assembly of the large subunit of the ribosome.

  16. Dissection of an old protein reveals a novel application: domain D of Staphylococcus aureus Protein A (sSpAD as a secretion - tag

    Directory of Open Access Journals (Sweden)

    Paal Michael

    2010-11-01

    Full Text Available Abstract Background Escherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous. Results We discovered that domain D of the Staphylococcus aureus protein A was sufficient for the secretion of various target proteins into the periplasmic space of E. coli. Our experiments indicated the Sec pathway as the mode of secretion, although N-terminal processing was not observed. Furthermore, the solubility of recombinant fusion proteins was improved for proteins prone to aggregation. The tag allowed a straightforward affinity purification of recombinant fusion protein via an IgG column, which was exemplified for the target protein human superoxide dismutase 1 (SOD. Conclusions In this work we present a new secretion tag that combines several advantages for the production of recombinant proteins in E. coli. Domain D of S. aureus protein A protects the protein of interest against N-terminal degradation, increases target protein solubility and enables a straight-forward purification of the recombinant protein using of IgG columns.

  17. The Staphylococcus aureus Protein IsdH Inhibits Host Hemoglobin Scavenging to Promote Heme Acquisition by the Pathogen.

    Science.gov (United States)

    Sæderup, Kirstine Lindhardt; Stødkilde, Kristian; Graversen, Jonas Heilskov; Dickson, Claire F; Etzerodt, Anders; Hansen, Søren Werner Karlskov; Fago, Angela; Gell, David; Andersen, Christian Brix Folsted; Moestrup, Søren Kragh

    2016-11-11

    Hemolysis is a complication in septic infections with Staphylococcus aureus, which utilizes the released Hb as an iron source. S. aureus can acquire heme in vitro from hemoglobin (Hb) by a heme-sequestering mechanism that involves proteins from the S. aureus iron-regulated surface determinant (Isd) system. However, the host has its own mechanism to recapture the free Hb via haptoglobin (Hp) binding and uptake of Hb-Hp by the CD163 receptor in macrophages. It has so far remained unclear how the Isd system competes with this host iron recycling system in situ to obtain the important nutrient. By binding and uptake studies, we now show that the IsdH protein, which serves as an Hb receptor in the Isd system, directly interferes with the CD163-mediated clearance by binding the Hb-Hp complex and inhibiting CD163 recognition. Analysis of truncated IsdH variants including one or more of three near iron transporter domains, IsdH(N1), IsdH(N2), and IsdH(N3), revealed that Hb binding of IsdH(N1) and IsdH(N2) accounted for the high affinity for Hb-Hp complexes. The third near iron transporter domain, IsdH(N3), exhibited redox-dependent heme extraction, when Hb in the Hb-Hp complex was in the oxidized met form but not in the reduced oxy form. IsdB, the other S. aureus Hb receptor, failed to extract heme from Hb-Hp, and it was a poor competitor for Hb-Hp binding to CD163. This indicates that Hb recognition by IsdH, but not by IsdB, sterically inhibits the receptor recognition of Hb-Hp. This function of IsdH may have an overall stimulatory effect on S. aureus heme acquisition and growth. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Structural and dynamical characterization of a biologically active unfolded fibronectin-binding protein from Staphylococcus aureus.

    Science.gov (United States)

    Penkett, C J; Redfield, C; Jones, J A; Dodd, I; Hubbard, J; Smith, R A; Smith, L J; Dobson, C M

    1998-12-01

    A 130-residue fragment (D1-D4) taken from a fibronectin-binding protein of Staphylococcus aureus, which contains four fibronectin-binding repeats and is unfolded but biologically active at neutral pH, has been studied extensively by NMR spectroscopy. Using heteronuclear multidimensional techniques, the conformational properties of D1-D4 have been defined at both a global and a local level. Diffusion studies give an average effective radius of 26.2 +/- 0.1 A, approximately 75% larger than that expected for a globular protein of this size. Analysis of chemical shift, 3JHNalpha coupling constant, and NOE data show that the experimental parameters agree well overall with values measured in short model peptides and with predictions from a statistical model for a random coil. Sequences where specific features give deviations from these predictions for a random coil have however been identified. These arise from clustering of hydrophobic side chains and electrostatic interactions between charged groups. 15N relaxation studies demonstrate that local fluctuations of the chain are the dominant motional process that gives rise to relaxation of the 15N nuclei, with a persistence length of approximately 7-10 residues for the segmental motion. The consequences of the structural and dynamical properties of this unfolded protein for its biological role of binding to fibronectin have been considered. It is found that the regions of the sequence involved in binding have a high propensity for populating extended conformations, a feature that would allow a number of both charged and hydrophobic groups to be presented to fibronectin for highly specific binding.

  19. Evaluation of a lysostaphin-fusion protein as a dry-cow therapy for Staphylococcus aureus mastitis in dairy cattle.

    Science.gov (United States)

    Hoernig, K J; Donovan, D M; Pithua, P; Williams, F; Middleton, J R

    2016-06-01

    This study evaluated the efficacy of a recombinant lysostaphin fused to a protein transduction domain (rLYS-PTD) as a dry-cow therapy for the treatment of experimentally induced chronic, subclinical Staphylococcus aureus mastitis. Twenty-two Holstein dairy cows were experimentally infected with Staph. aureus in a single pair of diagonal mammary quarters approximately 45d before dry off. Staphylococcus aureus-infected mammary quarters of cows were randomly assigned to 1 of 2 treatment groups at dry off: (1) 279mg of rLYS-PTD in 50mL of vehicle (n=11 cows; 22 quarters) or (2) 50mL of vehicle solution (n=11 cows; 22 quarters) by intramammary infusion. All cows were followed for 30d postpartum to determine cure rates using bacteriologic culture, somatic cell counts, and clinical mastitis scores. No cures were recorded in either the treatment or control groups. Milk somatic cell count, bacterial colony counts, and mastitis scores did not significantly differ between treatment groups. In conclusion, rLYS-PTD was not an effective dry-cow therapeutic for chronic, subclinical Staph. aureus mastitis at the tested dose and formulation.

  20. Structural reorganization of the bacterial cell-division protein FtsZ from Staphylococcus aureus.

    Science.gov (United States)

    Matsui, Takashi; Yamane, Junji; Mogi, Nobuyuki; Yamaguchi, Hiroto; Takemoto, Hiroshi; Yao, Min; Tanaka, Isao

    2012-09-01

    FtsZ is a key molecule in bacterial cell division. In the presence of GTP, it polymerizes into tubulin-like protofilaments by head-to-tail association. Protofilaments of FtsZ seem to adopt a straight or a curved conformation in relation to the bound nucleotide. However, although several bacterial and archaeal FtsZ structures have been determined, all of the structures reported previously are considered to have a curved conformation. In this study, structures of FtsZ from Staphylococcus aureus (SaFtsZ) were determined in apo, GDP-bound and inhibitor-complex forms and it was found that SaFtsZ undergoes marked conformational changes. The accumulated evidence suggests that the GDP-bound structure has the features of the straight form. The structural change between the curved and straight forms shows intriguing similarity to the eukaryotic cytoskeletal protein tubulin. Furthermore, the structure of the apo form showed an unexpectedly large conformational change in the core region. FtsZ has also been recognized as a novel target for antibacterial drugs. The structure of the complex with the inhibitor PC190723, which has potent and selective antistaphylococcal activity, indicated that the inhibitor binds at the cleft between the two subdomains.

  1. Structure and substrate recognition of the Staphylococcus aureus protein tyrosine phosphatase PtpA.

    Science.gov (United States)

    Vega, Carolina; Chou, Seemay; Engel, Katherine; Harrell, Maria E; Rajagopal, Lakshmi; Grundner, Christoph

    2011-10-14

    Phosphosignaling through pSer/pThr/pTyr is emerging as a common signaling mechanism in prokaryotes. The human pathogen Staphylococcus aureus produces two low-molecular-weight protein tyrosine phosphatases (PTPs), PtpA and PtpB, with unknown functions. To provide the structural context for understanding PtpA function and substrate recognition, establish PtpA's structural relations within the PTP family, and provide a framework for the design of specific inhibitors, we solved the crystal structure of PtpA at 1 Å resolution. While PtpA adopts the common, conserved PTP fold and shows close overall similarity to eukaryotic PTPs, several features in the active site and surface organization are unique and can be explored to design selective inhibitors. A peptide bound in the active site mimics a phosphotyrosine substrate, affords insight into substrate recognition, and provides a testable substrate prediction. Genetic deletion of ptpA or ptpB does not affect in vitro growth or cell wall integrity, raising the possibility that PtpA and PtpB have specialized functions during infection.

  2. The Staphylococcus aureus NuoL-like protein MpsA contributes to the generation of membrane potential.

    Science.gov (United States)

    Mayer, Sonja; Steffen, Wojtek; Steuber, Julia; Götz, Friedrich

    2015-03-01

    In aerobic microorganisms, the entry point of respiratory electron transfer is represented by the NADH:quinone oxidoreductase. The enzyme couples the oxidation of NADH with the reduction of quinone. In the type 1 NADH:quinone oxidoreductase (Ndh1), this reaction is accompanied by the translocation of cations, such as H(+) or Na(+). In Escherichia coli, cation translocation is accomplished by the subunit NuoL, thus generating membrane potential (Δψ). Some microorganisms achieve NADH oxidation by the alternative, nonelectrogenic type 2 NADH:quinone oxidoreductase (Ndh2), which is not cation translocating. Since these enzymes had not been described in Staphylococcus aureus, the goal of this study was to identify proteins operating in the NADH:quinone segment of its respiratory chain. We demonstrated that Ndh2 represents a NADH:quinone oxidoreductase in S. aureus. Additionally, we identified a hypothetical protein in S. aureus showing sequence similarity to the proton-translocating subunit NuoL of complex I in E. coli: the NuoL-like protein MpsA. Mutants with deletion of the nuoL-like gene mpsA and its corresponding operon, mpsABC (mps for membrane potential-generating system), exhibited a small-colony-variant-like phenotype and were severely affected in Δψ and oxygen consumption rates. The MpsABC proteins did not confer NADH oxidation activity. Using an Na(+)/H(+) antiporter-deficient E. coli strain, we could show that MpsABC constitute a cation-translocating system capable of Na(+) transport. Our study demonstrates that MpsABC represent an important functional system of the respiratory chain of S. aureus that acts as an electrogenic unit responsible for the generation of Δψ.

  3. Staphylococcus aureus Manganese Transport Protein C (MntC) Is an Extracellular Matrix- and Plasminogen-Binding Protein

    Science.gov (United States)

    Salazar, Natália; Castiblanco-Valencia, Mónica Marcela; da Silva, Ludmila Bezerra; de Castro, Íris Arantes; Monaris, Denize; Masuda, Hana Paula; Barbosa, Angela Silva; Arêas, Ana Paula Mattos

    2014-01-01

    Infections caused by Staphylococcus aureus – particularly nosocomial infections - represent a great concern. Usually, the early stage of pathogenesis consists on asymptomatic nasopharynx colonization, which could result in dissemination to other mucosal niches or invasion of sterile sites, such as blood. This pathogenic route depends on scavenging of nutrients as well as binding to and disrupting extracellular matrix (ECM). Manganese transport protein C (MntC), a conserved manganese-binding protein, takes part in this infectious scenario as an ion-scavenging factor and surprisingly as an ECM and coagulation cascade binding protein, as revealed in this work. This study showed a marked ability of MntC to bind to several ECM and coagulation cascade components, including laminin, collagen type IV, cellular and plasma fibronectin, plasminogen and fibrinogen by ELISA. The MntC binding to plasminogen appears to be related to the presence of surface-exposed lysines, since previous incubation with an analogue of lysine residue, ε-aminocaproic acid, or increasing ionic strength affected the interaction between MntC and plasminogen. MntC-bound plasminogen was converted to active plasmin in the presence of urokinase plasminogen activator (uPA). The newly released plasmin, in turn, acted in the cleavage of the α and β chains of fibrinogen. In conclusion, we describe a novel function for MntC that may help staphylococcal mucosal colonization and establishment of invasive disease, through the interaction with ECM and coagulation cascade host proteins. These data suggest that this potential virulence factor could be an adequate candidate to compose an anti-staphylococcal human vaccine formulation. PMID:25409527

  4. The Chaperone ClpX Stimulates Expression of Staphylococcus aureus Protein A by Rot Dependent and Independent Pathways

    DEFF Research Database (Denmark)

    Jelsbak, Lotte; Ingmer, Hanne; Valihrach, Lukás;

    2010-01-01

    The Clp ATPases (Hsp100) constitute a family of closely related proteins that have protein reactivating and remodelling activities typical of molecular chaperones. In Staphylococcus aureus the ClpX chaperone is essential for virulence and for transcription of spa encoding Protein A. The present...... study was undertaken to elucidate the mechanism by which ClpX stimulates expression of Protein A. For this purpose, we prepared antibodies directed against Rot, an activator of spa transcription, and demonstrated that cells devoid of ClpX contain three-fold less Rot than wild-type cells. By varying Rot...... expression from an inducible promoter we showed that expression of Protein A requires a threshold level of Rot. In the absence of ClpX the Rot content is reduced below this threshold level, hence, explaining the substantially reduced Protein A expression in the clpX mutant. Experiments addressed...

  5. Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus.

    Science.gov (United States)

    Poupel, Olivier; Moyat, Mati; Groizeleau, Julie; Antunes, Luísa C S; Gribaldo, Simonetta; Msadek, Tarek; Dubrac, Sarah

    2016-01-01

    The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene

  6. Laser-assisted synthesis of Staphylococcus aureus protein-capped silicon quantum dots as bio-functional nanoprobes

    Science.gov (United States)

    Bagga, K.; Barchanski, A.; Intartaglia, R.; Dante, S.; Marotta, R.; Diaspro, A.; Sajti, C. L.; Brandi, F.

    2013-06-01

    A novel approach for nanofabricating protein-functionalized luminescent silicon nanoparticles based on infrared ultrafast laser ablation of silicon in an aqueous solution of Staphylococcus aureus protein A is reported. It is demonstrated that 8 nm protein A-capped silicon quantum dots with blue-green photoemissive properties are generated. The conjugation efficiency studies reveal a high percentage of protein A attached to the Si nanoparticle surface through physical adsorption phenomena during the in situ laser process. The biological functionality of laser-generated Staphylococcus aureus protein A-capped Si nanoparticles is investigated. Confocal and electron microscopy together with energy dispersive x-ray spectroscopy analysis show that these Si-based bio-nanostructures selectively bind IgG in the cells. Cell viability studies reveal that these protein A-capped Si nanoparticles are suitable for biological applications, demonstrating their potential as universal secondary biomarkers for in vivo applications such as long-term, real-time cell labeling, cell staining and controlled drug delivery.

  7. The Staphylococcus aureus extracellular adherence protein promotes bacterial internalization by keratinocytes independent of fibronectin-binding proteins.

    Science.gov (United States)

    Bur, Stephanie; Preissner, Klaus T; Herrmann, Mathias; Bischoff, Markus

    2013-08-01

    Staphylococcus aureus, the leading causal pathogen of skin infections, is strongly associated with skin atopy, and a number of bacterial adhesins allow the microbe to adhere to and invade eukaryotic cells. One of these adhesive molecules is the multifunctional extracellular adherence protein (Eap), which is overexpressed in situ in authentic human wounds and was shown to delay wound healing in experimental models. Yet, its role during invasion of keratinocytes is not clearly defined. By using a gentamicin/lysostaphin protection assay we demonstrate here that preincubation of HaCaT cells or primary keratinocytes with Eap results in a concentration-dependent significant increase in staphylococcal adhesion, followed by an even more pronounced internalization of bacteria by eukaryotic cells. Flow cytometric analysis revealed that Eap increased both the number of infected eukaryotic cells and the bacterial load per infected cell. Moreover, treatment of keratinocytes with Eap strongly enhanced the internalization of coagulase-negative staphylococci, as well as of E. coli, and markedly promoted staphylococcal invasion into extended-culture keratinocytes, displaying expression of keratin 10 and involucrin as differentiation markers. Thus, wound-related staphylococcal Eap may provide a major cellular invasin function, thereby enhancing the pathogen's ability to hide from the host immune system during acute and chronic skin infection.

  8. Protection of mice against Staphylococcus aureus infection by a recombinant protein ClfA-IsdB-Hlg as a vaccine candidate.

    Science.gov (United States)

    Delfani, Somayeh; Mohabati Mobarez, Ashraf; Imani Fooladi, Abbas Ali; Amani, Jafar; Emaneini, Mohammad

    2016-02-01

    Staphylococcus aureus is one of the most important causes of nosocomial infections. An effective vaccine to prevent S. aureus infections is urgently required due to the dramatic increase in the number of antibiotic-resistant strains. In this report, we evaluated a newly recombinant protein composed of selected antigenic regions of clumping factor A (ClfA), iron surface determinant B (IsdB) and gamma hemolysin B (HlgB) of S. aureus and sequence coding for hydrophobic linkers between three domains. The recombinant gene was constructed in pET-28a (+) and expressed in Escherichia coli BL21. In addition, sequence coding for a His(6)-tag was added followed by a hybrid procedure of nickel chelate protein purification. Immunization of BALB/c mice with the recombinant protein ClfA-IsdB-Hlg evoked antigen-specific antibodies that could opsonize S. aureus cells, enhancing in vitro phagocytosis by macrophages. Vaccination with the recombinant protein also reduced the bacterial load recovered from mice spleen samples and increased survival following the intraperitoneal challenge with pathogenic S. aureus compared to the control mice. Our results showed that the recombinant protein ClfA-IsdB-Hlg is a promising vaccine candidate for the prevention of S. aureus bacteremia infections.

  9. Soluble CD163 masks fibronectin-binding protein A-mediated inflammatory activation of Staphylococcus aureus infected monocytes.

    Science.gov (United States)

    Kneidl, Jessica; Mysore, Vijayashree; Geraci, Jennifer; Tuchscherr, Lorena; Löffler, Bettina; Holzinger, Dirk; Roth, Johannes; Barczyk-Kahlert, Katarzyna

    2014-03-01

    Binding to fibronectin (FN) is a crucial pathogenic factor of Staphylococcus aureus mediated by fibronectin-binding protein A (FnBP-A) and extracellular adherence protein (Eap). Recently, we have shown that binding of soluble CD163 (sCD163) to FN linked to these molecules exhibits anti-microbial effects by enhancing phagocytosis and killing activity of S. aureus-infected monocytes. However, it remained unclear whether sCD163 also influences the monocytic activation status. Using genetically modified staphylococcal strains we now identified FnBP-A, but not Eap, as activator of the inflammatory response of monocytes to infection. FnBP-A-mediated inflammatory activation was masked by sCD163 binding to S. aureus promoting efficient pathogen elimination. Thus, sCD163 protects monocytes from overwhelming activation upon staphylococcal infection by dampening the secretion of pro-inflammatory cytokines TNFα, IL-1β, IL-6 and IL-8 and DAMP molecule MRP8/14. Moreover, sCD163 limited expression of pro-apoptotic transcription factor NR4A1 induced during S. aureus infection and inhibited induction of chemokine CXCL2promoting survival of staphylococci in vivo. sCD163-mediated effects were not due to general immunosuppression since MAP kinase activation and ROS production were unaltered during infection of monocytes with sCD163-bound bacteria. Thus, sCD163 promotes a specific defence of the immune system against FnBP-A-mediated inflammatory activation enabling successful pathogen elimination, tissue recovery and resolution of inflammation.

  10. Identification of Functional Regulatory Residues of the β-Lactam Inducible Penicillin Binding Protein in Methicillin-Resistant Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Andreas N. Mbah

    2013-01-01

    Full Text Available Resistance to methicillin by Staphylococcus aureus is a persistent clinical problem worldwide. A mechanism for resistance has been proposed in which methicillin resistant Staphylococcus aureus (MRSA isolates acquired a new protein called β-lactam inducible penicillin binding protein (PBP-2′. The PBP-2′ functions by substituting other penicillin binding proteins which have been inhibited by β-lactam antibiotics. Presently, there is no structural and regulatory information on PBP-2′ protein. We conducted a complete structural and functional regulatory analysis of PBP-2′ protein. Our analysis revealed that the PBP-2′ is very stable with more hydrophilic amino acids expressing antigenic sites. PBP-2′ has three striking regulatory points constituted by first penicillin binding site at Ser25, second penicillin binding site at Ser405, and finally a single metallic ligand binding site at Glu657 which binds to Zn2+ ions. This report highlights structural features of PBP-2′ that can serve as targets for developing new chemotherapeutic agents and conducting site direct mutagenesis experiments.

  11. Identification of Functional Regulatory Residues of the β -Lactam Inducible Penicillin Binding Protein in Methicillin-Resistant Staphylococcus aureus.

    Science.gov (United States)

    Mbah, Andreas N; Isokpehi, Raphael D

    2013-01-01

    Resistance to methicillin by Staphylococcus aureus is a persistent clinical problem worldwide. A mechanism for resistance has been proposed in which methicillin resistant Staphylococcus aureus (MRSA) isolates acquired a new protein called β -lactam inducible penicillin binding protein (PBP-2'). The PBP-2' functions by substituting other penicillin binding proteins which have been inhibited by β -lactam antibiotics. Presently, there is no structural and regulatory information on PBP-2' protein. We conducted a complete structural and functional regulatory analysis of PBP-2' protein. Our analysis revealed that the PBP-2' is very stable with more hydrophilic amino acids expressing antigenic sites. PBP-2' has three striking regulatory points constituted by first penicillin binding site at Ser25, second penicillin binding site at Ser405, and finally a single metallic ligand binding site at Glu657 which binds to Zn(2+) ions. This report highlights structural features of PBP-2' that can serve as targets for developing new chemotherapeutic agents and conducting site direct mutagenesis experiments.

  12. Clonal distribution of bone sialoprotein-binding protein gene among Staphylococcus aureus isolates associated with bloodstream infections.

    Science.gov (United States)

    Wiśniewska, Katarzyna; Piórkowska, Anna; Kasprzyk, Joanna; Bronk, Marek; Świeć, Krystyna

    2014-11-01

    Staphylococcus aureus is a leading cause of bloodstream infections (BSI) and diseases that may be caused by hematogenous spread. The staphylococcal adhesin, for which the association with the infections emerging as a complication of septicemia has been well documented, is a bone sialoprotein-binding protein (Bbp). The aim of the study was to assess the prevalence of a bbp gene in S. aureus bloodstream isolates associated with BSI and to investigate to what degree the distribution of this gene is linked to the clonality of the population. Spa typing, used in order to explore the genetic population structure of the isolates, yielded 29 types. Six spa clusters and seven singletons were identified. The most frequent was spa clonal complex CC021 associated with MLST CC30 (38%). The bbp gene was found in 47% of isolates. Almost all isolates (95%) clustered in spa clonal complex CC021 were positive for this gene. All isolates carrying the bbp gene were sensitive to methicillin, and if clustered in the spa CC021, belonged to agr group III. Our study shows that Bbp is not strictly associated with BSI. However, one may conclude that for clonally related S. aureus strains most commonly causing BSI, the risk of Bbp-mediated complications of septicemia is expected to be higher than for other strains.

  13. Method for generation of peptide-specific IgY antibodies directed to Staphylococcus aureus extracellular fibrinogen binding protein epitope.

    Science.gov (United States)

    Walczak, Maciej; Grzywa, Renata; Łupicka-Słowik, Agnieszka; Skoreński, Marcin; Bobrek, Kamila; Nowak, Daria; Boivin, Stephane; Brown, Eric L; Oleksyszyn, Józef; Sieńczyk, Marcin

    2015-09-01

    The IgY antibodies offer an attractive alternative to mammalian IgGs in research, diagnosis and medicine. The isolation of immunoglobulin Y from the egg yolks is efficient and economical, causing minimal suffering to animals. Here we present the methodology for the production of IgY antibodies specific to Staphylococcus aureus fibrinogen binding protein (Efb) and its peptidyl epitope (spanning residues 127-140). The Efb is an extracellular, adhesion protein which binds both human fibrinogen and complement C3 protein thus contributing to the high infectious potential of this pathogen. The selected epitope of Efb protein is responsible for the interaction with C3. The immunochemical characterization of both anti-Efb and epitope-specific IgY antibodies revealed their similar avidity, titer, and reactivity profile, although some differences in the hen's immune response to administered antigens is discussed.

  14. A fibronectin-binding protein (FbpA) of Weissella cibaria inhibits colonization and infection of Staphylococcus aureus in mammary glands.

    Science.gov (United States)

    Wang, Liangliang; Si, Wei; Xue, Huping; Zhao, Xin

    2017-01-26

    Staphylococcus aureus (S. aureus) is a frequent cause of infections in both humans and animals. Probiotics are known to inhibit colonization of pathogens on host tissues. However, mechanisms for the inhibition are still elusive due to complex host-microbe and microbe-microbe interactions. Here, we show that reduced abilities of S. aureus to infect mammary glands in the presence of Weissella cibaria (W. cibaria) were correlated with its poor adherence to mammary epithelial cells. Such inhibition by W. cibaria isolates was at least partially attributed to a fibronectin-binding protein (FbpA) on this lactic acid bacterium. Three W. cibaria isolates containing fbpA had higher inhibitory abilities than other three LAB isolates without the gene. The fbpA-deficient mutant of W. cibaria isolate LW1, LW1ΔfbpA, lost the inhibitory activity to reduce the adhesion of S. aureus to mammary epithelial cells and was less able to reduce the colonization of S. aureus in mammary glands. Expression of FbpA to the surface of LW1ΔfbpA reversed its inhibitory activities. Furthermore, addition of purified FbpA inhibited S. aureus biofilm formation. Our results suggest that W. cibaria FbpA hinders S. aureus colonization and infection through interfering with the S. aureus invasion pathway mediated by fibronectin-binding proteins and inhibiting biofilm formation of S. aureus.

  15. Affinity of ceftaroline and other beta-lactams for penicillin-binding proteins from Staphylococcus aureus and Streptococcus pneumoniae.

    Science.gov (United States)

    Kosowska-Shick, K; McGhee, P L; Appelbaum, P C

    2010-05-01

    We compared the affinities of ceftaroline for all penicillin-binding proteins (PBPs) with those of ceftriaxone and cefotaxime in 6 Staphylococcus aureus and 7 Streptococcus pneumoniae isolates with various resistance phenotypes. Ceftaroline MICs were pneumoniae. Ceftaroline affinities for penicillin-susceptible S. pneumoniae strains were in the order PBP2X and -3 > PBP1A, -1B, and -2A > PBP2B, and ceftaroline had >or=4-fold higher 50% inhibitory concentrations (IC(50)s) (0.1 to 4 microg/ml) for PBP2X, -2A, -2B, and -3 than those for the other cephalosporins tested. Among 3 penicillin-resistant S. pneumoniae strains, ceftaroline had a high affinity for PBP2X (IC(50), 0.1 to 1 microg/ml), a primary target for cephalosporin PBP binding activity, and high affinities for PBP2B (IC(50), 0.5 to 4 microg/ml) and PBP1A (IC(50), 0.125 to 0.25 microg/ml) as well, both of which are also known as major targets for PBP binding activity of cephalosporins. Ceftaroline PBP affinities in methicillin-susceptible S. aureus strains were greater than or equal to those of the 3 other beta-lactams tested. Ceftaroline bound to PBP2a in methicillin-resistant S. aureus (IC(50), 0.01 to 1 microg/ml) with up to 256-fold-higher affinity than those of other agents. Ceftaroline demonstrated very good PBP affinity against all S. aureus and S. pneumoniae strains tested, including resistant isolates.

  16. Staphylococcus aureus Colonization of the Mouse Gastrointestinal Tract Is Modulated by Wall Teichoic Acid, Capsule, and Surface Proteins.

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    Yoshiki Misawa

    2015-07-01

    Full Text Available Staphylococcus aureus colonizes the nose, throat, skin, and gastrointestinal (GI tract of humans. GI carriage of S. aureus is difficult to eradicate and has been shown to facilitate the transmission of the bacterium among individuals. Although staphylococcal colonization of the GI tract is asymptomatic, it increases the likelihood of infection, particularly skin and soft tissue infections caused by USA300 isolates. We established a mouse model of persistent S. aureus GI colonization and characterized the impact of selected surface antigens on colonization. In competition experiments, an acapsular mutant colonized better than the parental strain Newman, whereas mutants defective in sortase A and clumping factor A showed impaired ability to colonize the GI tract. Mutants lacking protein A, clumping factor B, poly-N-acetyl glucosamine, or SdrCDE showed no defect in colonization. An S. aureus wall teichoic acid (WTA mutant (ΔtagO failed to colonize the mouse nose or GI tract, and the tagO and clfA mutants showed reduced adherence in vitro to intestinal epithelial cells. The tagO mutant was recovered in lower numbers than the wild type strain in the murine stomach and duodenum 1 h after inoculation. This reduced fitness correlated with the in vitro susceptibility of the tagO mutant to bile salts, proteases, and a gut-associated defensin. Newman ΔtagO showed enhanced susceptibility to autolysis, and an autolysin (atl tagO double mutant abrogated this phenotype. However, the atl tagO mutant did not survive better in the mouse GI tract than the tagO mutant. Our results indicate that the failure of the tagO mutant to colonize the GI tract correlates with its poor adherence and susceptibility to bactericidal factors within the mouse gut, but not to enhanced activity of its major autolysin.

  17. New shuttle vector-based expression system to generate polyhistidine-tagged fusion proteins in Staphylococcus aureus and Escherichia coli.

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    Schwendener, Sybille; Perreten, Vincent

    2015-05-01

    Four Staphylococcus aureus-Escherichia coli shuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-Pcap-HC and pTSSCm-Pcap contain the strong constitutive promoter of S. aureus type 1 capsule gene 1A (Pcap) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (rgs-his6). All plasmids contained the backbone derived from pBUS1, including the E. coli origin ColE1, five copies of terminator rrnB T1, and tetracycline resistance marker tet(L) for S. aureus and E. coli. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand origin oriL from pUB110 (pBUS1-HC and pBUS1-Pcap-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-Pcap). The new constructs displayed increased plasmid yield and segregational stability in S. aureus. Furthermore, pBUS1-Pcap-HC and pTSSCm-Pcap offer the potential to generate C-terminal RGS-His6 translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links the rgs-his6 codons to the 3' end of the target gene. The generation of the rgs-his6 codon-fusion, gene expression, and protein purification were demonstrated in both S. aureus and E. coli using the macrolide-lincosamide-streptogramin B resistance gene erm(44) inserted downstream of Pcap. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells.

  18. Involvement of major facilitator superfamily proteins SfaA and SbnD in staphyloferrin secretion in Staphylococcus aureus.

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    Hannauer, Mélissa; Sheldon, Jessica R; Heinrichs, David E

    2015-03-12

    A paucity of information exists concerning the mechanism(s) by which bacteria secrete siderophores into the extracellular compartment. We investigated the role of SfaA and SbnD, two major facilitator superfamily (MFS)-type efflux proteins, in the secretion of the Staphylococcus aureus siderophores staphyloferrin A (SA) and staphyloferrin B (SB), respectively. Deletion of sfaA resulted in a drastic reduction of SA secreted into the supernatant with a corresponding accumulation of SA in the cytoplasm and a significant growth defect in cells devoid of SB synthesis. In contrast, sbnD mutants showed transiently lowered levels of secreted SB, suggesting the involvement of additional efflux mechanisms.

  19. Characterization of EssB, a protein required for secretion of ESAT-6 like proteins in Staphylococcus aureus

    OpenAIRE

    Chen Yi-Hsing; Anderson Mark; Hendrickx Antoni PA; Missiakas Dominique

    2012-01-01

    Abstract Background Staphylococcus aureus secretes EsxA and EsxB, two small polypeptides of the WXG100 family of proteins. Genetic analyses have shown that production and secretion of EsxA and EsxB require an intact ESAT-6 Secretion System (ESS), a cluster of genes that is conserved in many Firmicutes and encompasses esxA and esxB . Here, we characterize EssB, one of the proteins encoded by the ESS cluster. EssB is highly conserved in Gram-positive bacteria and belongs to the Cluster of ...

  20. The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules

    NARCIS (Netherlands)

    Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

    Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including

  1. The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules

    NARCIS (Netherlands)

    Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

    2006-01-01

    Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including

  2. Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains

    NARCIS (Netherlands)

    Kneuper, Holger; Cao, Zhen Ping; Twomey, Kate B; Zoltner, Martin; Jäger, Franziska; Cargill, James S; Chalmers, James; van der Kooi - Pol, Magda; van Dijl, Jan Maarten; Ryan, Robert P; Hunter, William N; Palmer, Tracy

    2014-01-01

    The Type VII protein secretion system, found in Gram-positive bacteria, secretes small proteins, containing a conserved W-x-G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but r

  3. Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains

    NARCIS (Netherlands)

    Kneuper, Holger; Cao, Zhen Ping; Twomey, Kate B; Zoltner, Martin; Jäger, Franziska; Cargill, James S; Chalmers, James; van der Kooi - Pol, Magda; van Dijl, Jan Maarten; Ryan, Robert P; Hunter, William N; Palmer, Tracy

    The Type VII protein secretion system, found in Gram-positive bacteria, secretes small proteins, containing a conserved W-x-G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but

  4. Staphylococcus aureus FepA and FepB proteins drive heme iron utilization in Escherichia coli.

    Science.gov (United States)

    Turlin, Evelyne; Débarbouillé, Michel; Augustyniak, Katarzyna; Gilles, Anne-Marie; Wandersman, Cécile

    2013-01-01

    EfeUOB-like tripartite systems are widespread in bacteria and in many cases they are encoded by genes organized into iron-regulated operons. They consist of: EfeU, a protein similar to the yeast iron permease Ftrp1; EfeO, an extracytoplasmic protein of unknown function and EfeB, also an extracytoplasmic protein with heme peroxidase activity, belonging to the DyP family. Many bacterial EfeUOB systems have been implicated in iron uptake, but a prefential iron source remains undetermined. Nevertheless, in the case of Escherichia coli, the EfeUOB system has been shown to recognize heme and to allow extracytoplasmic heme iron extraction via a deferrochelation reaction. Given the high level of sequence conservations between EfeUOB orthologs, we hypothesized that heme might be the physiological iron substrate for the other orthologous systems. To test this hypothesis, we undertook characterization of the Staphylococcus aureus FepABC system. Results presented here indicate: i) that the S. aureus FepB protein binds both heme and PPIX with high affinity, like EfeB, the E. coli ortholog; ii) that it has low peroxidase activity, comparable to that of EfeB; iii) that both FepA and FepB drive heme iron utilization, and both are required for this activity and iv) that the E. coli FepA ortholog (EfeO) cannot replace FepA in FepB-driven iron release from heme indicating protein specificity in these activities. Our results show that the function in heme iron extraction is conserved in the two orthologous systems.

  5. Quantitative proteomic view on secreted, cell surface-associated, and cytoplasmic proteins of the methicillin-resistant human pathogen Staphylococcus aureus under iron-limited conditions.

    Science.gov (United States)

    Hempel, Kristina; Herbst, Florian-Alexander; Moche, Martin; Hecker, Michael; Becher, Dörte

    2011-04-01

    Staphylococcus aureus is capable of colonizing and infecting humans by its arsenal of surface-exposed and secreted proteins. Iron-limited conditions in mammalian body fluids serve as a major environmental signal to bacteria to express virulence determinants. Here we present a comprehensive, gel-free, and GeLC-MS/MS-based quantitative proteome profiling of S. aureus under this infection-relevant situation. (14)N(15)N metabolic labeling and three complementing approaches were combined for relative quantitative analyses of surface-associated proteins. The surface-exposed and secreted proteome profiling approaches comprise trypsin shaving, biotinylation, and precipitation of the supernatant. By analysis of the outer subproteomic and cytoplasmic protein fraction, 1210 proteins could be identified including 221 surface-associated proteins. Thus, access was enabled to 70% of the predicted cell wall-associated proteins, 80% of the predicted sortase substrates, two/thirds of lipoproteins and more than 50% of secreted and cytoplasmic proteins. For iron-deficiency, 158 surface-associated proteins were quantified. Twenty-nine proteins were found in altered amounts showing particularly surface-exposed proteins strongly induced, such as the iron-regulated surface determinant proteins IsdA, IsdB, IsdC and IsdD as well as lipid-anchored iron compound-binding proteins. The work presents a crucial subject for understanding S. aureus pathophysiology by the use of methods that allow quantitative surface proteome profiling.

  6. The carriage of the serine-aspartate repeat protein-encoding sdr genes among Staphylococcus aureus lineages.

    Science.gov (United States)

    Liu, Huanle; Lv, Jingnan; Qi, Xiuqin; Ding, Yu; Li, Dan; Hu, Longhua; Wang, Liangxing; Yu, Fangyou

    2015-01-01

    The serine-aspartate repeat proteins (Sdr) are members of a family of surface proteins and contribute to the pathogenicity of Staphylococcus aureus. Among 288 S. aureus isolates including 158 and 130 associated with skin and soft tissue infections and bloodstream infection, respectively; 275 (95.5%) were positive for at least one of three sdr genes tested. The positivity rates for sdrC, sdrD, and sdrE among S. aureus isolates were 87.8% (253/288), 63.9% (184/288), and 68.1% (196/288), respectively. 224 (77.8%) of 288 isolates were concomitantly positive for two or three sdr genes. There was an association between carriage of sdrE and methicillin-resistant S. aureus (MRSA) isolates, while the carriage rates of sdrC and sdrD in MRSA isolates were similar to those in methicillin-sensitive S. aureus (MSSA) isolates. The prevalence of co-existence of sdrC and sdrE among MRSA isolates was significantly higher than that among MSSA isolates (p<0.05). All ST1, ST5, ST7, and ST25 isolates were positive for sdrD. While all ST121 and ST398 isolates were negative for sdrD. All ST59 and ST88 isolates were positive for sdrE. All ST1 isolates were concomitantly positive for sdrC and sdrD. Concomitant carriage of sdrC, sdrD, and sdrE was found among all ST5, 75.0% (9/12) of ST1, 69.2% (9/13) of ST6, 78.6% (11/14) of ST25, and 90.9% (20/22) of ST88 isolates. sdrD was linked to CC5, CC7 and CC88 isolates, especially CC88 isolates. There was a strong association between the presence of sdrE and CC59, CC88, and CC5 isolates. A significant correlation between concomitant carriage of sdrC, sdrD, and sdrE and CC88 isolates was found. sdrC-positive, sdrD-positive and sdrE-negative gene profile was significantly associated with CC7 clone. There was an association between sdrC-positive, sdrD-negative, and sdrE-positive gene profile and CC59 isolates. A correlation between sdrC-positive, sdrD-negative, and sdrE-negative gene profile and CC121 clone was found. More CC59 isolates carried sdr

  7. Proteome analyses of cellular proteins in methicillin-resistant Staphylococcus aureus treated with rhodomyrtone, a novel antibiotic candidate.

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    Wipawadee Sianglum

    Full Text Available The ethanolic extract from Rhodomyrtus tomentosa leaf exhibited good antibacterial activities against both methicillin-resistant Staphylococcus aureus (MRSA and S. aureus ATCC 29213. Its minimal inhibitory concentration (MIC values ranged from 31.25-62.5 µg/ml, and the minimal bactericidal concentration (MBC was 250 µg/ml. Rhodomyrtone, an acylphloroglucinol derivative, was 62.5-125 times more potent at inhibiting the bacteria than the ethanolic extract, the MIC and MBC values were 0.5 µg/ml and 2 µg/ml, respectively. To provide insights into antibacterial mechanisms involved, the effects of rhodomyrtone on cellular protein expression of MRSA have been investigated using proteomic approaches. Proteome analyses revealed that rhodomyrtone at subinhibitory concentration (0.174 µg/ml affected the expression of several major functional classes of whole cell proteins in MRSA. The identified proteins involve in cell wall biosynthesis and cell division, protein degradation, stress response and oxidative stress, cell surface antigen and virulence factor, and various metabolic pathways such as amino acid, carbohydrate, energy, lipid, and nucleotide metabolism. Transmission electron micrographs confirmed the effects of rhodomyrtone on morphological and ultrastructural alterations in the treated bacterial cells. Biological processes in cell wall biosynthesis and cell division were interrupted. Prominent changes including alterations in cell wall, abnormal septum formation, cellular disintegration, and cell lysis were observed. Unusual size and shape of staphylococcal cells were obviously noted in the treated MRSA. These pioneer findings on proteomic profiling and phenotypic features of rhodomyrtone-treated MRSA may resolve its antimicrobial mechanisms which could lead to the development of a new effective regimen for the treatment of MRSA infections.

  8. Proteomic analyses of nucleoid-associated proteins in Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus.

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    Ryosuke L Ohniwa

    Full Text Available BACKGROUND: The bacterial nucleoid contains several hundred kinds of nucleoid-associated proteins (NAPs, which play critical roles in genome functions such as transcription and replication. Several NAPs, such as Hu and H-NS in Escherichia coli, have so far been identified. METHODOLOGY/PRINCIPAL FINDINGS: Log- and stationary-phase cells of E. coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus were lysed in spermidine solutions. Nucleoids were collected by sucrose gradient centrifugation, and their protein constituents analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS. Over 200 proteins were identified in each species. Envelope and soluble protein fractions were also identified. By using these data sets, we obtained lists of contaminant-subtracted proteins enriched in the nucleoid fractions (csNAP lists. The lists do not cover all of the NAPs, but included Hu regardless of the growth phases and species. In addition, the csNAP lists of each species suggested that the bacterial nucleoid is equipped with the species-specific set of global regulators, oxidation-reduction enzymes, and fatty acid synthases. This implies bacteria individually developed nucleoid associated proteins toward obtaining similar characteristics. CONCLUSIONS/SIGNIFICANCE: Ours is the first study to reveal hundreds of NAPs in the bacterial nucleoid, and the obtained data set enabled us to overview some important features of the nucleoid. Several implications obtained from the present proteomic study may make it a landmark for the future functional and evolutionary study of the bacterial nucleoid.

  9. Frequency of Fibronectin Binding Protein A and Panton-Valentine Leukocidin in Methicillin-Resistant Staphylococcus aureus Collected From Educational Hospitals in Qazvin, Iran

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    Taromian

    2016-02-01

    Full Text Available Background Staphylococcus aureus is one of the most important organisms involved in nosocomial infection acquired by patients. In recent years, the appearance of methicillin-resistant S. aureus (MRSA has turned the treatment of these infections into a serious challenge. Surface proteins, such as fibronectin binding proteins (FnBP, and the ability to produce Panton-Valentine leukocidin (PVL are important factors in pathogenesis of this organism. Objectives The purpose of this study was to determine the prevalence of disease-associated genes in the clinical isolates of S. aureus encoding FNB and PVL, collected from the educational hospitals of Qazvin, Iran. Patients and Methods This was a descriptive, cross-sectional study in which a total of 103 isolates of methicillin-resistant S. aureus were collected from hospitalized patients in teaching hospitals of Qazvin, during 2013 - 2014. Initially, the identification of isolates was performed according to the standard laboratory methods, followed by confirming the presence of the femA gene, a gene specific to S. aureus. Later, the prevalence of virulence genes (fnb and pvl was investigated by the PCR method, using specific primers. PCR products were sequenced to confirm the presence of the target genes. Results The results of this study showed that among 103 isolates of S. aureus resistant to methicillin, 88 isolates were positive for the presence of the pvl and fnb genes, with the fnb gene present in 86 (83.5% isolates and the pvl gene only in 2 (1.9% isolates. Conclusions The results of the present study indicate the presence of the pvl and fnb genes in the strains of S. aureus isolated from clinical specimens collected from the patients admitted to teaching hospitals in Qazvin. Considering the clinical significance of these organisms, and their potential in threatening public health systems, the identification, treatment, and infection control management of patients infected with these organisms is

  10. Green fluorescent protein (GFP)-based overexpression screening and characterization of AgrC, a Receptor protein of quorum sensing in Staphylococcus aureus.

    Science.gov (United States)

    Wang, Lina; Quan, Chunshan; Liu, Baoquan; Xu, Yongbin; Zhao, Pengchao; Xiong, Wen; Fan, Shengdi

    2013-09-06

    Staphylococcus aureus AgrC is an important component of the agr quorum-sensing system. AgrC is a membrane-embedded histidine kinase that is thought to act as a sensor for the recognition of environmental signals and the transduction of signals into the cytoplasm. However, the difficulty of expressing and purifying functional membrane proteins has drastically hindered in-depth understanding of the molecular structures and physiological functions of these proteins. Here, we describe the high-yield expression and purification of AgrC, and analyze its kinase activity. A C-terminal green fluorescent protein (GFP) fusion to AgrC served as a reporter for monitoring protein expression levels in real time. Protein expression levels were analyzed by the microscopic assessment of the whole-cell fluorescence. The expressed AgrC-GFP protein with a C-terminal His-tagged was purified using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) at yields of ≥ 10 mg/L, following optimization. We also assessed the effects of different detergents on membrane solubilization and AgrC kinase activity, and polyoxyethylene-(23)-lauryl-ether (Brij-35) was identified as the most suitable detergent. Furthermore, the secondary structural stability of purified AgrC was analyzed using circular dichroism (CD) spectroscopy. This study may serve as a general guide for improving the yields of other membrane protein preparations and selecting the appropriate detergent to stabilize membrane proteins for biophysical and biochemical analyses.

  11. Green Fluorescent Protein (GFP-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureus

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    Shengdi Fan

    2013-09-01

    Full Text Available Staphylococcus aureus AgrC is an important component of the agr quorum-sensing system. AgrC is a membrane-embedded histidine kinase that is thought to act as a sensor for the recognition of environmental signals and the transduction of signals into the cytoplasm. However, the difficulty of expressing and purifying functional membrane proteins has drastically hindered in-depth understanding of the molecular structures and physiological functions of these proteins. Here, we describe the high-yield expression and purification of AgrC, and analyze its kinase activity. A C-terminal green fluorescent protein (GFP fusion to AgrC served as a reporter for monitoring protein expression levels in real time. Protein expression levels were analyzed by the microscopic assessment of the whole-cell fluorescence. The expressed AgrC-GFP protein with a C-terminal His-tagged was purified using immobilized metal affinity chromatography (IMAC and size exclusion chromatography (SEC at yields of ≥10 mg/L, following optimization. We also assessed the effects of different detergents on membrane solubilization and AgrC kinase activity, and polyoxyethylene-(23-lauryl-ether (Brij-35 was identified as the most suitable detergent. Furthermore, the secondary structural stability of purified AgrC was analyzed using circular dichroism (CD spectroscopy. This study may serve as a general guide for improving the yields of other membrane protein preparations and selecting the appropriate detergent to stabilize membrane proteins for biophysical and biochemical analyses.

  12. The extracellular adherence protein from Staphylococcus aureus inhibits the classical and lectin pathways of complement by blocking formation of the C3 proconvertase.

    Science.gov (United States)

    Woehl, Jordan L; Stapels, Daphne A C; Garcia, Brandon L; Ramyar, Kasra X; Keightley, Andrew; Ruyken, Maartje; Syriga, Maria; Sfyroera, Georgia; Weber, Alexander B; Zolkiewski, Michal; Ricklin, Daniel; Lambris, John D; Rooijakkers, Suzan H M; Geisbrecht, Brian V

    2014-12-15

    The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of small inhibitory proteins. A number of these proteins inhibit the complement system, which labels bacteria for phagocytosis and generates inflammatory chemoattractants. Although the majority of staphylococcal complement inhibitors act on the alternative pathway to block the amplification loop, only a few proteins act on the initial recognition cascades that constitute the classical pathway (CP) and lectin pathway (LP). We screened a collection of recombinant, secreted staphylococcal proteins to determine whether S. aureus produces other molecules that inhibit the CP and/or LP. Using this approach, we identified the extracellular adherence protein (Eap) as a potent, specific inhibitor of both the CP and LP. We found that Eap blocked CP/LP-dependent activation of C3, but not C4, and that Eap likewise inhibited deposition of C3b on the surface of S. aureus cells. In turn, this significantly diminished the extent of S. aureus opsonophagocytosis and killing by neutrophils. This combination of functional properties suggested that Eap acts specifically at the level of the CP/LP C3 convertase (C4b2a). Indeed, we demonstrated a direct, nanomolar-affinity interaction of Eap with C4b. Eap binding to C4b inhibited binding of both full-length C2 and its C2b fragment, which indicated that Eap disrupts formation of the CP/LP C3 proconvertase (C4b2). As a whole, our results demonstrate that S. aureus inhibits two initiation routes of complement by expression of the Eap protein, and thereby define a novel mechanism of immune evasion.

  13. Protein S-Bacillithiolation Functions in Thiol Protection and Redox Regulation of the Glyceraldehyde-3-Phosphate Dehydrogenase Gap in Staphylococcus aureus Under Hypochlorite Stress.

    Science.gov (United States)

    Imber, Marcel; Huyen, Nguyen Thi Thu; Pietrzyk-Brzezinska, Agnieszka J; Loi, Vu Van; Hillion, Melanie; Bernhardt, Jörg; Thärichen, Lena; Kolšek, Katra; Saleh, Malek; Hamilton, Chris J; Adrian, Lorenz; Gräter, Frauke; Wahl, Markus C; Antelmann, Haike

    2017-01-18

    Bacillithiol (BSH) is the major low-molecular-weight thiol of the human pathogen Staphylococcus aureus. In this study, we used OxICAT and Voronoi redox treemaps to quantify hypochlorite-sensitive protein thiols in S. aureus USA300 and analyzed the role of BSH in protein S-bacillithiolation. The OxICAT analyses enabled the quantification of 228 Cys residues in the redox proteome of S. aureus USA300. Hypochlorite stress resulted in >10% increased oxidation of 58 Cys residues (25.4%) in the thiol redox proteome. Among the highly oxidized sodium hypochlorite (NaOCl)-sensitive proteins are five S-bacillithiolated proteins (Gap, AldA, GuaB, RpmJ, and PpaC). The glyceraldehyde-3-phosphate (G3P) dehydrogenase Gap represents the most abundant S-bacillithiolated protein contributing 4% to the total Cys proteome. The active site Cys151 of Gap was very sensitive to overoxidation and irreversible inactivation by hydrogen peroxide (H2O2) or NaOCl in vitro. Treatment with H2O2 or NaOCl in the presence of BSH resulted in reversible Gap inactivation due to S-bacillithiolation, which could be regenerated by the bacilliredoxin Brx (SAUSA300_1321) in vitro. Molecular docking was used to model the S-bacillithiolated Gap active site, suggesting that formation of the BSH mixed disulfide does not require major structural changes. Conclusion and Innovation: Using OxICAT analyses, we identified 58 novel NaOCl-sensitive proteins in the pathogen S. aureus that could play protective roles against the host immune defense and include the glycolytic Gap as major target for S-bacillithiolation. S-bacillithiolation of Gap did not require structural changes, but efficiently functions in redox regulation and protection of the active site against irreversible overoxidation in S. aureus. Antioxid. Redox Signal. 00, 000-000.

  14. Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains.

    Science.gov (United States)

    Kneuper, Holger; Cao, Zhen Ping; Twomey, Kate B; Zoltner, Martin; Jäger, Franziska; Cargill, James S; Chalmers, James; van der Kooi-Pol, Magdalena M; van Dijl, Jan Maarten; Ryan, Robert P; Hunter, William N; Palmer, Tracy

    2014-09-01

    The Type VII protein secretion system, found in Gram-positive bacteria, secretes small proteins, containing a conserved W-x-G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but required for virulence. In this study we show that there are unexpected differences in the organization of the ess gene cluster between closely related strains of S. aureus. We further show that in laboratory growth medium different strains of S. aureus secrete the EsxA and EsxC substrate proteins at different growth points, and that the Ess system in strain Newman is inactive under these conditions. Systematic deletion analysis in S. aureus RN6390 is consistent with the EsaA, EsaB, EssA, EssB, EssC and EsxA proteins comprising core components of the secretion machinery in this strain. Finally we demonstrate that the Ess secretion machinery of two S. aureus strains, RN6390 and COL, is important for nasal colonization and virulence in the murine lung pneumonia model. Surprisingly, however, the secretion system plays no role in the virulence of strain SA113 under the same conditions.

  15. The Staphylococcus aureus Global Regulator MgrA Modulates Clumping and Virulence by Controlling Surface Protein Expression.

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    Heidi A Crosby

    2016-05-01

    Full Text Available Staphylococcus aureus is a human commensal and opportunistic pathogen that causes devastating infections in a wide range of locations within the body. One of the defining characteristics of S. aureus is its ability to form clumps in the presence of soluble fibrinogen, which likely has a protective benefit and facilitates adhesion to host tissue. We have previously shown that the ArlRS two-component regulatory system controls clumping, in part by repressing production of the large surface protein Ebh. In this work we show that ArlRS does not directly regulate Ebh, but instead ArlRS activates expression of the global regulator MgrA. Strains lacking mgrA fail to clump in the presence of fibrinogen, and clumping can be restored to an arlRS mutant by overexpressing either arlRS or mgrA, indicating that ArlRS and MgrA constitute a regulatory pathway. We used RNA-seq to show that MgrA represses ebh, as well as seven cell wall-associated proteins (SraP, Spa, FnbB, SasG, SasC, FmtB, and SdrD. EMSA analysis showed that MgrA directly represses expression of ebh and sraP. Clumping can be restored to an mgrA mutant by deleting the genes for Ebh, SraP and SasG, suggesting that increased expression of these proteins blocks clumping by steric hindrance. We show that mgrA mutants are less virulent in a rabbit model of endocarditis, and virulence can be partially restored by deleting the genes for the surface proteins ebh, sraP, and sasG. While mgrA mutants are unable to clump, they are known to have enhanced biofilm capacity. We demonstrate that this increase in biofilm formation is partially due to up-regulation of SasG, a surface protein known to promote intercellular interactions. These results confirm that ArlRS and MgrA constitute a regulatory cascade, and that they control expression of a number of genes important for virulence, including those for eight large surface proteins.

  16. Crystal structure of Staphylococcus aureus exfoliative toxin D-like protein: Structural basis for the high specificity of exfoliative toxins.

    Science.gov (United States)

    Mariutti, Ricardo B; Souza, Tatiana A C B; Ullah, Anwar; Caruso, Icaro P; de Moraes, Fábio R; Zanphorlin, Leticia M; Tartaglia, Natayme R; Seyffert, Nubia; Azevedo, Vasco A; Le Loir, Yves; Murakami, Mário T; Arni, Raghuvir K

    2015-11-01

    Exfoliative toxins are serine proteases secreted by Staphylococcus aureus that are associated with toxin-mediated staphylococcal syndromes. To date, four different serotypes of exfoliative toxins have been identified and 3 of them (ETA, ETB, and ETD) are linked to human infection. Among these toxins, only the ETD structure remained unknown, limiting our understanding of the structural determinants for the functional differentiation between these toxins. We recently identified an ETD-like protein associated to S. aureus strains involved in mild mastitis in sheep. The crystal structure of this ETD-like protein was determined at 1.95 Å resolution and the structural analysis provide insights into the oligomerization, stability and specificity and enabled a comprehensive structural comparison with ETA and ETB. Despite the highly conserved molecular architecture, significant differences in the composition of the loops and in both the N- and C-terminal α-helices seem to define ETD-like specificity. Molecular dynamics simulations indicate that these regions defining ET specificity present different degrees of flexibility and may undergo conformational changes upon substrate recognition and binding. DLS and AUC experiments indicated that the ETD-like is monomeric in solution whereas it is present as a dimer in the asymmetric unit indicating that oligomerization is not related to functional differentiation among these toxins. Differential scanning calorimetry and circular dichroism assays demonstrated an endothermic transition centered at 52 °C, and an exothermic aggregation in temperatures up to 64 °C. All these together provide insights about the mode of action of a toxin often secreted in syndromes that are not associated with either ETA or ETB.

  17. Inhibition of formyl peptide receptor in high-grade astrocytoma by CHemotaxis Inhibitory Protein of S. aureus.

    Science.gov (United States)

    Boer, J C; Domanska, U M; Timmer-Bosscha, H; Boer, I G J; de Haas, C J C; Joseph, J V; Kruyt, F A E; de Vries, E G E; den Dunnen, W F A; van Strijp, J A G; Walenkamp, A M E

    2013-02-19

    High-grade astrocytomas are malignant brain tumours that infiltrate the surrounding brain tissue and have a poor prognosis. Activation of formyl peptide receptor (FPR1) on the human astrocytoma cell line U87 promotes cell motility, growth and angiogenesis. We therefore investigated the FPR1 inhibitor, Chemotaxis Inhibitory Protein of S. aureus (CHIPS), as a potential anti-astrocytoma drug. FPR1 expression was studied immunohistochemically in astrocytomas WHO grades I-IV. With intracellular calcium mobilisation and migration assays, human ligands were tested for their ability to activate FPR1 on U87 cells and on a cell line derived from primary astrocytoma grade IV patient material. Thereafter, we selectively inhibited these ligand-induced responses of FPR1 with an anti-inflammatory compound called Chemotaxis Inhibitory Protein of S. aureus (CHIPS). U87 xenografts in NOD-SCID mice served to investigate the effects of CHIPS in vivo. FPR1 was expressed in 29 out of 32 (90%) of all grades of astrocytomas. Two human mitochondrial-derived formylated peptides, formyl-methionil-leucine-lysine-isoleucine-valine (fMLKLIV) and formyl-methionil-methionil-tyrosine-alanine-leucine-phenylalanine (fMMYALF), were potent activators of FPR1 on tumour cells. Ligand-induced responses of FPR1-expressing tumour cells could be inhibited with FPR1 inhibitor CHIPS. Treatment of tumour-bearing mice with CHIPS slightly reduced tumour growth and improved survival as compared to non-treated animals (P=0.0019). Targeting FPR1 with CHIPS reduces cell motility and tumour cell activation, and prolongs the survival of tumour-bearing mice. This strategy could be explored in future research to improve treatment results for astrocytoma patients.

  18. Crustin protein Amk1 from black tiger shrimp (Penaeus monodon inhibits Vibrio harveyi and Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Moltira Tonganunt1*

    2008-05-01

    Full Text Available A crustin gene (Amk1 was identified from a haemocyte library of the black tiger shrimp, Penaeus monodon. The full-length cDNA consists of 411 bp encoding a deduced precursor of 136 amino acids with a signal peptide of 17 aminoacids. Amk1 contains a hydrophobic and a Gly-rich region at the N-terminus and a 12 conserved cysteine domain (6-DSC at the C-terminus. Transcripts of Amk1 are mainly detected in haemocytes and gills by RT-PCR analysis. A recombinant Amk1was overexpressed and purified from Escherichia coli. This has a molecular mass of 43.66 kDa with a predicted pI of 8.23. Antibacterial assays demonstrated that recombinant Amk1 exhibited antibacterial activity against Gram-positive and Gramnegativebacteria with strong inhibition against Staphylococcus aureus and Vibrio harveyi.

  19. Staphylococcus aureus isolates encode variant staphylococcal enterotoxin B proteins that are diverse in superantigenicity and lethality.

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    Petra L Kohler

    Full Text Available Staphylococcus aureus produces superantigens (SAgs that bind and cross-link T cells and APCs, leading to activation and proliferation of immune cells. SAgs bind to variable regions of the β-chains of T cell receptors (Vβ-TCRs, and each SAg binds a unique subset of Vβ-TCRs. This binding leads to massive cytokine production and can result in toxic shock syndrome (TSS. The most abundantly produced staphylococcal SAgs and the most common causes of staphylococcal TSS are TSS toxin-1 (TSST-1, and staphylococcal enterotoxins B and C (SEB and SEC, respectively. There are several characterized variants of humans SECs, designated SEC1-4, but only one variant of SEB has been described. Sequencing the seb genes from over 20 S. aureus isolates show there are at least five different alleles of seb, encoding forms of SEB with predicted amino acid substitutions outside of the predicted immune-cell binding regions of the SAgs. Examination of purified, variant SEBs indicates that these amino acid substitutions cause differences in proliferation of rabbit splenocytes in vitro. Additionally, the SEBs varied in lethality in a rabbit model of TSS. The SEBs were diverse in their abilities to cause proliferation of human peripheral blood mononuclear cells, and differed in their activation of subsets of T cells. A soluble, high-affinity Vβ-TCR, designed to neutralize the previously characterized variant of SEB (SEB1, was able to neutralize the variant SEBs, indicating that this high-affinity peptide may be useful in treating a variety of SEB-mediated illnesses.

  20. Investigation of signal transduction routes within the sensor/transducer protein BlaR1 of Staphylococcus aureus.

    Science.gov (United States)

    Staude, Michael W; Frederick, Thomas E; Natarajan, Sivanandam V; Wilson, Brian D; Tanner, Carol E; Ruggiero, Steven T; Mobashery, Shahriar; Peng, Jeffrey W

    2015-03-03

    The transmembrane antibiotic sensor/signal transducer protein BlaR1 is part of a cohort of proteins that confer β-lactam antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA) [Fisher, J. F., Meroueh, S. O., and Mobashery, S. (2005) Chem. Rev. 105, 395-424; Llarrull, L. I., Fisher, J. F., and Mobashery, S. (2009) Antimicrob. Agents Chemother. 53, 4051-4063; Llarrull, L. I., Toth, M., Champion, M. M., and Mobashery, S. (2011) J. Biol. Chem. 286, 38148-38158]. Specifically, BlaR1 regulates the inducible expression of β-lactamases that hydrolytically destroy β-lactam antibiotics. The resistance phenotype starts with β-lactam antibiotic acylation of the BlaR1 extracellular domain (BlaRS). The acylation activates the cytoplasmic protease domain through an obscure signal transduction mechanism. Here, we compare protein dynamics of apo versus antibiotic-acylated BlaRS using nuclear magnetic resonance. Our analyses reveal inter-residue interactions that relay acylation-induced perturbations within the antibiotic-binding site to the transmembrane helix regions near the membrane surface. These are the first insights into the process of signal transduction by BlaR1.

  1. Changes in the Expression of Biofilm-Associated Surface Proteins in Staphylococcus aureus Food-Environmental Isolates Subjected to Sublethal Concentrations of Disinfectants.

    Science.gov (United States)

    Cincarova, Lenka; Polansky, Ondrej; Babak, Vladimir; Kulich, Pavel; Kralik, Petr

    2016-01-01

    Sublethal concentrations (sub-MICs) of certain disinfectants are no longer effective in removing biofilms from abiotic surfaces and can even promote the formation of biofilms. Bacterial cells can probably adapt to these low concentrations of disinfectants and defend themselves by way of biofilm formation. In this paper, we report on three Staphylococcus aureus biofilm formers (strong B+++, moderate B++, and weak B+) that were cultivated with sub-MICs of commonly used disinfectants, ethanol or chloramine T, and quantified using Syto9 green fluorogenic nucleic acid stain. We demonstrate that 1.25-2.5% ethanol and 2500 μg/mL chloramine T significantly enhanced S. aureus biofilm formation. To visualize differences in biofilm compactness between S. aureus biofilms in control medium, 1.25% ethanol, or 2500 μg/mL chloramine T, scanning electron microscopy was used. To describe changes in abundance of surface-exposed proteins in ethanol- or chloramine T-treated biofilms, surface proteins were prepared using a novel trypsin shaving approach and quantified after dimethyl labeling by LC-LTQ/Orbitrap MS. Our data show that some proteins with adhesive functions and others with cell maintenance functions and virulence factor EsxA were significantly upregulated by both treatments. In contrast, immunoglobulin-binding protein A was significantly downregulated for both disinfectants. Significant differences were observed in the effect of the two disinfectants on the expression of surface proteins including some adhesins, foldase protein PrsA, and two virulence factors.

  2. The phage lytic proteins from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 display multiple active catalytic domains and do not trigger staphylococcal resistance.

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    Lorena Rodríguez-Rubio

    Full Text Available The increase in antibiotic resistance world-wide revitalized the interest in the use of phage lysins to combat pathogenic bacteria. In this work, we analyzed the specific cleavage sites on the staphylococcal peptidoglycan produced by three phage lytic proteins. The investigated cell wall lytic enzymes were the endolysin LysH5 derived from the S. aureus bacteriophage vB_SauS-phi-IPLA88 (phi-IPLA88 and two fusion proteins between lysostaphin and the virion-associated peptidoglycan hydrolase HydH5 (HydH5SH3b and HydH5Lyso. We determined that all catalytic domains present in these proteins were active. Additionally, we tested for the emergence of resistant Staphylococcus aureus to any of the three phage lytic proteins constructs. Resistant S. aureus could not be identified after 10 cycles of bacterial exposure to phage lytic proteins either in liquid or plate cultures. However, a quick increase in lysostaphin resistance (up to 1000-fold in liquid culture was observed. The lack of resistant development supports the use of phage lytic proteins as future therapeutics to treat staphylococcal infections.

  3. Detection of ST772 Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus (Bengal Bay clone and ST22 S. aureus isolates with a genetic variant of elastin binding protein in Nepal

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    R.H. Pokhrel

    2016-05-01

    Full Text Available Genetic characteristics were analysed for recent clinical isolates of methicillin-resistant and -susceptible Staphylococcus aureus (MRSA and MSSA respectively in Kathmandu, Nepal. MRSA isolates harbouring Panton-Valentine leukocidin (PVL genes were classified into ST1, ST22 and ST88 with SCCmec-IV and ST772 with SCCmec-V (Bengal Bay clone, while PVL-positive MSSA into ST22, ST30 and ST772. ST22 isolates (PVL-positive MRSA and MSSA, PVL-negative MRSA possessed a variant of elastin binding protein gene (ebpS with an internal deletion of 180 bp, which was similar to that reported for ST121 S. aureus previously outside Nepal. Phylogenetic analysis indicated that the ebpS variant in ST22 might have occurred independently of ST121 strains. This is the first report of ST772 PVL-positive MRSA in Nepal and detection of the deletion variant of ebpS in ST22 S. aureus.

  4. Surface-associated proteins of Staphylococcus aureus: their possible roles in virulence

    OpenAIRE

    Foster, Timothy; McDevitt, D

    1994-01-01

    textabstractA class of proteins that are associated with the cell surface of Gram-positive bacteria has been recognised. Common structural features which are implicated in the proper secretion and attachment of these proteins to the cell surface occur in the C-termini. N-terminal domains interact with the host by binding to soluble host proteins, to matrix proteins or to host cells. They probably have important roles in pathogenicity by allowing bacteria to avoid host defences and by acting a...

  5. Surface-associated proteins of Staphylococcus aureus: their possible roles in virulence

    NARCIS (Netherlands)

    T.J. Foster (Timothy); D. McDevitt

    1994-01-01

    textabstractA class of proteins that are associated with the cell surface of Gram-positive bacteria has been recognised. Common structural features which are implicated in the proper secretion and attachment of these proteins to the cell surface occur in the C-termini. N-terminal domains interact wi

  6. Surface-associated proteins of Staphylococcus aureus: their possible roles in virulence

    NARCIS (Netherlands)

    T.J. Foster (Timothy); D. McDevitt

    1994-01-01

    textabstractA class of proteins that are associated with the cell surface of Gram-positive bacteria has been recognised. Common structural features which are implicated in the proper secretion and attachment of these proteins to the cell surface occur in the C-termini. N-terminal domains interact

  7. Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins

    Science.gov (United States)

    A partial-thickness epidermal explant model was colonized with GFP-expressing S. aureus and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. Oxygen concentration in explants and H2O2 in media was quantified using microelectrodes. The re...

  8. Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin alpha5beta1

    NARCIS (Netherlands)

    Sinha, B; François, P P; Nüsse, O; Foti, M; Hartford, O M; Vaudaux, P; Foster, T J; Lew, D P; Herrmann, M; Krause, K H

    The ability of Staphylococcus aureus to invade mammalian cells may explain its capacity to colonize mucosa and to persist in tissues after bacteraemia. To date, the underlying molecular mechanisms of cellular invasion by S. aureus are unknown, despite its high prevalence and difficulties in

  9. Population variation in anti-S. aureus IgG isotypes influences surface protein A mediated immune subversion.

    Science.gov (United States)

    Whitehouse, Julia; Flaxman, Amy; Rollier, Christine; O'Shea, Matthew K; Fallowfield, Joanne; Lindsay, Michael; Gunner, Frances; Knox, Kyle; Wyllie, David H; Yamaguchi, Yuko

    2016-04-04

    Staphylococcus aureus is a pathogen which causes life-threatening infection, the incidence of which rises during adult life. This, together with the emergence of drug-resistant strains and the expansion of more susceptible elderly populations, represents the rationale for the ongoing development of S. aureus vaccines targeting adult populations. Humoral responses to S. aureus naturally develop early in life, influence susceptibility to infection, and potentially influence the effect of vaccination. Despite this, the nature of pre-existing anti-S. aureus antibodies in healthy adult populations is not fully characterised. Immunoglobulin levels against S. aureus surface antigens were measured by a filter membrane enzyme-linked immunosorbent assay using fixed ΔSpA S. aureus as an antigen in serum samples obtained from three clinical cohorts comprising 133 healthy adult volunteers from 19 to 65 years of age. Functional capacity of antibody was also assessed, using antibody-mediated attachment of FITC-stained S. aureus to differentiated HL-60 cells. Wide variation in the concentrations of immunoglobulins recognising S. aureus surface antigens was observed among individuals in all three cohorts. There was a decline of anti-S. aureus IgG1 with age, and a similar trend was observed in IgM, but not in IgA or other IgG sub-classes. Antibody mediated bacterial attachment to cells was associated with IgG1 and IgG3 concentrations in serum. The presence of SpA on the bacterial cell surface reduced antibody-mediated binding of bacteria to phagocytes in serum with low, but not high, levels of naturally occurring anti-S. aureus IgG3 antibodies. Naturally acquired immunoglobulin responses to S. aureus are heterogeneous in populations and their concentrations alter during adulthood. Elevated IgG1 or IgG3 titres against S. aureus enhance S. aureus recognition by phagocytosis and may be correlates of natural protection and/or vaccine efficacy in adult populations. Copyright © 2016 The

  10. Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein from Staphylococcus aureus peptidoglycan.

    Science.gov (United States)

    Vieira, Diana; Figueiredo, Teresa A; Verma, Anil; Sobral, Rita G; Ludovice, Ana M; de Lencastre, Hermínia; Trincao, Jose

    2014-05-01

    Amidation of peptidoglycan is an essential feature in Staphylococcus aureus that is necessary for resistance to β-lactams and lysozyme. GatD, a 27 kDa type I glutamine amidotransferase-like protein, together with MurT ligase, catalyses the amidation reaction of the glutamic acid residues of the peptidoglycan of S. aureus. The native and the selenomethionine-derivative proteins were crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol, sodium acetate and calcium acetate. The crystals obtained diffracted beyond 1.85 and 2.25 Å, respectively, and belonged to space group P212121. X-ray diffraction data sets were collected at Diamond Light Source (on beamlines I02 and I04) and were used to obtain initial phases.

  11. Carbon and amide detect backbone assignment methods of a novel repeat protein from the staphylocoagulase in S. aureus.

    Science.gov (United States)

    Voehler, Markus; Ashoka, Maddur Appajaiah; Meiler, Jens; Bock, Paul E

    2017-08-17

    The C-terminal repeat domain of staphylocoagulase that is secreted by the S. aureus is believed to play an important role interacting with fibrinogen and promotes blood clotting. To study this interaction by NMR, full assignment of each amide residue in the HSQC spectrum was required. Despite of the short sequence of the repeat construct, the HSQC spectrum contained a substantial amount of overlapped and exchange broadened resonances, indicating little secondary or tertiary structure. This caused severe problems while using the conventional, amide based NMR method for the backbone assignment. With the growing interest in small apparently disordered proteins, these issues are being faced more frequently. An alternative strategy to improve the backbone assignment capability involved carbon direct detection methods. Circumventing the amide proton detection offers a larger signal dispersion and more uniform signal intensity. For peptides with higher concentrations and in combination with the cold carbon channels of new cryoprobes, higher fields, and sufficiently long relaxation times, the disadvantage of the lower sensitivity of the (13)C nucleus can be overcome. Another advantage of this method is the assignment of the proline backbone residues. Complete assignment with the carbon-detected strategy was achieved with a set of only two 3D, one 2D, and a HNCO measurement, which was necessary to translate the information to the HSQC spectrum.

  12. Neutrophil bactericidal activity against Staphylococcus aureus adherent on biological surfaces. Surface-bound extracellular matrix proteins activate intracellular killing by oxygen-dependent and -independent mechanisms.

    OpenAIRE

    Hermann, M.; Jaconi, M E; Dahlgren, C; Waldvogel, F A; Stendahl, O; Lew, D P

    1990-01-01

    The activation patterns of surface adherent neutrophils are modulated via interaction of extracellular matrix proteins with neutrophil integrins. To evaluate neutrophil bactericidal activity, Staphylococcus aureus adherent to biological surfaces were incubated with neutrophils and serum, and the survival of surface bacteria was determined. When compared to albumin-coated surfaces, the bactericidal activity of neutrophils adherent to purified human extracellular matrix was markedly enhanced (m...

  13. Extracellular Fibrinogen-binding Protein (Efb) from Staphylococcus aureus Inhibits the Formation of Platelet-Leukocyte Complexes.

    Science.gov (United States)

    Posner, Mareike G; Upadhyay, Abhishek; Abubaker, Aisha Alsheikh; Fortunato, Tiago M; Vara, Dina; Canobbio, Ilaria; Bagby, Stefan; Pula, Giordano

    2016-02-05

    Extracellular fibrinogen-binding protein (Efb) from Staphylococcus aureus inhibits platelet activation, although its mechanism of action has not been established. In this study, we discovered that the N-terminal region of Efb (Efb-N) promotes platelet binding of fibrinogen and that Efb-N binding to platelets proceeds via two independent mechanisms: fibrinogen-mediated and fibrinogen-independent. By proteomic analysis of Efb-interacting proteins within platelets and confirmation by pulldown assays followed by immunoblotting, we identified P-selectin and multimerin-1 as novel Efb interaction partners. The interaction of both P-selectin and multimerin-1 with Efb is independent of fibrinogen. We focused on Efb interaction with P-selectin. Excess of P-selectin extracellular domain significantly impaired Efb binding by activated platelets, suggesting that P-selectin is the main receptor for Efb on the surface of activated platelets. Efb-N interaction with P-selectin inhibited P-selectin binding to its physiological ligand, P-selectin glycoprotein ligand-1 (PSGL-1), both in cell lysates and in cell-free assays. Because of the importance of P-selectin-PSGL-1 binding in the interaction between platelets and leukocytes, we tested human whole blood and found that Efb abolishes the formation of platelet-monocyte and platelet-granulocyte complexes. In summary, we present evidence that in addition to its documented antithrombotic activity, Efb can play an immunoregulatory role via inhibition of P-selectin-PSGL-1-dependent formation of platelet-leukocyte complexes.

  14. Changes in the Expression of Biofilm-Associated Surface Proteins in Staphylococcus aureus Food-Environmental Isolates Subjected to Sublethal Concentrations of Disinfectants

    Directory of Open Access Journals (Sweden)

    Lenka Cincarova

    2016-01-01

    Full Text Available Sublethal concentrations (sub-MICs of certain disinfectants are no longer effective in removing biofilms from abiotic surfaces and can even promote the formation of biofilms. Bacterial cells can probably adapt to these low concentrations of disinfectants and defend themselves by way of biofilm formation. In this paper, we report on three Staphylococcus aureus biofilm formers (strong B+++, moderate B++, and weak B+ that were cultivated with sub-MICs of commonly used disinfectants, ethanol or chloramine T, and quantified using Syto9 green fluorogenic nucleic acid stain. We demonstrate that 1.25–2.5% ethanol and 2500 μg/mL chloramine T significantly enhanced S. aureus biofilm formation. To visualize differences in biofilm compactness between S. aureus biofilms in control medium, 1.25% ethanol, or 2500 μg/mL chloramine T, scanning electron microscopy was used. To describe changes in abundance of surface-exposed proteins in ethanol- or chloramine T-treated biofilms, surface proteins were prepared using a novel trypsin shaving approach and quantified after dimethyl labeling by LC-LTQ/Orbitrap MS. Our data show that some proteins with adhesive functions and others with cell maintenance functions and virulence factor EsxA were significantly upregulated by both treatments. In contrast, immunoglobulin-binding protein A was significantly downregulated for both disinfectants. Significant differences were observed in the effect of the two disinfectants on the expression of surface proteins including some adhesins, foldase protein PrsA, and two virulence factors.

  15. A novel mode of regulation of the Staphylococcus aureus Vancomycin-resistance-associated response regulator VraR mediated by Stk1 protein phosphorylation.

    Science.gov (United States)

    Canova, Marc J; Baronian, Grégory; Brelle, Solène; Cohen-Gonsaud, Martin; Bischoff, Markus; Molle, Virginie

    2014-04-25

    The Staphylococcus aureus Vancomycin-resistance-associated response regulator VraR is known as an important response regulator, member of the VraTSR three-component signal transduction system that modulates the expression of the cell wall stress stimulon in response to a number of different cell wall active antibiotics. Given its crucial role in regulating gene expression in response to antibiotic challenges, VraR must be tightly regulated. We report here for the first time in S. aureus convergence of two major signal transduction systems, serine/threonine protein kinase and two (three)-component systems. We demonstrate that VraR can be phosphorylated by the staphylococcal Ser/Thr protein kinase Stk1 and that phosphorylation negatively affects its DNA-binding properties. Mass spectrometric analyses and site-directed mutagenesis identified Thr106, Thr119, Thr175 and Thr178 as phosphoacceptors. A S. aureus ΔvraR mutant expressing a VraR derivative that mimics constitutive phosphorylation, VraR_Asp, still exhibited markedly decreased antibiotic resistance against different cell wall active antibiotics, when compared to the wild-type, suggesting that VraR phosphorylation may represent a novel and presumably more general mechanism of regulation of the two (three)-component systems in staphylococci.

  16. Staphylococcus aureus mutants lacking cell wall-bound protein A found in isolates from bacteraemia, MRSA infection and a healthy nasal carrier.

    Science.gov (United States)

    Sørum, Marit; Sangvik, Maria; Stegger, Marc; Olsen, Renate S; Johannessen, Mona; Skov, Robert; Sollid, Johanna U E

    2013-02-01

    Staphylococcus aureus is a major human pathogen and a multitude of virulence factors enables it to cause infections, from superficial lesions to life-threatening systemic conditions. Staphylococcal protein A (SpA) is a surface protein contributing to S. aureus pathogenesis by interfering with immune responses and activating inflammation. Seven isolates with frameshift mutations in the spa repeat region were investigated to determine whether these mutations lead to truncation and secretion of SpA into the extracellular environment. Five isolates originated from blood cultures, one from an MRSA infection and one from a persistent nasal carrier. Full-length spa genes from the seven isolates were sequenced, and Western blot experiments were performed to localize SpA. Three isolates had identical deviating 25-bp spa repeats, but all isolates displayed different repeat successions. The DNA sequence revealed that the frameshift mutations created premature stop codons in all seven isolates, resulting in truncated SpA of different lengths, however, all lacking the XC region with the C-terminal sorting signal. SpA was detected by Western blot in six of the seven isolates, mainly extracellularly. Our findings demonstrate that S. aureus isolates with truncated SpA, not anchored to the cell wall, can still be found in bacteraemia, infection and among carriers.

  17. The Staphylococcus aureus protein-coding gene gdpS modulates sarS expression via mRNA-mRNA interaction.

    Science.gov (United States)

    Chen, Chuan; Zhang, Xu; Shang, Fei; Sun, Haipeng; Sun, Baolin; Xue, Ting

    2015-08-01

    Staphylococcus aureus is an important Gram-positive pathogen responsible for numerous diseases ranging from localized skin infections to life-threatening systemic infections. The virulence of S. aureus is essentially determined by a wide spectrum of factors, including cell wall-associated proteins and secreted toxins that are precisely controlled in response to environmental changes. GGDEF domain protein from Staphylococcus (GdpS) is the only conserved staphylococcal GGDEF domain protein that is involved not in c-di-GMP synthesis but in the virulence regulation of S. aureus NCTC8325. Our previous study showed that the inactivation of gdpS generates an extensive change of virulence factors together with, in particular, a major Spa (protein A) surface protein. As reported, sarS is a direct positive regulator of spa. The decreased transcript levels of sarS in the gdpS mutant compared with the parental NCTC8325 strain suggest that gdpS affects spa through interaction with sarS. In this study, site mutation and complementary experiments showed that the translation product of gdpS was not involved in the regulation of transcript levels of sarS. We found that gdpS functioned through direct RNA-RNA base pairing with the 5' untranslated region (5'UTR) of sarS mRNA and that a putative 18-nucleotide region played a significant role in the regulatory process. Furthermore, the mRNA half-life analysis of sarS in the gdpS mutant showed that gdpS positively regulates the mRNA levels of sarS by contributing to the stabilization of sarS mRNA, suggesting that gdpS mRNA may regulate spa expression in an RNA-dependent pathway.

  18. Analysis of skin and secretions of Dybowski's frogs (Rana dybowskii) exposed to Staphylococcus aureus or Escherichia coli identifies immune response proteins.

    Science.gov (United States)

    Xiao, Xiang-Hong; Miao, Hui-Min; Xu, Yi-Gang; Zhang, Jing-Yu; Chai, Long-Hui; Xu, Jia-Jia

    2014-04-01

    The aim of the present study was to investigate responses in Dybowski's frogs (Rana dybowskii) exposed to bacteria, using proteomic and transcriptomic approaches. Staphylococcus aureus and Escherichia coli were used as representative Gram-positive and Gram-negative bacteria, respectively, in an infectious challenge model. Frog skin and skin secretions were collected and protein expression in infected frogs compared to control frogs by two-dimensional gel electrophoresis, silver staining, and image analysis. Proteins that demonstrated differential expression were analysed by mass spectrometry and identified by searching protein databases. More than 180 protein spots demonstrated differential expression in E. coli- or S. aureus-challenged groups and, of these, more than 55 spots were up- or down-regulated at least sixfold, post-infection. Proteins with a potential function in the immune response were identified, such as stathmin 1a, annexin A1, superoxide dismutase A, C-type lectin, lysozyme, antimicrobial peptides, cofilin-1-B, mannose receptor, histone H4, prohormone convertase 1, carbonyl reductase 1 and some components of the Toll-like receptor (TLR) signalling pathway. These molecules are potential candidates for further investigation of immune mechanisms in R. dybowskii; in particular, TLR-mediated responses, which might be activated in frogs exposed to pathogenic bacteria as part of innate immune defence, but which might also impact on adaptive immunity to infection.

  19. Comparative proteomic analysis of extracellular proteins expressed by various clonal types of Staphylococcus aureus and during planktonic growth and biofilm development

    Directory of Open Access Journals (Sweden)

    Salman Sahab Atshan

    2015-06-01

    Full Text Available Staphylococcus aureus is well known for its biofilm formation with rapid emergence of new clones circulating worldwide. The main objectives of the study were 1 to identify possible differences in protein expression among various and closely related clonal types of S. aureus, 2 to establish the differences in protein expression in terms of size of protein spots and its intensities between bacteria which are grown statically (biofilm formation with that of under aeration and agitation, and 3 to compare the differences in protein expression as a function of time (in hours. In this study, we selected six clinical isolates comprising two similar (MRSA-527 and MRSA-524 and four different (MRSA-139, MSSA-12E, MSSA-22d, and MSSA-10E types identified by spa typing, MLST and SCCmec typing. We performed 2D gel migration comparison. Also, two MRSA isolates (527 and 139 were selected to determine quantitative changes in the level of extracellular proteins at different biofilm growth time points of 12 h, 24 h, and 48 h. The study was done using a strategy that combines 2-DGE and LC-MS/MS analysis for absolute quantification and identification of the extracellular proteins. The 2DGE revealed that the proteomic profiles for the isolates belonging to the similar spa, MLST and SCCmec types were still quite different. Among the extracellular proteins secreted at different time points of biofilm formation, significant changes in protein expression were observed at 48 h incubation as compared to the exponential growth at 12 h incubation. The main conclusion of the work is that the authors do observe differences among isolates, and growth conditions do influence the protein content at different time points of biofilm formation.

  20. Staphylococcus aureus protein A binding to osteoblast tumour necrosis factor receptor 1 results in activation of nuclear factor kappa B and release of interleukin-6 in bone infection.

    Science.gov (United States)

    Claro, Tânia; Widaa, Amro; McDonnell, Cormac; Foster, Timothy J; O'Brien, Fergal J; Kerrigan, Steven W

    2013-01-01

    Staphylococcus aureus is the major pathogen among the staphylococci and the most common cause of bone infections. These infections are mainly characterized by bone destruction and inflammation, and are often debilitating and very difficult to treat. Previously we demonstrated that S. aureus protein A (SpA) can bind to osteoblasts, which results in inhibition of osteoblast proliferation and mineralization, apoptosis, and activation of osteoclasts. In this study we used small interfering RNA (siRNA) to demonstrate that osteoblast tumour necrosis factor receptor-1 (TNFR-1) is responsible for the recognition of and binding to SpA. TNFR-1 binding to SpA results in the activation of nuclear factor kappa B (NFκB). In turn, NFκB translocates to the nucleus of the osteoblast, which leads to release of interleukin 6 (IL-6). Silencing TNFR-1 in osteoblasts or disruption of the spa gene in S. aureus prevented both NFκB activation and IL-6 release. As well as playing a key role in proinflammatory reactions, IL-6 is also an important osteotropic factor. Release of IL-6 from osteoblasts results in the activation of the bone-resorbing cells, the osteoclasts. Consistent with our results described above, both silencing TNFR-1 in osteoblasts and disruption of spa in S. aureus prevented osteoclast activation. These studies are the first to demonstrate the importance of the TNFR-1-SpA interaction in bone infection, and may help explain the mechanism through which osteoclasts become overactivated, leading to bone destruction. Anti-inflammatory drug therapy could be used either alone or in conjunction with antibiotics to treat osteomyelitis or for prophylaxis in high-risk patients.

  1. Mitogen-activated protein kinases (MAPKs) regulate IL-6 over-production during concomitant influenza virus and Staphylococcus aureus infection

    Science.gov (United States)

    Klemm, Carolin; Bruchhagen, Christin; van Krüchten, Andre; Niemann, Silke; Löffler, Bettina; Peters, Georg; Ludwig, Stephan; Ehrhardt, Christina

    2017-01-01

    Bacterial super-infections are a major complication of influenza virus (IV) infections and often lead to severe pneumonia. One hallmark of IV-associated Staphylococcus aureus (S. aureus) infection is rapid progression to a serious disease outcome. Changes in immune and inflammatory host responses increase morbidity and complicate efficient therapy. A key player during inflammation is the multifunctional cytokine IL-6. Although increased IL-6 levels have been observed after severe disease upon IV and/or bacterial super-infection, the underlying molecular mechanisms still remain to be elucidated. In the present study, we focused on cellular signalling pathways regulating IL-6 production upon IV/S. aureus super-infection. Additionally, infection with viable bacteria was mimicked by lipoteichoic acid stimulation in this model. Analyses of cellular signalling mechanisms revealed synergistically increased activation of the MAPK p38 as well as enhanced phosphorylation of the MAPKs ERK1/2 and JNK in the presence of super-infecting bacteria. Interestingly, inhibition of MAPK activity indicated a strong dependence of IL-6 expression on p38 and ERK1/2, while the MAPK JNK seems not to be involved. Thus, our results provide new molecular insights into the regulation of IL-6, a marker of severe disease, which might contribute to the lethal synergism of IV and S. aureus. PMID:28195157

  2. Immunization routes in cattle impact the levels and neutralizing capacity of antibodies induced against S. aureus immune evasion proteins

    NARCIS (Netherlands)

    Boerhout, Eveline; Vrieling, Manouk; Benedictus, Lindert; Daemen, Ineke; Ravesloot, Lars; Rutten, Victor; Nuijten, Piet; van Strijp, Jos; Koets, Ad; Eisenberg, Susanne

    2015-01-01

    Vaccines against S. aureus bovine mastitis are scarce and show limited protection only. All currently available vaccines are applied via the parenteral (usually intramuscular) route. It is unknown, however, whether this route is the most suitable to specifically increase intramammary immunity to

  3. Immunization routes in cattle impact the levels and neutralizing capacity of antibodies induced against S. aureus immune evasion proteins

    NARCIS (Netherlands)

    Boerhout, Eveline; Vrieling, Manouk; Benedictus, Lindert; Daemen, Ineke; Ravesloot, Lars; Rutten, Victor; Nuijten, Piet; Strijp, Van Jos; Koets, Ad; Eisenberg, Susanne

    2015-01-01

    Vaccines against S. aureus bovine mastitis are scarce and show limited protection only. All currently available vaccines are applied via the parenteral (usually intramuscular) route. It is unknown, however, whether this route is the most suitable to specifically increase intramammary immunity to

  4. Lemierre's syndrome presenting to the ED: rapidly fatal sepsis caused by methicillin-susceptible Staphylococcus aureus Staphylococcus protein A type t044.

    Science.gov (United States)

    Pitsiou, Georgia; Kachrimanidou, Melina; Papa, Anna; Kioumis, Ioannis; Paspala, Asimina; Boutou, Afroditi; Vlachou, Stamatina; Tsorlini, Eleni; Argyropoulou-Pataka, Paraskevi

    2013-01-01

    We describe the case of a fatal septic illness in a previously healthy young man caused by community-acquired methicillin-susceptible Staphylococcus aureus of Staphylococcus protein A (spa) type t044. The patient developed a devastating Lemierre-like syndrome with extensive thrombosis of inferior vena cava and iliac veins with multiple metastatic septic emboli of the lungs. He presented to the emergency department with rapidly progressing sepsis followed by multiple organ dysfunction syndrome. Recognition of such virulent community-acquired strains is of great importance because they could prove to be emerging pathogens for life-threatening diseases.

  5. The Staphylococcus aureus protein Sbi acts as a complement inhibitor and forms a tripartite complex with host complement Factor H and C3b.

    Directory of Open Access Journals (Sweden)

    Katrin Haupt

    2008-12-01

    Full Text Available The Gram-positive bacterium Staphylococcus aureus, similar to other pathogens, binds human complement regulators Factor H and Factor H related protein 1 (FHR-1 from human serum. Here we identify the secreted protein Sbi (Staphylococcus aureus binder of IgG as a ligand that interacts with Factor H by a-to our knowledge-new type of interaction. Factor H binds to Sbi in combination with C3b or C3d, and forms tripartite SbiratioC3ratioFactor H complexes. Apparently, the type of C3 influences the stability of the complex; surface plasmon resonance studies revealed a higher stability of C3d complexed to Sbi, as compared to C3b or C3. As part of this tripartite complex, Factor H is functionally active and displays complement regulatory activity. Sbi, by recruiting Factor H and C3b, acts as a potent complement inhibitor, and inhibits alternative pathway-mediated lyses of rabbit erythrocytes by human serum and sera of other species. Thus, Sbi is a multifunctional bacterial protein, which binds host complement components Factor H and C3 as well as IgG and beta(2-glycoprotein I and interferes with innate immune recognition.

  6. Crystal structure of YwpF from Staphylococcus aureus reveals its architecture comprised of a β-barrel core domain resembling type VI secretion system proteins and a two-helix pair.

    Science.gov (United States)

    Lee, Sang Jae; Lee, Kyu-Yeon; Lee, Ki-Young; Kim, Dong-Gyun; Kim, Soon-Jong; Lee, Bong-Jin

    2015-04-01

    The ywpF gene (SAV2097) of the Staphylococcus aureus strain Mu50 encodes the YwpF protein, which may play a role in antibiotic resistance. Here, we report the first crystal structure of the YwpF superfamily from S. aureus at 2.5-Å resolution. The YwpF structure consists of two regions: an N-terminal core β-barrel domain that shows structural similarity to type VI secretion system (T6SS) proteins (e.g., Hcp1, Hcp3, and EvpC) and a C-terminal two-helix pair. Although the monomer structure of S. aureus YwpF resembles those of T6SS proteins, the dimer/tetramer model of S. aureus YwpF is distinct from the functionally important hexameric ring of T6SS proteins. We therefore suggest that the S. aureus YwpF may have a different function compared to T6SS proteins. © 2015 Wiley Periodicals, Inc.

  7. Catabolite control protein E (CcpE) is a LysR-type transcriptional regulator of tricarboxylic acid cycle activity in Staphylococcus aureus.

    Science.gov (United States)

    Hartmann, Torsten; Zhang, Bo; Baronian, Grégory; Schulthess, Bettina; Homerova, Dagmar; Grubmüller, Stephanie; Kutzner, Erika; Gaupp, Rosmarie; Bertram, Ralph; Powers, Robert; Eisenreich, Wolfgang; Kormanec, Jan; Herrmann, Mathias; Molle, Virginie; Somerville, Greg A; Bischoff, Markus

    2013-12-13

    The tricarboxylic acid cycle (TCA cycle) is a central metabolic pathway that provides energy, reducing potential, and biosynthetic intermediates. In Staphylococcus aureus, TCA cycle activity is controlled by several regulators (e.g. CcpA, CodY, and RpiRc) in response to the availability of sugars, amino acids, and environmental stress. Developing a bioinformatic search for additional carbon catabolite-responsive regulators in S. aureus, we identified a LysR-type regulator, catabolite control protein E (CcpE), with homology to the Bacillus subtilis CcpC regulator. Inactivation of ccpE in S. aureus strain Newman revealed that CcpE is a positive transcriptional effector of the first two enzymes of the TCA cycle, aconitase (citB) and to a lesser extent citrate synthase (citZ). Consistent with the transcriptional data, aconitase activity dramatically decreased in the ccpE mutant relative to the wild-type strain. The effect of ccpE inactivation on citB transcription and the lesser effect on citZ transcription were also reflected in electrophoretic mobility shift assays where CcpE bound to the citB promoter but not the citZ promoter. Metabolomic studies showed that inactivation of ccpE resulted in increased intracellular concentrations of acetate, citrate, lactate, and alanine, consistent with a redirection of carbon away from the TCA cycle. Taken together, our data suggest that CcpE is a major direct positive regulator of the TCA cycle gene citB.

  8. IruO is a reductase for heme degradation by IsdI and IsdG proteins in Staphylococcus aureus.

    Science.gov (United States)

    Loutet, Slade A; Kobylarz, Marek J; Chau, Crystal H T; Murphy, Michael E P

    2013-09-06

    Staphylococcus aureus is a common hospital- and community-acquired bacterium that can cause devastating infections and is often multidrug-resistant. Iron acquisition is required by S. aureus during an infection, and iron acquisition pathways are potential targets for therapies. The gene NWMN2274 in S. aureus strain Newman is annotated as an oxidoreductase of the diverse pyridine nucleotide-disulfide oxidoreductase (PNDO) family. We show that NWMN2274 is an electron donor to IsdG and IsdI catalyzing the degradation of heme, and we have renamed this protein IruO. Recombinant IruO is a FAD-containing NADPH-dependent reductase. In the presence of NADPH and IruO, either IsdI or IsdG degraded bound heme 10-fold more rapidly than with the chemical reductant ascorbic acid. Varying IsdI-heme substrate and monitoring loss of the heme Soret band gave a K(m) of 15 ± 4 μM, a k(cat) of 5.2 ± 0.7 min(-1), and a k(cat)/K(m) of 5.8 × 10(3) M(-1) s(-1). From HPLC and electronic spectra, the major heme degradation products are 5-oxo-δ-bilirubin and 15-oxo-β-bilirubin (staphylobilins), as observed with ascorbic acid. Although heme degradation by IsdI or IsdG can occur in the presence of H2O2, the addition of catalase and superoxide dismutase did not disrupt NADPH/IruO heme degradation reactions. The degree of electron coupling between IruO and IsdI or IsdG remains to be determined. Homologs of IruO were identified by sequence similarity in the genomes of Gram-positive bacteria that possess IsdG-family heme oxygenases. A phylogeny of these homologs identifies a distinct clade of pyridine nucleotide-disulfide oxidoreductases likely involved in iron uptake systems. IruO is the likely in vivo reductant required for heme degradation by S. aureus.

  9. Expression of acute phase proteins and inflammatory cytokines in mouse mammary gland following Staphylococcus aureus challenge and in response to milk accumulation

    DEFF Research Database (Denmark)

    Nazemi, Sasan; Aalbæk, Bent; Kjelgaard-Hansen, Mads

    2014-01-01

    We used a mouse model of pathogenic (Staphylococcus aureus) and non-pathogenic (teat sealing) mammary inflammation to investigate mRNA expression of several inflammatory cytokines and acute phase proteins (APP) in mammary tissue and liver, and the appearance of some of these factors in plasma...... in combination might provide a tool for diagnostic discrimination between mastitis caused by pathogenic invasion and milk accumulation, and hence allow for better targeting of antibiotic therapy. In comparison with mammary expression, expression of cytokines in liver tissue was up-regulated to a similar...... or lesser extent, whilst expression of APP was up-regulated to a much greater extent. The first appearance of increased cytokine and APP concentrations in plasma and of milk amyloid A (MAA) in milk occurred in advance of the measurable up-regulation of expression, hence their origin cannot be stated...

  10. 金黄色葡萄球菌膜蛋白EbpS%The membrane protein EbpS of Staphylococcus Aureus

    Institute of Scientific and Technical Information of China (English)

    李传芬; 胡成进

    2009-01-01

    金黄色葡萄球菌(简称:金葡菌,Staphylococcus aureus)是细菌性感染的主要致病菌,主要通过其表面特异的弹性蛋白结合蛋白(elastin-binding protein of Staphylococcus arueus,EbpS)与宿主胞外基质结合引起宿主的感染.EbpS蛋白在不同的环境中呈现不同的结构.这种结构的变化将会对进一步揭示金葡菌侵染宿主的机制产生极其重要的意义.

  11. Neutrophil bactericidal activity against Staphylococcus aureus adherent on biological surfaces. Surface-bound extracellular matrix proteins activate intracellular killing by oxygen-dependent and -independent mechanisms.

    Science.gov (United States)

    Hermann, M; Jaconi, M E; Dahlgren, C; Waldvogel, F A; Stendahl, O; Lew, D P

    1990-09-01

    The activation patterns of surface adherent neutrophils are modulated via interaction of extracellular matrix proteins with neutrophil integrins. To evaluate neutrophil bactericidal activity, Staphylococcus aureus adherent to biological surfaces were incubated with neutrophils and serum, and the survival of surface bacteria was determined. When compared to albumin-coated surfaces, the bactericidal activity of neutrophils adherent to purified human extracellular matrix was markedly enhanced (mean survival: 34.2% +/- 9.0% of albumin, P less than 0.0001) despite similar efficient ingestion of extracellular bacteria. Enhancement of killing was observed when surfaces were coated with purified constituents of extracellular matrix, i.e., fibronectin, fibrinogen, laminin, vitronectin, or type IV collagen. In addition to matrix proteins, the tetrapeptide RGDS (the sequence recognized by integrins) crosslinked to surface bound albumin was also active (survival: 74.5% +/- 5.5% of albumin, P less than 0.02), and fibronectin-increased killing was inhibited by soluble RGDS. Chemiluminescence measurements and experiments with CGD neutrophils revealed that both oxygen-dependent and -independent bactericidal mechanisms are involved. In conclusion, matrix proteins enhance intracellular bactericidal activity of adherent neutrophils, presumably by integrin recognition of RGDS-containing ligands. These results indicate a role for extracellular matrix proteins in the enhancement of the host defense against pyogenic infections.

  12. 金黄色葡萄球菌乳房炎奶牛血浆的比较蛋白质组研究%Differential Proteomics Analysis of Plasma Protein from Staphylococcus aureus Mastitic and Healthy Dairy Cows

    Institute of Scientific and Technical Information of China (English)

    杨永新; 曹随忠; 张勇; 程广龙; 赵辉玲; 赵兴绪

    2011-01-01

    为了分析金黄色葡萄球菌(Staphylococcus aureus)自然感染引起的隐性乳房炎奶牛血浆蛋白的表达变化,经细菌培养分离鉴定了乳中金黄色葡萄球菌,选择其感染的奶牛.采用二维凝胶电泳技术分离了临床健康奶牛和乳房炎奶牛的血浆蛋白,考马斯亮蓝G-250染色后PDQuest 8.0软件检测差异表达蛋白点,高效液相色谱串联离子阱质谱鉴定.结果发现,金黄色葡萄球菌乳房炎奶牛血浆中有10个蛋白点的表达量发生改变,其中6个蛋白点经质谱鉴定为结合珠蛋白、转甲状腺素蛋白和α1酸性糖蛋白等4种蛋白.金黄色葡萄球菌感染可造成奶牛血浆结合珠蛋白、α1酸性糖蛋白和血清淀粉样蛋白A的表达量增加.ELISA法测定血浆结合珠蛋白的结果也发现,金黄色葡萄球菌感染奶牛血浆结合珠蛋白水平显著高于健康牛(P<0.01).结果提示乳房炎奶牛血浆蛋白的变化可为揭示金黄色葡萄球菌感染乳腺炎症的应答提供依据.%The purpose of our study was to investigate the changes of plasma protein from the cows infected with Staphylococcus aureus. Dairy cows shown to be S. aureus-positive on bacteriological culture of milk from all 4 quarters, were classified as S. aureus subclinical mastitis. Two-dimensional gel electrophoresis was used to separate plasma protein from S. aureus subclinical mastitis and healthy dairy cows, and stained with Coomassie Blue G-250 solution. Differential expression proteins were detected by PDQuest 8.0 software and identified by ion trap mass spectrometer. The results showed that the ten protein spots were changed in cows subclinically infected with S. aureus mastitis, and six spots were identified as four proteins including haptoglobin, alpha 1 acid glycoprotein, transthyretin and serum amyloid protein A. Haptoglobin, alpha 1 acid glycoprotein, and serum amyloid protein A were up-regulated in plasma from S. aureus infected cows. ELISA detection of

  13. The Staphylococcus aureus Chaperone PrsA Is a New Auxiliary Factor of Oxacillin Resistance Affecting Penicillin-Binding Protein 2A.

    Science.gov (United States)

    Jousselin, Ambre; Manzano, Caroline; Biette, Alexandra; Reed, Patricia; Pinho, Mariana G; Rosato, Adriana E; Kelley, William L; Renzoni, Adriana

    2015-12-28

    Expression of the methicillin-resistant S. aureus (MRSA) phenotype results from the expression of the extra penicillin-binding protein 2A (PBP2A), which is encoded by mecA and acquired horizontally on part of the SCCmec cassette. PBP2A can catalyze dd-transpeptidation of peptidoglycan (PG) because of its low affinity for β-lactam antibiotics and can functionally cooperate with the PBP2 transglycosylase in the biosynthesis of PG. Here, we focus upon the role of the membrane-bound PrsA foldase protein as a regulator of β-lactam resistance expression. Deletion of prsA altered oxacillin resistance in three different SCCmec backgrounds and, more importantly, caused a decrease in PBP2A membrane amounts without affecting mecA mRNA levels. The N- and C-terminal domains of PrsA were found to be critical features for PBP2A protein membrane levels and oxacillin resistance. We propose that PrsA has a role in posttranscriptional maturation of PBP2A, possibly in the export and/or folding of newly synthesized PBP2A. This additional level of control in the expression of the mecA-dependent MRSA phenotype constitutes an opportunity to expand the strategies to design anti-infective agents.

  14. Complex structure and biochemical characterization of the Staphylococcus aureus cyclic diadenylate monophosphate (c-di-AMP)-binding protein PstA, the founding member of a new signal transduction protein family.

    Science.gov (United States)

    Campeotto, Ivan; Zhang, Yong; Mladenov, Miroslav G; Freemont, Paul S; Gründling, Angelika

    2015-01-30

    Signaling nucleotides are integral parts of signal transduction systems allowing bacteria to cope with and rapidly respond to changes in the environment. The Staphylococcus aureus PII-like signal transduction protein PstA was recently identified as a cyclic diadenylate monophosphate (c-di-AMP)-binding protein. Here, we present the crystal structures of the apo- and c-di-AMP-bound PstA protein, which is trimeric in solution as well as in the crystals. The structures combined with detailed bioinformatics analysis revealed that the protein belongs to a new family of proteins with a similar core fold but with distinct features to classical PII proteins, which usually function in nitrogen metabolism pathways in bacteria. The complex structure revealed three identical c-di-AMP-binding sites per trimer with each binding site at a monomer-monomer interface. Although distinctly different from other cyclic-di-nucleotide-binding sites, as the half-binding sites are not symmetrical, the complex structure also highlighted common features for c-di-AMP-binding sites. A comparison between the apo and complex structures revealed a series of conformational changes that result in the ordering of two anti-parallel β-strands that protrude from each monomer and allowed us to propose a mechanism on how the PstA protein functions as a signaling transduction protein.

  15. Triclosan promotes Staphylococcus aureus nasal colonization.

    Science.gov (United States)

    Syed, Adnan K; Ghosh, Sudeshna; Love, Nancy G; Boles, Blaise R

    2014-04-08

    The biocide triclosan is used in many personal care products, including toothpastes, soaps, clothing, and medical equipment. Consequently, it is present as a contaminant in the environment and has been detected in some human fluids, including serum, urine, and milk. Staphylococcus aureus is an opportunistic pathogen that colonizes the noses and throats of approximately 30% of the population. Colonization with S. aureus is known to be a risk factor for several types of infection. Here we demonstrate that triclosan is commonly found in the nasal secretions of healthy adults and the presence of triclosan trends positively with nasal colonization by S. aureus. We demonstrate that triclosan can promote the binding of S. aureus to host proteins such as collagen, fibronectin, and keratin, as well as inanimate surfaces such as plastic and glass. Lastly, triclosan-exposed rats are more susceptible to nasal colonization with S. aureus. These data reveal a novel factor that influences the ability of S. aureus to bind surfaces and alters S. aureus nasal colonization. IMPORTANCE Triclosan has been used as a biocide for over 40 years, but the broader effects that it has on the human microbiome have not been investigated. We demonstrate that triclosan is present in nasal secretions of a large portion of a test population and its presence correlates with Staphylococcus aureus nasal colonization. Triclosan also promotes the binding of S. aureus to human proteins and increases the susceptibility of rats to nasal colonization by S. aureus. These findings are significant because S. aureus colonization is a known risk factor for the development of several types of infections. Our data demonstrate the unintended consequences of unregulated triclosan use and contribute to the growing body of research demonstrating inadvertent effects of triclosan on the environment and human health.

  16. The extracellular adherence protein (Eap) of Staphylococcus aureus acts as a proliferation and migration repressing factor that alters the cell morphology of keratinocytes.

    Science.gov (United States)

    Eisenbeis, Janina; Peisker, Henrik; Backes, Christian S; Bur, Stephanie; Hölters, Sebastian; Thewes, Nicolas; Greiner, Markus; Junker, Christian; Schwarz, Eva C; Hoth, Markus; Junker, Kerstin; Preissner, Klaus T; Jacobs, Karin; Herrmann, Mathias; Bischoff, Markus

    2017-02-01

    Staphyloccocus aureus is a major human pathogen and a common cause for superficial and deep seated wound infections. The pathogen is equipped with a large arsenal of virulence factors, which facilitate attachment to various eukaryotic cell structures and modulate the host immune response. One of these factors is the extracellular adherence protein Eap, a member of the "secretable expanded repertoire adhesive molecules" (SERAM) protein family that possesses adhesive and immune modulatory properties. The secreted protein was previously shown to impair wound healing by interfering with host defense and neovascularization. However, its impact on keratinocyte proliferation and migration, two major steps in the re-epithelialization process of wounds, is not known. Here, we report that Eap affects the proliferation and migration capacities of keratinocytes by altering their morphology and adhesive properties. In particular, treatment of non-confluent HaCaT cell cultures with Eap resulted in cell morphology changes as well as a significant reduction in cell proliferation and migration. Eap-treated HaCaT cells changed their appearance from an oblong via a trapezoid to an astral-like shape, accompanied by decreases in cell volume and cell stiffness, and exhibited significantly increased cell adhesion. Eap had a similar influence on endothelial and cancer cells, indicative for a general effect of Eap on eukaryotic cell morphology and functions. Specifically, Eap was found to interfere with growth factor-stimulated activation of the mitogen-activated protein kinase (MAPK) pathway that is known to be responsible for cell shape modulation, induction of proliferation and migration of epithelial cells. Western blot analyses revealed that Eap blocked the phosphorylation of extracellular signal-regulated kinase 1 and 2 (Erk1/2) in keratinocyte growth factor (KGF)-stimulated HaCaT cells. Together, these data add another antagonistic mechanism of Eap in wound healing, whereby the

  17. Etudes structurales du ribosome de Staphylococcus aureus

    OpenAIRE

    Khusainov, Iskander

    2015-01-01

    The ribosome is a large cellular machinery that performs the protein synthesis in every living cell. Therefore, the ribosome is one of the major targets of naturally produced antibiotics, which can kill bacterial cells by blocking protein synthesis. However, some bacteria are resistant to these antibiotics due to small modifications of their ribosomes. Among them, Staphylococcus aureus (S. aureus) is a severe pathogen that causes numerous infections in humans. The crystal structures of comple...

  18. Structure of a conserved hypothetical protein SA1388 from S. aureus reveals a capped hexameric toroid with two PII domain lids and a dinuclear metal center

    Energy Technology Data Exchange (ETDEWEB)

    Saikatendu, Kumar Singh; Zhang, Xuejun; Kinch, Lisa; Leybourne, Matthew; Grishin, Nick V.; Zhang, Hong (Texas-D); (U. of Texas-SMED)

    2009-01-26

    The protein encoded by the SA1388 gene from Staphylococcus aureus was chosen for structure determination to elucidate its domain organization and confirm our earlier remote homology based prediction that it housed a nitrogen regulatory PII protein-like domain. SA1388 was predicted to contain a central PII-like domain and two flanking regions, which together belong to the NIF3-like protein family. Proteins like SA1388 remain a poorly studied group and their structural characterization could guide future investigations aimed at understanding their function. The structure of SA1388 has been solved to 2.0{angstrom} resolution by single wavelength anomalous dispersion phasing method using selenium anomalous signals. It reveals a canonical NIF3-like fold containing two domains with a PII-like domain inserted in the middle of the polypeptide. The N and C terminal halves of the NIF3-like domains are involved in dimerization, while the PII domain forms trimeric contacts with symmetry related monomers. Overall, the NIF3-like domains of SA1388 are organized as a hexameric toroid similar to its homologs, E. coli ybgI and the hypothetical protein SP1609 from Streptococcus pneumoniae. The openings on either side of the toroid are partially covered by trimeric 'lids' formed by the PII domains. The junction of the two NIF3 domains has two zinc ions bound at what appears to be a histidine rich active site. A well-defined electron density corresponding to an endogenously bound ligand of unknown identity is observed in close proximity to the metal site. SA1388 is the third member of the NIF3-like family of proteins to be structurally characterized, the other two also being hypothetical proteins of unknown function. The structure of SA1388 confirms our earlier prediction that the inserted domain that separates the two NIF3 domains adopts a PII-like fold and reveals an overall capped toroidal arrangement for the protein hexamer. The six PII-like domains form two trimeric

  19. Structure of a conserved hypothetical protein SA1388 from S. aureus reveals a capped hexameric toroid with two PII domain lids and a dinuclear metal center

    Directory of Open Access Journals (Sweden)

    Leybourne Matthew

    2006-12-01

    Full Text Available Abstract Background The protein encoded by the SA1388 gene from Staphylococcus aureus was chosen for structure determination to elucidate its domain organization and confirm our earlier remote homology based prediction that it housed a nitrogen regulatory PII protein-like domain. SA1388 was predicted to contain a central PII-like domain and two flanking regions, which together belong to the NIF3-like protein family. Proteins like SA1388 remain a poorly studied group and their structural characterization could guide future investigations aimed at understanding their function. Results The structure of SA1388 has been solved to 2.0Å resolution by single wavelength anomalous dispersion phasing method using selenium anomalous signals. It reveals a canonical NIF3-like fold containing two domains with a PII-like domain inserted in the middle of the polypeptide. The N and C terminal halves of the NIF3-like domains are involved in dimerization, while the PII domain forms trimeric contacts with symmetry related monomers. Overall, the NIF3-like domains of SA1388 are organized as a hexameric toroid similar to its homologs, E. coli ybgI and the hypothetical protein SP1609 from Streptococcus pneumoniae. The openings on either side of the toroid are partially covered by trimeric "lids" formed by the PII domains. The junction of the two NIF3 domains has two zinc ions bound at what appears to be a histidine rich active site. A well-defined electron density corresponding to an endogenously bound ligand of unknown identity is observed in close proximity to the metal site. Conclusion SA1388 is the third member of the NIF3-like family of proteins to be structurally characterized, the other two also being hypothetical proteins of unknown function. The structure of SA1388 confirms our earlier prediction that the inserted domain that separates the two NIF3 domains adopts a PII-like fold and reveals an overall capped toroidal arrangement for the protein hexamer. The

  20. Crystal structures of penicillin-binding protein 3 (PBP3) from methicillin-resistant Staphylococcus aureus in the apo and cefotaxime-bound forms.

    Science.gov (United States)

    Yoshida, Hisashi; Kawai, Fumihiro; Obayashi, Eiji; Akashi, Satoko; Roper, David I; Tame, Jeremy R H; Park, Sam-Yong

    2012-10-26

    Staphylococcus aureus is a widespread Gram-positive opportunistic pathogen, and a methicillin-resistant form (MRSA) is particularly difficult to treat clinically. We have solved two crystal structures of penicillin-binding protein (PBP) 3 (PBP3) from MRSA, the apo form and a complex with the β-lactam antibiotic cefotaxime, and used electrospray mass spectrometry to measure its sensitivity to a variety of penicillin derivatives. PBP3 is a class B PBP, possessing an N-terminal non-penicillin-binding domain, sometimes called a dimerization domain, and a C-terminal transpeptidase domain. The model shows a different orientation of its two domains compared to earlier models of other class B PBPs and a novel, larger N-domain. Consistent with the nomenclature of "dimerization domain", the N-terminal region forms an apparently tight interaction with a neighboring molecule related by a 2-fold symmetry axis in the crystal structure. This dimer form is predicted to be highly stable in solution by the PISA server, but mass spectrometry and analytical ultracentrifugation provide unequivocal evidence that the protein is a monomer in solution.

  1. Electron microscopy and computational studies of Ebh, a giant cell-wall-associated protein from Staphylococcus aureus.

    Science.gov (United States)

    Sakamoto, Sou; Tanaka, Yoshikazu; Tanaka, Isao; Takei, Toshiaki; Yu, Jian; Kuroda, Makoto; Yao, Min; Ohta, Toshiko; Tsumoto, Kouhei

    2008-11-14

    Ebh, a giant protein found in staphylococci, contains several domains, including a large central region with 52 imperfect repeats of a domain composed of 126 amino acids. We used electron microscopy to observe the rod-like structure of a partial Ebh protein containing 10 repeating units. This is the first report of the direct observation of an Ebh structure containing a large number of repeating units, although structures containing one, two, or four repeating units have been reported. The observed structure of the partial Ebh protein was distorted and had a length of ca. 520A and a width of ca. 21A. The observed structures were consistent with those deduced from crystal structure analysis, suggesting that the Ebh domains are connected to form a rod-like structure. The crystal structure data revealed distorted, string-like features in the simulated structure of the whole-length Ebh protein. Superposition of fragments of the simulated whole-length structure of the Ebh protein onto each electron micrograph showed a high level of correlation between the observed and calculated structures. These results suggest that Ebh is composed of highly flexible filate molecules. The highly repetitive structure and the associated unique structural flexibility of Ebh support the proposed function of this protein, i.e. binding to sugars in the cell wall. This binding might result in intra-cell-wall cross-linking that contributes to the rigidity of bacterial cells.

  2. Staphylococcus aureus and Pregnancy

    Science.gov (United States)

    Staphylococcus aureus and Pregnancy In every pregnancy, a woman starts out with a 3-5% chance of having a ... This sheet talks about whether exposure to staphylococcus aureus may increase the risk for birth defects over ...

  3. Staphylococcus aureus and Pregnancy

    Science.gov (United States)

    Staphylococcus aureus (Staph Infection) In every pregnancy, a woman starts out with a 3-5% chance of having ... risk. This sheet talks about whether exposure to staphylococcus aureus may increase the risk for birth defects over ...

  4. Structural and enzymatic analysis of TarM glycosyltransferase from Staphylococcus aureus reveals an oligomeric protein specific for the glycosylation of wall teichoic acid.

    Science.gov (United States)

    Koç, Cengiz; Gerlach, David; Beck, Sebastian; Peschel, Andreas; Xia, Guoqing; Stehle, Thilo

    2015-04-10

    Anionic glycopolymers known as wall teichoic acids (WTAs) functionalize the peptidoglycan layers of many Gram-positive bacteria. WTAs play central roles in many fundamental aspects of bacterial physiology, and they are important determinants of pathogenesis and antibiotic resistance. A number of enzymes that glycosylate WTA in Staphylococcus aureus have recently been identified. Among these is the glycosyltransferase TarM, a component of the WTA de novo biosynthesis pathway. TarM performs the synthesis of α-O-N-acetylglycosylated poly-5'-phosphoribitol in the WTA structure. We have solved the crystal structure of TarM at 2.4 Å resolution, and we have also determined a structure of the enzyme in complex with its substrate UDP-GlcNAc at 2.8 Å resolution. The protein assembles into a propeller-like homotrimer in which each blade contains a GT-B-type glycosyltransferase domain with a typical Rossmann fold. The enzymatic reaction retains the stereochemistry of the anomeric center of the transferred GlcNAc-moiety on the polyribitol backbone. TarM assembles into a trimer using a novel trimerization domain, here termed the HUB domain. Structure-guided mutagenesis experiments of TarM identify residues critical for enzyme activity, assign a putative role for the HUB in TarM function, and allow us to propose a likely reaction mechanism.

  5. Human immunoglobulin G recognizing fibrinogen-binding surface proteins is protective against both Staphylococcus aureus and Staphylococcus epidermidis infections in vivo.

    Science.gov (United States)

    Vernachio, John H; Bayer, Arnold S; Ames, Brenda; Bryant, Dawn; Prater, Bradley D; Syribeys, Peter J; Gorovits, Elena L; Patti, Joseph M

    2006-02-01

    A human donor-selected immunoglobulin G for intravenous injection (IGIV) product with elevated titers against the staphylococcal fibrinogen-binding MSCRAMM proteins ClfA and SdrG (INH-A21) was tested in vitro and in vivo. INH-A21 contained a significantly increased ability to inhibit the fibrinogen-binding activity of recombinant forms of both ClfA and SdrG. Evaluation of the opsonizing potential of INH-A21 was evaluated using fluorescently labeled bacteria; this assay indicated an increase in phagocytic activity compared to normal IGIV. The prophylactic efficacy of INH-A21 against an intraperitoneal challenge of methicillin-resistant Staphylococcus epidermidis (MRSE) was evaluated in a neonatal rat model. INH-A21 was also evaluated for prophylactic and therapeutic efficacy in a rabbit model of catheter-induced aortic valve infective endocarditis caused by either MRSE or methicillin-resistant Staphylococcus aureus (MRSA). Results from the in vivo models demonstrated potent prophylactic and therapeutic efficacy against both MRSE and MRSA. These data suggest that INH-A21 may be an important tool for the prevention and treatment of staphylococcal infections, especially in high-risk populations.

  6. Staphylococcus aureus toxins.

    Science.gov (United States)

    Otto, Michael

    2014-02-01

    Staphylococcus aureus is a dangerous pathogen that causes a variety of severe diseases. The virulence of S. aureus is defined by a large repertoire of virulence factors, among which secreted toxins play a preeminent role. Many S. aureus toxins damage biological membranes, leading to cell death. In particular, S. aureus produces potent hemolysins and leukotoxins. Among the latter, some were recently identified to lyse neutrophils after ingestion, representing an especially powerful weapon against bacterial elimination by innate host defense. Furthermore, S. aureus secretes many factors that inhibit the complement cascade or prevent recognition by host defenses. Several further toxins add to this multi-faceted program of S. aureus to evade elimination in the host. This review will give an overview over S. aureus toxins focusing on recent advances in our understanding of how leukotoxins work in receptor-mediated or receptor-independent fashions.

  7. Crystal structure and substrate specificity of the [beta]-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Qiu, Xiayang; Choudhry, Anthony E.; Janson, Cheryl A.; Grooms, Michael; Daines, Robert A.; Lonsdale, John T.; Khandekar, Sanjay S. (GSK)

    2010-07-20

    {beta}-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 {angstrom} resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rank order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- >> acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.

  8. The auxiliary protein complex SaePQ activates the phosphatase activity of sensor kinase SaeS in the SaeRS two-component system of Staphylococcus aureus

    OpenAIRE

    Jeong, Do-Won; Cho, Hoonsik; Jones, Marcus B.; Shatzkes, Kenneth; Sun, Fei; Ji, Quanjiang; Liu, Qian; Scott N Peterson; He, Chuan; Bae, Taeok

    2012-01-01

    In bacterial two-component regulatory systems (TCSs), dephosphorylation of phosphorylated response regulators is essential for resetting the activated systems to the pre-activation state. However, in the SaeRS TCS, a major virulence TCS of Staphylococcus aureus, the mechanism for dephosphorylation of the response regulator SaeR has not been identified. Here we report that two auxiliary proteins from the sae operon, SaeP and SaeQ, form a protein complex with the sensor kinase SaeS and activate...

  9. 金黄色葡萄球菌SarS蛋白参与压力应激的初步研究%SarS protein plays a role in stress response in Staphylococcus aureus

    Institute of Scientific and Technical Information of China (English)

    谭小蓉; 刘玉; 牟春花; 高亚萍; 董洁; 杨光

    2013-01-01

    Objective To investigate the regulatory function of Staphylococcus aureus accessory regulator S (SarS) protein in 5. aureus via insertion mutant construction. Methods The sarS inactive mutant was constructed by homologous re -combination in 5. aureus 8325 -4. The level of extra-toxins production was detected in MDBK cells , and the growth curves under different stress conditions were measured . Results The sarS mutant was successfully constructed in 5. aureus 8325 -4. Compared with its parent strain , the level of extra-toxins and the growth of the sarS mutant were not altered. However, it was found that the growth rate of sarS mutant was significantly decreased under several stress conditions compared with its parent strain. Conclusion SarS plays an important role in stress responses in 5. aureus.%目的 通过构建金黄色葡萄球菌(Staphylococcus aureus,简称金葡菌)中SarS蛋白突变株,研究SarS蛋白在金葡菌中的调控功能.方法 利用同源重组的方法构建金葡菌8325-4来源的sarS插入突变菌株,通过MDBK细胞模型检测突变株外毒素水平,以及其在高盐、高温等条件下的生长情况.结果 成功构建了金葡菌8325-4来源的sarS插入突变菌株.进一步的表型检测结果显示,与野生型菌株相比,sarS突变株在高温、高渗、重金属等应激条件下的生长速度明显下降,但是细菌外毒素对MDBK细胞的杀伤作用没有明显的改变.结论 SarS蛋白在金葡菌抵抗外界压力应激中发挥着重要的功能.

  10. The staphylococcal accessory regulator, SarA, is an RNA-binding protein that modulates the mRNA turnover properties of late-exponential and stationary phase Staphylococcus aureus cells

    Directory of Open Access Journals (Sweden)

    John M Morrison

    2012-03-01

    Full Text Available The modulation of mRNA turnover is gaining recognition as a mechanism by which Staphylococcus aureus regulates gene expression, but the factors that orchestrate alterations in transcript degradation are poorly understood. In that regard, we previously found that 138 mRNA species, including the virulence factors protein A (spa and collagen binding protein (cna, are stabilized in a sarA-dependent manner during exponential phase growth, suggesting that SarA protein may directly or indirectly effect the RNA turnover properties of these transcripts. Herein, we expanded our characterization of the effects of sarA on mRNA turnover during late exponential and stationary phases of growth. Results revealed that the locus affects the RNA degradation properties of cells during both growth phases. Further, using gel mobility shift assays and RIP-ChIP, it was found that SarA protein is capable of binding mRNA species that it stabilizes both in vitro and within bacterial cells. Taken together, these results suggest that SarA post-transcriptionally regulates S. aureus gene expression in a manner that involves binding to and consequently altering the mRNA turnover properties of target transcripts.

  11. Characterization of Staphylococcus aureus Primosomal DnaD Protein: Highly Conserved C-Terminal Region Is Crucial for ssDNA and PriA Helicase Binding but Not for DnaA Protein-Binding and Self-Tetramerization.

    Directory of Open Access Journals (Sweden)

    Yen-Hua Huang

    Full Text Available The role of DnaD in the recruitment of replicative helicase has been identified. However, knowledge of the DNA, PriA, and DnaA binding mechanism of this protein for the DnaA- and PriA-directed replication primosome assemblies is limited. We characterized the DNA-binding properties of DnaD from Staphylococcus aureus (SaDnaD and analyzed its interactions with SaPriA and SaDnaA. The gel filtration chromatography analysis of purified SaDnaD and its deletion mutant proteins (SaDnaD1-195, SaDnaD1-200 and SaDnaD1-204 showed a stable tetramer in solution. This finding indicates that the C-terminal region aa 196-228 is not crucial for SaDnaD oligomerization. SaDnaD forms distinct complexes with ssDNA of different lengths. In fluorescence titrations, SaDnaD bound to ssDNA with a binding-site size of approximately 32 nt. A stable complex of SaDnaD1-195, SaDnaD1-200, and SaDnaD1-204 with ssDNA dT40 was undetectable, indicating that the C-terminal region of SaDnaD (particularly aa 205-228 is crucial for ssDNA binding. The SPR results revealed that SaDnaD1-195 can interact with SaDnaA but not with SaPriA, which may indicate that DnaD has different binding sites for PriA and DnaA. Both SaDnaD and SaDnaDY176A mutant proteins, but not SaDnaD1-195, can significantly stimulate the ATPase activity of SaPriA. Hence, the stimulation effect mainly resulted from direct contact within the protein-protein interaction, not via the DNA-protein interaction. Kinetic studies revealed that the SaDnaD-SaPriA interaction increases the Vmax of the SaPriA ATPase fivefold without significantly affecting the Km. These results indicate that the conserved C-terminal region is crucial for ssDNA and PriA helicase binding, but not for DnaA protein-binding and self-tetramerization.

  12. Evaluation of distiller’s dried grains with solubles (DDGS) from different grain sources as dietary protein for hybrid tilapia, Oreochromis niloticus x O. Aureus

    Science.gov (United States)

    The effects of distiller’s dried grains with solubles (DDGS) from different sources on growth performance, hematology, and immunity of hybrid tilapia, Oreochromis niloticus x O. aureus, were evaluated. Sex-reversed, all-male hybrid tilapia (3.72 ± 0.08 g initial weight) were fed diets in which 30% o...

  13. Signatures of cytoplasmic proteins in the exoproteome distinguish community- and hospital-associated methicillin-resistant Staphylococcus aureus USA300 lineages

    DEFF Research Database (Denmark)

    Mekonnen, Solomon A; Palma Medina, Laura M; Glasner, Corinna

    2017-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is the common name for a heterogeneous group of highly drug-resistant staphylococci. Two major MRSA classes are distinguished based on epidemiology, namely community-associated (CA) and hospital-associated (HA) MRSA. Notably, the distinction of CA...

  14. Staphylococcus aureus in cheese

    Directory of Open Access Journals (Sweden)

    Dubravka Samaržija

    2007-04-01

    Full Text Available Growth of Staphylococcus aureus in cheese during production and storage can lead to production of enterotoxins responsible for human diseases. Due to specificity of those bacteria and complexity of cheese as a grown media, sometimes in the field it is very difficult to estimate an initial risk assessment of the S. aureus surveying in different cheese varieties. Moreover, the literature data on frequency and proportion of enterotoxigenic strains that cause cheese contamination are significantly different. The purpose of the present review is to objectively assess the risk of the potential occurrences of S. aureus in cheese and significance with respect to safety. The basic characteristics of Staphylococcus aureus, their presence in cheese and its potential risk for health are briefly reviewed. The own results of study relating to the presence of S. aureus in traditional (autochthonous hard sheep cheese made from raw milk are also discussed in this review.

  15. Distribution of mecA gene amongst Staphylococcus aureus isolates ...

    African Journals Online (AJOL)

    Dr Olaleye

    Χ2= 16.74; p < 0.05), beta lactamase producing S. aureus strains (Χ2= 29.21; p < 0.05) and ... (Archer, 1998), and MRSA strains being group of S. ... aureus produce a new penicillin binding protein (PBP), ..... our observation, Martin-Lopez et al.

  16. Desiccation tolerance in Staphylococcus aureus.

    Science.gov (United States)

    Chaibenjawong, Plykaeow; Foster, Simon J

    2011-02-01

    Staphylococcus aureus is a multidrug-resistant pathogen that not only causes a diverse array of human diseases, but also is able to survive in potentially dry and stressful environments, such as the human nose, on skin and on inanimate surfaces such as clothing and surfaces. This study investigated parameters governing desiccation tolerance of S. aureus and identified several components involved in the process. Initially, the role of environmental parameters such as temperature, growth phase, cell density, desiccation time and protectants in desiccation tolerance were determined. This established a robust model of desiccation tolerance in which S. aureus has the ability to survive on dry plastic surfaces for more than 1,097 days. Using a combination of a random screen and defined mutants, clpX, sigB and yjbH were identified as being required for desiccation tolerance. ClpX is a part of the ATP-dependent ClpXP protease, important for protein turnover, and YjbH has a proposed linked function. SigB is an accessory sigma factor with a role in generalized stress resistance. Understanding the molecular mechanisms that govern desiccation tolerance may determine the break points to be exploited to prevent the spread of this dangerous pathogen in hospitals and communities.

  17. Efektivitas Ekstrak Daun Jambu Biji Buah Putih Terhadap Pertumbuhan Staphylococcus aureus Dari Abses Dan Staphylococcus aureus (ATCC® 29213™)

    OpenAIRE

    Sinurat, Jojor

    2016-01-01

    Daun jambu biji mengandung senyawa aktif seperti tanin, triterpenoid, flavonoid, saponin yang mempunyai efek antibakteri. Mekanisme tanin sebagai antibakteri dengan mengkerutkan dinding sel dan membran sel, inaktivasi enzim, inaktivasi fungsi materi genetik bakteri. Flavonoid merusak sel bakteri, denaturasi protein, inaktivasi enzim dan menyebabkan lisis. Triterpenoid dan saponin menghambat pertumbuhan Staphylococcus aureus dengan cara merusak struktur membran sel. Staphylococcus aureus adala...

  18. Cytokine and acute phase protein mRNA expression in liver tissue from pigs with severe sepsis caused by intravenous inoculation of Staphylococcus aureus

    DEFF Research Database (Denmark)

    Nielsen, Ole Lerberg; Olsen, Helle Gerda; Iburg, Tine

    2010-01-01

    The aim was to substantiate previous findings of hepatic dysfunction in a porcine model of Staphylococcus aureus induced severe sepsis. Nine pigs were inoculated intravenously once or twice with 108S. aureus per kilogram body weight and killed 12, 24 and 48 h later. Three pigs served as controls....... Blood was sampled for bacteriology, haematology and clinical pathology. Tissues were collected at necropsy for bacteriology, gene expression analysis and histology. Bacterial counts in blood remained low, decreased in the lungs, liver and spleen, but increased in bone. All infected pigs developed sepsis...... of IL-6, IL-8, IL-1β, and CRP. N o increase could be detected in the IL-1α or TNFα liver-mRNA levels. IL-6, IL-8 and IL-1β expression peaked at 24 hours (2-5 fold compared to the control group). In conclusion, the increased liver cytokine mRNA levels indicate a local hepatic, non-infectious inflammatory...

  19. Lysine N[superscript zeta]-Decarboxylation Switch and Activation of the [beta]-Lactam Sensor Domain of BlaR1 Protein of Methicillin-resistant Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Borbulevych, Oleg; Kumarasiri, Malika; Wilson, Brian; Llarrull1, Leticia I.; Lee, Mijoon; Hesek, Dusan; Shi, Qicun; Peng, Jeffrey; Baker, Brian M.; Mobashery, Shahriar (Notre)

    2012-10-29

    The integral membrane protein BlaR1 of methicillin-resistant Staphylococcus aureus senses the presence of {beta}-lactam antibiotics in the milieu and transduces the information to the cytoplasm, where the biochemical events that unleash induction of antibiotic resistance mechanisms take place. We report herein by two-dimensional and three-dimensional NMR experiments of the sensor domain of BlaR1 in solution and by determination of an x-ray structure for the apo protein that Lys-392 of the antibiotic-binding site is posttranslationally modified by N{sup {zeta}}-carboxylation. Additional crystallographic and NMR data reveal that on acylation of Ser-389 by antibiotics, Lys-392 experiences N{sup {zeta}}-decarboxylation. This unique process, termed the lysine N{sup {zeta}}-decarboxylation switch, arrests the sensor domain in the activated ('on') state, necessary for signal transduction and all the subsequent biochemical processes. We present structural information on how this receptor activation process takes place, imparting longevity to the antibiotic-receptor complex that is needed for the induction of the antibiotic-resistant phenotype in methicillin-resistant S. aureus.

  20. Staphylococcal superantigen-like protein 3 binds to the Toll-like receptor 2 extracellular domain and inhibits cytokine production induced by Staphylococcus aureus, cell wall component, or lipopeptides in murine macrophages.

    Science.gov (United States)

    Yokoyama, Ryosuke; Itoh, Saotomo; Kamoshida, Go; Takii, Takemasa; Fujii, Satoshi; Tsuji, Tsutomu; Onozaki, Kikuo

    2012-08-01

    Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins sharing structural similarity with superantigens, but no superantigenic activity. Corresponding host target proteins or receptors against a portion of SSLs in the family have been identified. In this study, we show that SSL3 specifically binds to Toll-like receptor 2 (TLR2) and inhibits the stimulation of macrophages by TLR2 ligands. An approximately 100-kDa protein was recovered by using recombinant His-tagged SSL3-conjugated Sepharose from the lysate of porcine spleen, and the protein was identified as porcine TLR2 by peptide mass fingerprinting analysis. The SSL3-conjugated Sepharose recovered human and mouse TLR2 but not TLR4 from human neutrophils and mouse macrophage RAW 264.7 cells, as well as a recombinant TLR2 extracellular domain chimera protein. The production levels of interleukin 12 (IL-12) from mouse macrophages treated with heat-killed Staphylococcus aureus and of tumor necrosis factor alpha (TNF-α) from RAW 264.7 cells induced by peptidoglycan or lipopeptide TLR2 ligands were strongly suppressed in the presence of SSL3. The mutation of consensus sialic acid-containing glycan-binding residues in SSL3 did not abrogate the binding ability to TLR2 or inhibitory activity on TLR2, indicating that the interaction of SSL3 with TLR2 was independent of the sialic acid-containing glycan-binding residues. These findings demonstrate that SSL3 is able to bind the extracellular domain of TLR2 and interfere with TLR2 function. The present study provides a novel mechanism of SSL3 in immune evasion of S. aureus via interfering with its recognition by innate immune cells.

  1. Profiling the surfacome of Staphylococcus aureus

    NARCIS (Netherlands)

    Dreisbach, Annette; Hempel, Kristina; Buist, Girbe; Hecker, Michael; Becher, Doerte; van Dijl, Jan Maarten

    2010-01-01

    Staphylococcus aureus is a widespread opportunistic pathogen that can cause a wide variety of life-threatening diseases. Especially for the colonization of human tissues and the development of invasiveness, surface-exposed proteins are of major importance. In the present studies, we optimized a prot

  2. Staphylococcus aureus survival in human blood.

    Science.gov (United States)

    Malachowa, Natalia; DeLeo, Frank R

    2011-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is abundant in hospitals and in the United States is a leading cause of mortality due to infectious agents. Community-associated MRSA (CA-MRSA) strains such as USA300, which typically cause disease outside of healthcare settings, are also prevalent in the United States. Although most CA-MRSA infections affect skin and soft tissue, the pathogen can enter the bloodstream and ultimately cause severe disease. In a recent paper, we used USA300-specific microarrays to generate a comprehensive view of the molecules that facilitate S. aureus immune evasion and survival in human blood. Notably, genes encoding proteins involved in iron-uptake and utilization and gamma-hemolysin (hlgABC) are highly up-regulated by USA300 during culture in human blood. Here we discuss the potential implication of these findings and the possible role of gamma-hemolysin in the success of S. aureus as a human pathogen.

  3. Synthetic peptide inhibitors of DNA replication in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Løbner-Olesen, Anders; Kjelstrup, Susanne

    of clinically important pathogens and is essential for bacterial proliferation. The bacterial replication apparatus fulfill the requirements for a good drug target. The replisome of S. aureus consists of 5 different subunits (2, PolC2, 4, δ and δ`) who’s organization depends on multiple protein-protein...... interactions. Centrally in the replisome is the -clamp where to multiple proteins binds through a conserved motif. We have identified the protein-protein interactions in the replisome of S. aureus by use of a bacterial two-hybrid system. A reverse bacterial two-hybrid system (R-BTH) based on Pyr...

  4. Inhibiting platelets aggregation could aggravate the acute infection caused by Staphylococcus aureus.

    Science.gov (United States)

    Zhang, Xin; Liu, Yu; Gao, Yaping; Dong, Jie; Mu, Chunhua; Lu, Qiang; Shao, Ningsheng; Yang, Guang

    2011-01-01

    Several fibrinogen binding proteins (Fibs) play important roles in the pathogenesis of Staphylococcus aureus (S. aureus). Most Fibs can promote the aggregation of platelets during infection, but the extracellular fibrinogen-binding protein (Efb) is an exception. It is reported that Efb can specifically bind fibrinogen and inhibit the aggregation of platelet with its N terminal. However, the biological significance of platelet aggregation inhibition in the infection caused by S. aureus is unclear until now. Here, we demonstrated that the persistence and aggregation of platelets were important for killing S. aureus in whole blood. It was found that the N terminal of Efb (EfbN) and platelets inhibitors could increase the survival of S. aureus in whole blood. The study in vivo also showed that EfbN and platelets inhibitors could reduce the killing of S. aureus and increase the lethality rate of S. aureus in the acute infection mouse model.

  5. Identification of the ClpX Regulon in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Jelsbak, Lotte; Thomsen, Line Elnif; Ingmer, Hanne

    Staphyloccous aureus is a major human pathogen capable of causing a wide spectrum of infections ranging from superficial wound infections to life-threatening endocarditis and toxic shock syndrome. Essential for S. aureus virulence is a large number of cell-surface-associated proteins and secreted...... we show here that almost 400 genes (15%) are influenced by the clpX deletion. Furthermore, ClpX not only regulates many virulence factors, but rather serves as a global regulator of central functions for S. aureus lifestyle and pathogenicity....

  6. Dispersal of Bap-mediated Staphylococcus aureus biofilm by proteinase K.

    Science.gov (United States)

    Kumar Shukla, Sudhir; Rao, Toleti Subba

    2013-02-01

    The dominant role of biofilm-associated protein (Bap) in Staphylococcus aureus biofilm development prompted us to investigate Bap as a potential target for proteinase-mediated biofilm dispersion. Biofilm assay in microtitre plates showed that proteinase K hampered the early adhesion of cells as well as biofilm development. Proteinase K treatment of 24- and 48-h-old biofilms showed enhanced dispersion of bap-positive S. aureus biofilm; however, proteinase K did not affect the bap-negative S. aureus biofilm. When antibiotics were used in combination with proteinase K, significant enhancement in antibiotic action was noticed against bap-positive S. aureus biofilm. This study establishes that antibiotics in combination with proteinase K can be used for controlling S. aureus biofilms in whose development Bap surface protein has a major role. We propose that Bap protein could be a potential target for therapeutic control of S. aureus infections (for example, bovine mastitis).

  7. Toxin-Antitoxin Systems of Staphylococcus aureus.

    Science.gov (United States)

    Schuster, Christopher F; Bertram, Ralph

    2016-05-05

    Toxin-antitoxin (TA) systems are small genetic elements found in the majority of prokaryotes. They encode toxin proteins that interfere with vital cellular functions and are counteracted by antitoxins. Dependent on the chemical nature of the antitoxins (protein or RNA) and how they control the activity of the toxin, TA systems are currently divided into six different types. Genes comprising the TA types I, II and III have been identified in Staphylococcus aureus. MazF, the toxin of the mazEF locus is a sequence-specific RNase that cleaves a number of transcripts, including those encoding pathogenicity factors. Two yefM-yoeB paralogs represent two independent, but auto-regulated TA systems that give rise to ribosome-dependent RNases. In addition, omega/epsilon/zeta constitutes a tripartite TA system that supposedly plays a role in the stabilization of resistance factors. The SprA1/SprA1AS and SprF1/SprG1 systems are post-transcriptionally regulated by RNA antitoxins and encode small membrane damaging proteins. TA systems controlled by interaction between toxin protein and antitoxin RNA have been identified in S. aureus in silico, but not yet experimentally proven. A closer inspection of possible links between TA systems and S. aureus pathophysiology will reveal, if these genetic loci may represent druggable targets. The modification of a staphylococcal TA toxin to a cyclopeptide antibiotic highlights the potential of TA systems as rather untapped sources of drug discovery.

  8. Toxin-Antitoxin Systems of Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Christopher F. Schuster

    2016-05-01

    Full Text Available Toxin-antitoxin (TA systems are small genetic elements found in the majority of prokaryotes. They encode toxin proteins that interfere with vital cellular functions and are counteracted by antitoxins. Dependent on the chemical nature of the antitoxins (protein or RNA and how they control the activity of the toxin, TA systems are currently divided into six different types. Genes comprising the TA types I, II and III have been identified in Staphylococcus aureus. MazF, the toxin of the mazEF locus is a sequence-specific RNase that cleaves a number of transcripts, including those encoding pathogenicity factors. Two yefM-yoeB paralogs represent two independent, but auto-regulated TA systems that give rise to ribosome-dependent RNases. In addition, omega/epsilon/zeta constitutes a tripartite TA system that supposedly plays a role in the stabilization of resistance factors. The SprA1/SprA1AS and SprF1/SprG1 systems are post-transcriptionally regulated by RNA antitoxins and encode small membrane damaging proteins. TA systems controlled by interaction between toxin protein and antitoxin RNA have been identified in S. aureus in silico, but not yet experimentally proven. A closer inspection of possible links between TA systems and S. aureus pathophysiology will reveal, if these genetic loci may represent druggable targets. The modification of a staphylococcal TA toxin to a cyclopeptide antibiotic highlights the potential of TA systems as rather untapped sources of drug discovery.

  9. Endovascular infections caused by methicillin-resistant Staphylococcus aureus are linked to clonal complex-specific alterations in binding and invasion domains of fibronectin-binding protein A as well as the occurrence of fnbB.

    Science.gov (United States)

    Xiong, Yan Q; Sharma-Kuinkel, Batu K; Casillas-Ituarte, Nadia N; Fowler, Vance G; Rude, Thomas; DiBartola, Alex C; Lins, Roberto D; Abdel-Hady, Wessam; Lower, Steven K; Bayer, Arnold S

    2015-12-01

    Endovascular infections caused by Staphylococcus aureus involve interactions with fibronectin present as extracellular matrix or surface ligand on host cells. We examined the expression, structure, and binding activity of the two major S. aureus fibronectin-binding proteins (FnBPA, FnBPB) in 10 distinct, methicillin-resistant clinical isolates from patients with either persistent or resolving bacteremia. The persistent bacteremia isolates (n = 5) formed significantly stronger bonds with immobilized fibronectin as determined by dynamic binding measurements performed with atomic force microscopy. Several notable differences were also observed when the results were grouped by clonal complex 5 (CC5) strains (n = 5) versus CC45 strains (n = 5). Fibronectin-binding receptors on CC5 formed stronger bonds with immobilized fibronectin (P < 0.001). The fnbA gene was expressed at higher levels in CC45, whereas fnbB was found in only CC5 isolates. The fnbB gene was not sequenced because all CC45 isolates lacked this gene. Instead, comparisons were made for fnbA, which was present in all 10 isolates. Sequencing of fnbA revealed discrete differences within high-affinity, fibronectin-binding repeats (FnBRs) of FnBPA that included (i) 5-amino-acid polymorphisms in FnBR-9, FnBR-10, and FnBR-11 involving charged or polar side chains, (ii) an extra, 38-amino-acid repeat inserted between FnBR-9 and FnBR-10 exclusively seen in CC45 isolates, and (iii) CC5 isolates had the SVDFEED epitope in FnBR-11 (a sequence shown to be essential for fibronectin binding), while this sequence was replaced in all CC45 isolates with GIDFVED (a motif known to favor host cell invasion at the cost of reduced fibronectin binding). These complementary sequence and binding data suggest that differences in fnbA and fnbB, particularly polymorphisms and duplications in FnBPA, give S. aureus two distinct advantages in human endovascular infections: (i) FnBPs similar to that of CC5 enhance ligand binding and

  10. Exfoliative Toxins of Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Michal Bukowski

    2010-05-01

    Full Text Available Staphylococcus aureus is an important pathogen of humans and livestock. It causes a diverse array of diseases, ranging from relatively harmless localized skin infections to life-threatening systemic conditions. Among multiple virulence factors, staphylococci secrete several exotoxins directly associated with particular disease symptoms. These include toxic shock syndrome toxin 1 (TSST-1, enterotoxins, and exfoliative toxins (ETs. The latter are particularly interesting as the sole agents responsible for staphylococcal scalded skin syndrome (SSSS, a disease predominantly affecting infants and characterized by the loss of superficial skin layers, dehydration, and secondary infections. The molecular basis of the clinical symptoms of SSSS is well understood. ETs are serine proteases with high substrate specificity, which selectively recognize and hydrolyze desmosomal proteins in the skin. The fascinating road leading to the discovery of ETs as the agents responsible for SSSS and the characterization of the molecular mechanism of their action, including recent advances in the field, are reviewed in this article.

  11. Staphylococcus aureus bacteremia.

    Science.gov (United States)

    Jensen, Allan Garlik

    2003-11-01

    Staphylococcus aureus bacteremia (SAB) is still associated with a high mortality, and knowledge on risk factors and the clinical and the therapeutic aspects of SAB is still limited. This thesis focuses on the clinical aspects of SAB and its metastatic infections. In a study of all patients with bacteremia in Copenhagen County October 1992 through April 1993 (study I) we emphasized previous findings, that S. aureus is one of the most frequent pathogens in bacteremia, and in a case control study also in Copenhagen County 1994-95 (study II) we demonstrated, that not only an inserted central venous catheter and nasal S. aureus carriage but also hyponatremia and anemia are important risk factors for hospital-acquired SAB (study II). Studies on the treatment of SAB have pointed out, that the eradication of a primary is important, but there are only limited clinical studies dealing with antibiotic treatment. By logistic regression analysis, we were able to demonstrate that focus eradication is essential, but also that treatment with dicloxacillin 1 g x 4 or 2 g x 3 are superior to 1 g x 3 (studie III), indicating that the time for serum concentration above the Minimal Inhibitory Concentration (MIC) for the bacteria plays a role in the outcome of SAB treatment. S. aureus osteomyelitis secondary to SAB is frequently observed. No other countries, however, have a centralized registration, which make it possible to evaluate a large number of these patients. Since 1960, The Staphylococcal Laboratory, Statens Serum Institut in Copenhagen, has registrated selected clinical informations from nearly all patients with positive blood cultures of S. aureus. Based on this registration, we were able to show an increased number of S. aureus osteomyelitis among older patients and a decreased number of S. aureus osteomyelitis of femur and tibia among younger infants in the period 1980-90 (study IV). By reviewing the records of a large number of patients with vertebral S. aureus

  12. Spa Typing of Staphylococcus aureus Strains Isolated From Clinical Specimens of Patients With Nosocomial Infections in Tehran, Iran.

    Science.gov (United States)

    Goudarzi, Mehdi; Fazeli, Maryam; Goudarzi, Hossein; Azad, Mehdi; Seyedjavadi, Sima Sadat

    2016-07-01

    The incidence of nosocomial Staphylococcus aureus infection is increasing annually and becoming a true global challenge. The pattern of Staphylococcus aureus protein A (spa) types in different geographic regions is diverse. This study determined the prevalence of methicillin-resistant S. aureus and different spa types in S. aureus clinical isolates. During a six-month period, 90 S. aureus isolates were recovered from 320 clinical specimens. The in vitro susceptibility of various S. aureus isolates to 16 antibiotic discs was assessed using the Kirby-Bauer disk diffusion method. Molecular typing was carried out with S. aureus protein A typing via polymerase chain reaction. The frequency of methicillin-resistant S. aureus in our study was 88.9%. Twenty-three (25.5%) isolates were positive for panton-valentine leukocidin encoding genes. S. aureus presented a high resistance rate to ampicillin (100%) and penicillin (100%). No resistance was observed to vancomycin, teicoplanin, or linezolid. The rates of resistance to the majority of antibiotics tested varied between 23.3% and 82.2%. The rate of multidrug resistance among these clinical isolates was 93.3%. The 90 S. aureus isolates were classified into five S. aureus protein A types: t037 (33.3%), t030 (22.2%), t790 (16.7%), t969 (11.1%), and t044 (7.7%). Eight (8.9%) isolates were not typable using the S. aureus protein A typing method. We report a high methicillin-resistant S. aureus rate in our hospital. Additionally, t030 and t037 were the predominant spa-types among hospital-associated S. aureus. Our findings emphasize the need for continuous surveillance to prevent the dissemination of multidrug resistance among different S. aureus protein A types in Iran.

  13. Spa Typing of Staphylococcus aureus Strains Isolated From Clinical Specimens of Patients With Nosocomial Infections in Tehran, Iran

    Science.gov (United States)

    Goudarzi, Mehdi; Fazeli, Maryam; Goudarzi, Hossein; Azad, Mehdi; Seyedjavadi, Sima Sadat

    2016-01-01

    Background The incidence of nosocomial Staphylococcus aureus infection is increasing annually and becoming a true global challenge. The pattern of Staphylococcus aureus protein A (spa) types in different geographic regions is diverse. Objectives This study determined the prevalence of methicillin-resistant S. aureus and different spa types in S. aureus clinical isolates. Materials and Methods During a six-month period, 90 S. aureus isolates were recovered from 320 clinical specimens. The in vitro susceptibility of various S. aureus isolates to 16 antibiotic discs was assessed using the Kirby-Bauer disk diffusion method. Molecular typing was carried out with S. aureus protein A typing via polymerase chain reaction. Results The frequency of methicillin-resistant S. aureus in our study was 88.9%. Twenty-three (25.5%) isolates were positive for panton-valentine leukocidin encoding genes. S. aureus presented a high resistance rate to ampicillin (100%) and penicillin (100%). No resistance was observed to vancomycin, teicoplanin, or linezolid. The rates of resistance to the majority of antibiotics tested varied between 23.3% and 82.2%. The rate of multidrug resistance among these clinical isolates was 93.3%. The 90 S. aureus isolates were classified into five S. aureus protein A types: t037 (33.3%), t030 (22.2%), t790 (16.7%), t969 (11.1%), and t044 (7.7%). Eight (8.9%) isolates were not typable using the S. aureus protein A typing method. Conclusions We report a high methicillin-resistant S. aureus rate in our hospital. Additionally, t030 and t037 were the predominant spa-types among hospital-associated S. aureus. Our findings emphasize the need for continuous surveillance to prevent the dissemination of multidrug resistance among different S. aureus protein A types in Iran. PMID:27679706

  14. Epic Immune Battles of History: Neutrophils vs. Staphylococcus aureus

    Science.gov (United States)

    Guerra, Fermin E.; Borgogna, Timothy R.; Patel, Delisha M.; Sward, Eli W.; Voyich, Jovanka M.

    2017-01-01

    Neutrophils are the most abundant leukocytes in human blood and the first line of defense after bacteria have breached the epithelial barriers. After migration to a site of infection, neutrophils engage and expose invading microorganisms to antimicrobial peptides and proteins, as well as reactive oxygen species, as part of their bactericidal arsenal. Ideally, neutrophils ingest bacteria to prevent damage to surrounding cells and tissues, kill invading microorganisms with antimicrobial mechanisms, undergo programmed cell death to minimize inflammation, and are cleared away by macrophages. Staphylococcus aureus (S. aureus) is a prevalent Gram-positive bacterium that is a common commensal and causes a wide range of diseases from skin infections to endocarditis. Since its discovery, S. aureus has been a formidable neutrophil foe that has challenged the efficacy of this professional assassin. Indeed, proper clearance of S. aureus by neutrophils is essential to positive infection outcome, and S. aureus has developed mechanisms to evade neutrophil killing. Herein, we will review mechanisms used by S. aureus to modulate and evade neutrophil bactericidal mechanisms including priming, activation, chemotaxis, production of reactive oxygen species, and resolution of infection. We will also highlight how S. aureus uses sensory/regulatory systems to tailor production of virulence factors specifically to the triggering signal, e.g., neutrophils and defensins. To conclude, we will provide an overview of therapeutic approaches that may potentially enhance neutrophil antimicrobial functions. PMID:28713774

  15. Lessons from the Crystal Structure of the S. aureus Surface Protein Clumping Factor A in Complex With Tefibazumab, an Inhibiting Monoclonal Antibody

    Directory of Open Access Journals (Sweden)

    Vannakambadi K. Ganesh

    2016-11-01

    Full Text Available The Staphylococcus aureus fibrinogen binding MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules, ClfA (clumping factor A is an important virulence factor in staphylococcal infections and a component of several vaccines currently under clinical evaluation. The mouse monoclonal antibody aurexis (also called 12-9, and the humanized version tefibazumab are therapeutic monoclonal antibodies targeting ClfA that in combination with conventional antibiotics were effective in animal models but showed less impressive efficacy in a limited Phase II clinical trial. We here report the crystal structure and a biochemical characterization of the ClfA/tefibazumab (Fab complex. The epitope for tefibazumab is located to the “top” of the N3 subdomain of ClfA and partially overlaps with a previously unidentified second binding site for fibrinogen. A high-affinity binding of ClfA to fibrinogen involves both an interaction at the N3 site and the previously identified docking of the C-terminal segment of the fibrinogen γ-chain in the N2N3 trench. Although tefibazumab binds ClfA with high affinity we observe a modest IC50 value for the inhibition of fibrinogen binding to the MSCRAMM. This observation, paired with a common natural occurring variant of ClfA that is not effectively recognized by the mAb, may partly explain the modest effect tefibazumab showed in the initial clinic trail. This information will provide guidance for the design of the next generation of therapeutic anti-staphylococcal mAbs targeting ClfA.

  16. Differential expression profiling of proteomes of pathogenic and commensal strains of Staphylococcus aureus using SILAC

    OpenAIRE

    Manickam, Manisha

    2011-01-01

    Staphylococcus aureus (S. aureus) is the etiological agent of food-borne diseases, skin infections in humans and mastitis in bovines. S. aureus is also known to exist as a commensal on skin, nose and other mucosal surfaces of the host. This symbiotic association is a result of immune dampening or tolerance induced in the host by this pathogen. We proposed the variation in protein expression by commensal and pathogenic strain as an important factor behind the difference in pathogenicity. The i...

  17. The PRE-Derived NMR Model of the 38.8-kDa Tri-Domain IsdH Protein from Staphylococcus aureus Suggests That It Adaptively Recognizes Human Hemoglobin.

    Science.gov (United States)

    Sjodt, Megan; Macdonald, Ramsay; Spirig, Thomas; Chan, Albert H; Dickson, Claire F; Fabian, Marian; Olson, John S; Gell, David A; Clubb, Robert T

    2016-03-27

    Staphylococcus aureus is a medically important bacterial pathogen that, during infections, acquires iron from human hemoglobin (Hb). It uses two closely related iron-regulated surface determinant (Isd) proteins to capture and extract the oxidized form of heme (hemin) from Hb, IsdH and IsdB. Both receptors rapidly extract hemin using a conserved tri-domain unit consisting of two NEAT (near iron transporter) domains connected by a helical linker domain. To gain insight into the mechanism of extraction, we used NMR to investigate the structure and dynamics of the 38.8-kDa tri-domain IsdH protein (IsdH(N2N3), A326-D660 with a Y642A mutation that prevents hemin binding). The structure was modeled using long-range paramagnetic relaxation enhancement (PRE) distance restraints, dihedral angle, small-angle X-ray scattering, residual dipolar coupling and inter-domain NOE nuclear Overhauser effect data. The receptor adopts an extended conformation wherein the linker and N3 domains pack against each other via a hydrophobic interface. In contrast, the N2 domain contacts the linker domain via a hydrophilic interface and, based on NMR relaxation data, undergoes inter-domain motions enabling it to reorient with respect to the body of the protein. Ensemble calculations were used to estimate the range of N2 domain positions compatible with the PRE data. A comparison of the Hb-free and Hb-bound forms reveals that Hb binding alters the positioning of the N2 domain. We propose that binding occurs through a combination of conformational selection and induced-fit mechanisms that may promote hemin release from Hb by altering the position of its F helix.

  18. Penicillin Binding Protein 1 Is Important in the Compensatory Response of Staphylococcus aureus to Daptomycin-Induced Membrane Damage and Is a Potential Target for β-Lactam–Daptomycin Synergy

    Science.gov (United States)

    Berti, Andrew D.; Theisen, Erin; Sauer, John-Demian; Nonejuie, Poochit; Olson, Joshua; Pogliano, Joseph; Sakoulas, George; Nizet, Victor; Proctor, Richard A.

    2015-01-01

    The activity of daptomycin (DAP) against methicillin-resistant Staphylococcus aureus (MRSA) is enhanced in the presence of β-lactam antibiotics. This effect is more pronounced with β-lactam antibiotics that exhibit avid binding to penicillin binding protein 1 (PBP1). Here, we present evidence that PBP1 has a significant role in responding to DAP-induced stress on the cell. Expression of the pbpA transcript, encoding PBP1, was specifically induced by DAP exposure whereas expression of pbpB, pbpC, and pbpD, encoding PBP2, PBP3, and PBP4, respectively, remained unchanged. Using a MRSA COL strain with pbpA under an inducible promoter, increased pbpA transcription was accompanied by reduced susceptibility to, and killing by, DAP in vitro. Exposure to β-lactams that preferentially inactivate PBP1 was not associated with increased DAP binding, suggesting that synergy in the setting of anti-PBP1 pharmacotherapy results from increased DAP potency on a per-molecule basis. Combination exposure in an in vitro pharmacokinetic/pharmacodynamic model system with β-lactams that preferentially inactivate PBP1 (DAP-meropenem [MEM] or DAP-imipenem [IPM]) resulted in more-rapid killing than did combination exposure with DAP-nafcillin (NAF) (nonselective), DAP-ceftriaxone (CRO) or DAP-cefotaxime (CTX) (PBP2 selective), DAP-cefaclor (CEC) (PBP3 selective), or DAP-cefoxitin (FOX) (PBP4 selective). Compared to β-lactams with poor PBP1 binding specificity, exposure of S. aureus to DAP plus PBP1-selective β-lactams resulted in an increased frequency of septation and cell wall abnormalities. These data suggest that PBP1 activity may contribute to survival during DAP-induced metabolic stress. Therefore, targeted inactivation of PBP1 may enhance the antimicrobial efficiency of DAP, supporting the use of DAP–β-lactam combination therapy for serious MRSA infections, particularly when the β-lactam undermines the PBP1-mediated compensatory response. PMID:26525797

  19. Combining phagomagnetic separation with immunoassay for specific, fast and sensitive detection of Staphylococcus aureus.

    Science.gov (United States)

    Yan, Chenghui; Zhang, Yun; Yang, Hang; Yu, Junping; Wei, Hongping

    2017-08-01

    A Staphylococcus aureus (S. aureus)-specific lytic bacteriophage P-S. aureus-9, isolated from an environmental water sample, was assembled on magnetic beads for capturing S. aureus from samples through magnetic separation. Horseradish Peroxidase (HRP) labeled immunoglobulin (IgG) antibodies were used to detect the captured S. aureus by reacting with protein A on S. aureus followed by colorimetric signals, which were generated from the catalytic reaction between HRP and the substrate 3,3',5,5'-Tetramethylbenzidine (TMB). Under optimal conditions, the calibration curve was linear from 1.0×10(4) to 1.0×10(6)CFUmL(-1). The limit of detection (LOD) for the assay was 2.47×10(3)CFUmL(-1) and 8.86×10(3)CFUmL(-1) of S. aureus in PBS and apple juice, respectively. Moreover, the whole assay revealed outstanding specificity towards S. aureus, without any interference of common pathogenic bacteria, and can be completed within 90min without any pre-enrichment. As far as known, it was the first time to detect S. aureus based on the double site recognition of bacteriophage and mammal IgG. The novel approach has shown good potentials for a rapid, specific, cheap and simple detection of S. aureus in food samples. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Identification of the ClpX Regulon in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Jelsbak, Lotte; Thomsen, Line Elnif; Ingmer, Hanne;

    Staphyloccous aureus is a major human pathogen capable of causing a wide spectrum of infections ranging from superficial wound infections to life-threatening endocarditis and toxic shock syndrome. Essential for S. aureus virulence is a large number of cell-surface-associated proteins and secreted...... proteins. Results from our group have shown that the ClpXP proteolytic complex and the ClpX chaperone play central roles in regulating expression of many of these factors (2;3). By using DNA microarrays to compare transcription of strain 8325-4 (wt) and the isogenic ¿clpX strain during the transition phase...

  1. One-year mortality in coagulase-negative Staphylococcus and Staphylococcus aureus infective endocarditis

    DEFF Research Database (Denmark)

    Rasmussen, Rasmus V; Snygg-Martin, Ulrika; Olaison, Lars;

    2009-01-01

    The aim of this study was to investigate in-hospital mortality and 12-month mortality in patients with coagulase-negative Staphylococcus (CoNS) compared to Staphylococcus aureus (S. aureus) infective endocarditis (IE). We used a prospective cohort study of 66 consecutive CoNS and 170 S. aureus IE...... patients, collected at 2 tertiary university hospitals in Copenhagen (Denmark) and at 1 tertiary university hospital in Gothenburg (Sweden). Median (range) C-reactive protein at admission was higher in patients with S. aureus IE (150 mg/l (1-521) vs 94 mg/l (6-303); p

  2. The Bicomponent Pore-Forming Leucocidins of Staphylococcus aureus

    Science.gov (United States)

    Alonzo, Francis

    2014-01-01

    SUMMARY The ability to produce water-soluble proteins with the capacity to oligomerize and form pores within cellular lipid bilayers is a trait conserved among nearly all forms of life, including humans, single-celled eukaryotes, and numerous bacterial species. In bacteria, some of the most notable pore-forming molecules are protein toxins that interact with mammalian cell membranes to promote lysis, deliver effectors, and modulate cellular homeostasis. Of the bacterial species capable of producing pore-forming toxic molecules, the Gram-positive pathogen Staphylococcus aureus is one of the most notorious. S. aureus can produce seven different pore-forming protein toxins, all of which are believed to play a unique role in promoting the ability of the organism to cause disease in humans and other mammals. The most diverse of these pore-forming toxins, in terms of both functional activity and global representation within S. aureus clinical isolates, are the bicomponent leucocidins. From the first description of their activity on host immune cells over 100 years ago to the detailed investigations of their biochemical function today, the leucocidins remain at the forefront of S. aureus pathogenesis research initiatives. Study of their mode of action is of immediate interest in the realm of therapeutic agent design as well as for studies of bacterial pathogenesis. This review provides an updated perspective on our understanding of the S. aureus leucocidins and their function, specificity, and potential as therapeutic targets. PMID:24847020

  3. Targeting intracellular Staphylococcus aureus to lower recurrence of orthopaedic infection.

    Science.gov (United States)

    Dusane, Devendra H; Kyrouac, Douglas; Petersen, Iris; Bushrow, Luke; Calhoun, Jason H; Granger, Jeffrey F; Phieffer, Laura S; Stoodley, Paul

    2017-09-08

    Staphylococcus aureus is often found in orthopaedic infections and may be protected from commonly prescribed antibiotics by forming biofilms or growing intracellularly within osteoblasts. To investigate the effect of non-antibiotic compounds in conjunction with antibiotics to clear intracellular and biofilm forming S. aureus causing osteomyelitis. SAOS-2 osteoblast-like cell lines were infected with S. aureus BB1279. Antibiotics (vancomycin, VAN; and dicloxacillin, DICLOX), bacterial efflux pump inhibitors (piperine, PIP; carbonyl cyanide m-chlorophenyl hydrazone, CCCP) and bone morphogenetic protein (BMP-2) were evaluated individually and in combination to kill intracellular bacteria. We present direct evidence that after gentamicin killed extracellular planktonic bacteria and antibiotics had been stopped, seeding from the infected osteoblasts grew as biofilms. VAN was ineffective in treating the intracellular bacteria even at 10x MIC; however in presence of PIP or CCCP the intracellular S. aureus was significantly reduced. Bacterial efflux pump inhibitors (PIP and CCCP) were effective in enhancing permeability of antibiotics within the osteoblasts and facilitated killing of intracellular S. aureus. Confocal laser scanning microscopy (CLSM) showed increased uptake of propidium iodide within osteoblasts in presence of PIP and CCCP. BMP-2 had no effect on growth of S. aureus either alone or in combination with antibiotics. Combined application of antibiotics and natural agents could help in the treatment of osteoblast infected intracellular bacteria and biofilms associated with osteomyelitis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  4. Toll-Like Receptor 2 Stimulation of Osteoblasts Mediates Staphylococcus Aureus Induced Bone Resorption and Osteoclastogenesis through Enhanced RANKL

    Science.gov (United States)

    Kassem, Ali; Lindholm, Catharina; Lerner, Ulf H

    2016-01-01

    Severe Staphylococcus aureus (S. aureus) infections pose an immense threat to population health and constitute a great burden for the health care worldwide. Inter alia, S. aureus septic arthritis is a disease with high mortality and morbidity caused by destruction of the infected joints and systemic bone loss, osteoporosis. Toll-Like receptors (TLRs) are innate immune cell receptors recognizing a variety of microbial molecules and structures. S. aureus recognition via TLR2 initiates a signaling cascade resulting in production of various cytokines, but the mechanisms by which S. aureus causes rapid and excessive bone loss are still unclear. We, therefore, investigated how S. aureus regulates periosteal/endosteal osteoclast formation and bone resorption. S. aureus stimulation of neonatal mouse parietal bone induced ex vivo bone resorption and osteoclastic gene expression. This effect was associated with increased mRNA and protein expression of receptor activator of NF-kB ligand (RANKL) without significant change in osteoprotegerin (OPG) expression. Bone resorption induced by S. aureus was abolished by OPG. S. aureus increased the expression of osteoclastogenic cytokines and prostaglandins in the parietal bones but the stimulatory effect of S. aureus on bone resorption and Tnfsf11 mRNA expression was independent of these cytokines and prostaglandins. Stimulation of isolated periosteal osteoblasts with S. aureus also resulted in increased expression of Tnfsf11 mRNA, an effect lost in osteoblasts from Tlr2 knockout mice. S. aureus stimulated osteoclastogenesis in isolated periosteal cells without affecting RANKL-stimulated resorption. In contrast, S. aureus inhibited RANKL-induced osteoclast formation in bone marrow macrophages. These data show that S. aureus enhances bone resorption and periosteal osteoclast formation by increasing osteoblast RANKL production through TLR2. Our study indicates the importance of using different in vitro approaches for studies of how S

  5. NVC-422 inactivates Staphylococcus aureus toxins.

    Science.gov (United States)

    Jekle, Andreas; Yoon, Jungjoo; Zuck, Meghan; Najafi, Ramin; Wang, Lu; Shiau, Timothy; Francavilla, Charles; Rani, Suriani Abdul; Eitzinger, Christian; Nagl, Markus; Anderson, Mark; Debabov, Dmitri

    2013-02-01

    Bacterial pathogens have specific virulence factors (e.g., toxins) that contribute significantly to the virulence and infectivity of microorganisms within the human hosts. Virulence factors are molecules expressed by pathogens that enable colonization, immunoevasion, and immunosuppression, obtaining nutrients from the host or gaining entry into host cells. They can cause pathogenesis by inhibiting or stimulating certain host functions. For example, in systemic Staphylococcus aureus infections, virulence factors such as toxic shock syndrome toxin 1 (TSST-1), staphylococcal enterotoxin A (SEA), and staphylococcal enterotoxin B (SEB) cause sepsis or toxic shock by uncontrolled stimulation of T lymphocytes and by triggering a cytokine storm. In vitro, these superantigens stimulate the proliferation of human peripheral blood mononuclear cells (PBMC) and the release of many cytokines. NVC-422 (N,N-dichloro-2,2-dimethyltaurine) is a broad-spectrum, fast-acting topical anti-infective agent against microbial pathogens, including antibiotic-resistant microbes. Using mass spectrometry, we demonstrate here that NVC-422 oxidizes methionine residues of TSST-1, SEA, SEB, and exfoliative toxin A (ETA). Exposure of virulence factors to 0.1% NVC-422 for 1 h prevented TSST-1-, SEA-, SEB-, and ETA-induced cell proliferation and cytokine release. Moreover, NVC-422 also delayed and reduced the protein A- and clumping factor-associated agglutination of S. aureus cultures. These results show that, in addition to its well-described direct microbicidal activity, NVC-422 can inactivate S. aureus virulence factors through rapid oxidation of methionines.

  6. Immunopathogenesis of Staphylococcus aureus pulmonary infection

    Science.gov (United States)

    Parker, Dane; Prince, Alice

    2013-01-01

    Staphylococcus aureus is a common human pathogen highly evolved as both a component of the commensal flora and as a major cause of invasive infection. Severe respiratory infection due to staphylococci has been increasing due to the prevalence of more virulent USA300 CA-MRSA strains in the general population. The ability of S. aureus to adapt to the milieu of the respiratory tract has facilitated its emergence as a respiratory pathogen. Its metabolic versatility, the ability to scavenge iron, coordinate gene expression, and the horizontal acquisition of useful genetic elements have all contributed to its success as a component of the respiratory flora, in hospitalized patients, as a complication of influenza and in normal hosts. The expression of surface adhesins facilitates its persistence in the airways. In addition, the highly sophisticated interactions of the multiple S. aureus virulence factors, particularly the α-hemolysin and protein A, with diverse immune effectors in the lung such as ADAM10, TNFR1, EGFR, immunoglobulin, and complement all contribute to the pathogenesis of staphylococcal pneumonia. PMID:22037948

  7. Aspartate inhibits Staphylococcus aureus biofilm formation.

    Science.gov (United States)

    Yang, Hang; Wang, Mengyue; Yu, Junping; Wei, Hongping

    2015-04-01

    Biofilm formation renders Staphylococcus aureus highly resistant to conventional antibiotics and host defenses. Four D-amino acids (D-Leu, D-Met, D-Trp and D-Tyr) have been reported to be able to inhibit biofilm formation and disassemble established S. aureus biofilms. We report here for the first time that both D- and L-isoforms of aspartate (Asp) inhibited S. aureus biofilm formation on tissue culture plates. Similar biofilm inhibition effects were also observed against other staphylococcal strains, including S. saprophyticus, S. equorum, S. chromogenes and S. haemolyticus. It was found that Asp at high concentrations (>10 mM) inhibited the growth of planktonic N315 cells, but at subinhibitory concentrations decreased the cellular metabolic activity without influencing cell growth. The decreased cellular metabolic activity might be the reason for the production of less protein and DNA in the matrix of the biofilms formed in the presence of Asp. However, varied inhibition efficacies of Asp were observed for biofilms formed by clinical staphylococcal isolates. There might be mechanisms other than decreasing the metabolic activity, e.g. the biofilm phenotypes, affecting biofilm formation in the presence of Asp.

  8. Linezolid resistant Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Pavani Gandham

    2014-08-01

    Full Text Available Linezolid is the only antibiotic available as an oral formulation for resistant staphylococcal infections. It is effective in skin and soft tissue infections, nosocomial pneumonias including VAP, infective endocarditis and MRSA meningitis. It is also effective in the eradication of both nasal and throat colonization of MRSA. Its high bioavailability and post antibiotic effect, ease of switching to oral therapy during its use and the fact that it can be used in patients of all ages, also in patients with liver disease and poor kidney function and its increased effectiveness over glycopeptides makes this drug a precious drug in the treatment of resistant staphylococcal infections. Linezolid resistance in staphylococcus is defined as a linezolid MIC of and #8805;8 mg/L. Reported Linezolid resistance in India and elsewhere is 2-20%. There is clonal dissemination of Linezolid Resistant Staphylococcus aureus (LRSA within or across health care settings which demands continuous surveillance to determine the emergent risk of resistance strains and to establish guidelines for appropriate use. Clinical laboratories should confirm any LRSA preferably by a second method, prior to using linezolid for serious infections. Effective surveillance, more judicious use of this antibiotic, avoiding linezolid usage for empiric therapy in hospital acquired staphylococcus infections, optimization of the pharmacological parameters of the antibiotics in specific clinical situation, decreasing bacterial load by timely surgical debridement or drainage of collections, use of combination therapies would prevent the emergence of resistance to linezolid in staphylococcus aureus. [Int J Res Med Sci 2014; 2(4.000: 1253-1256

  9. Development and evaluation of IgY ImmunoCapture PCR ELISA for detection of Staphylococcus aureus enterotoxin A devoid of protein A interference.

    Science.gov (United States)

    Reddy, Prakash; Ramlal, Shylaja; Sripathy, Murali Harishchandra; Batra, Harsh Vardhan

    2014-06-01

    In the present study, a sensitive and specific IgY mediated ImmunoCapture-PCR-ELISA (IC-PCR-ELISA) was developed for the detection of staphylococcal enterotoxin A (SEA) from culture supernatants and suspected contaminated samples. Due to the virtue of avian immunoglobulins (IgY) to have the least affinity towards staphylococcal protein A (SpA) responsible for false positives, we employed anti-SEA IgY for capture of SEA toxin and revealed with SEA specific rabbit antibodies conjugated to a 524bp DNA marker. Biotin-11-dUTP was incorporated during PCR amplification and post PCR analysis was performed by PCR-ELISA. Unlike IgG immunocapture, IgY mediated immunocapture of SEA was free from false positives due to protein A. The developed assay was specific to SEA except for minor cross reactivity with staphylococcal enterotoxin E (SEE). Several raw milk samples were evaluated for the presence of SEA with and without enrichment. Three samples were found to be positive for SEA after enrichment for 8h. Though IC-PCR-ELISA for SEA showed 100% correlation with PCR analysis for sea gene, the assay was unique in terms of sensitivity of detecting ~10pg/ml of SEA toxin from spiked milk samples. Result of IC-PCR-ELISA was further confirmed by conventional methods of isolation and characterization. The presented method can be very useful for rapid analysis of milk samples for SEA contamination and can be further extended for detection of multiple SE's in different wells of same PCR plate using common DNA substrate.

  10. Staphylococcus aureus: methicillin-susceptible S. aureus to methicillin-resistant S. aureus and vancomycin-resistant S. aureus.

    Science.gov (United States)

    Rehm, Susan J; Tice, Alan

    2010-09-15

    The evolution of methicillin-resistant and vancomycin-resistant Staphylococcus aureus has demanded serious review of antimicrobial use and development of new agents and revised approaches to prevent and overcome drug resistance. Depending on local conditions and patient risk factors, empirical therapy of suspected S. aureus infection may require coverage of drug-resistant organisms with newer agents and novel antibiotic combinations. The question of treatment with inappropriate antibiotics raises grave concerns with regard to methicillin-resistant S. aureus selection, overgrowth, and increased virulence. Several strategies to reduce the nosocomial burden of resistance are suggested, including shortened hospital stays and outpatient parenteral antimicrobial therapy of the most serious infections.

  11. Decolonization of Staphylococcus aureus carriage.

    Science.gov (United States)

    Botelho-Nevers, E; Gagnaire, J; Verhoeven, P O; Cazorla, C; Grattard, F; Pozzetto, B; Berthelot, P; Lucht, F

    2017-09-01

    Staphylococcus aureus nasal colonization is a well-known independent risk factor for infection caused by this bacterium. Screening and decolonization of carriers have been proven effective in reducing S. aureus infections in some populations. However, a gap remains between what has been proven effective and what is currently done. We aimed to summarize recommendations and current knowledge of S. aureus decolonization to answer the following questions: Why? For whom? How? When? And what are the perspectives? Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  12. Líquen aureus "algesiogênico" "Algesiogenic" lichen aureus

    Directory of Open Access Journals (Sweden)

    Roberto Rheingantz da Cunha Filho

    2006-03-01

    Full Text Available Descreve-se caso de líquen aureus em paciente do sexo feminino, com 23 anos de idade que apresentava há dois anos lesão dolorosa, purpúrica, acastanhada tendendo por semelhante a cor de ferrugem e de aspecto liquenóide no antebraço. O exame anatomopatológico revelou denso infiltrado linfo-histiocitário na derme superior papilar, com extravasamento de hemácias. O líquen aureus é relativamente raro, sendo ainda mais raro o sintoma de dor.A case is described of lichen aureus in a 23 year old female with a 2-year history of painful, purpuric, rust-coloured to tan, lichenous lesion on forearm. A biopsy specimen demonstrated a dense lymphohistiocytic infiltrate in the upper dermis, with extravasation of red cells. The "algesiogenic" lichen aureus is a very rare dermatosis.

  13. Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity.

    Science.gov (United States)

    Graille, M; Stura, E A; Corper, A L; Sutton, B J; Taussig, M J; Charbonnier, J B; Silverman, G J

    2000-05-09

    Staphylococcus aureus produces a virulence factor, protein A (SpA), that contains five homologous Ig-binding domains. The interactions of SpA with the Fab region of membrane-anchored Igs can stimulate a large fraction of B cells, contributing to lymphocyte clonal selection. To understand the molecular basis for this activity, we have solved the crystal structure of the complex between domain D of SpA and the Fab fragment of a human IgM antibody to 2.7-A resolution. In the complex, helices II and III of domain D interact with the variable region of the Fab heavy chain (V(H)) through framework residues, without the involvement of the hypervariable regions implicated in antigen recognition. The contact residues are highly conserved in human V(H)3 antibodies but not in other families. The contact residues from domain D also are conserved among all SpA Ig-binding domains, suggesting that each could bind in a similar manner. Features of this interaction parallel those reported for staphylococcal enterotoxins that are superantigens for many T cells. The structural homology between Ig V(H) regions and the T-cell receptor V(beta) regions facilitates their comparison, and both types of interactions involve lymphocyte receptor surface remote from the antigen binding site. However, T-cell superantigens reportedly interact through hydrogen bonds with T-cell receptor V(beta) backbone atoms in a primary sequence-independent manner, whereas SpA relies on a sequence-restricted conformational binding with residue side chains, suggesting that this common bacterial pathogen has adopted distinct molecular recognition strategies for affecting large sets of B and T lymphocytes.

  14. spa typing for epidemiological surveillance of Staphylococcus aureus

    NARCIS (Netherlands)

    Hallin, Marie; Friedrich, Alexander W; Struelens, Marc J; Caugant, Dominique A.

    2009-01-01

    The spa typing method is based on sequencing of the polymorphic X region of the protein A gene (spa), present in all strains of Staphylococcus aureus. The X region is constituted of a variable number of 24-bp repeats flanked by well-conserved regions. This single-locus sequence-based typing method c

  15. Staphylococcus aureus redirects central metabolism to increase iron availability.

    Directory of Open Access Journals (Sweden)

    David B Friedman

    2006-08-01

    Full Text Available Staphylococcus aureus pathogenesis is significantly influenced by the iron status of the host. However, the regulatory impact of host iron sources on S. aureus gene expression remains unknown. In this study, we combine multivariable difference gel electrophoresis and mass spectrometry with multivariate statistical analyses to systematically cluster cellular protein response across distinct iron-exposure conditions. Quadruplicate samples were simultaneously analyzed for alterations in protein abundance and/or post-translational modification state in response to environmental (iron chelation, hemin treatment or genetic (Deltafur alterations in bacterial iron exposure. We identified 120 proteins representing several coordinated biochemical pathways that are affected by changes in iron-exposure status. Highlighted in these experiments is the identification of the heme-regulated transport system (HrtAB, a novel transport system which plays a critical role in staphylococcal heme metabolism. Further, we show that regulated overproduction of acidic end-products brought on by iron starvation decreases local pH resulting in the release of iron from the host iron-sequestering protein transferrin. These findings reveal novel strategies used by S. aureus to acquire scarce nutrients in the hostile host environment and begin to define the iron and heme-dependent regulons of S. aureus.

  16. spa typing for epidemiological surveillance of Staphylococcus aureus

    NARCIS (Netherlands)

    Hallin, Marie; Friedrich, Alexander W; Struelens, Marc J; Caugant, Dominique A.

    2009-01-01

    The spa typing method is based on sequencing of the polymorphic X region of the protein A gene (spa), present in all strains of Staphylococcus aureus. The X region is constituted of a variable number of 24-bp repeats flanked by well-conserved regions. This single-locus sequence-based typing method

  17. Staphylococcus aureus redirects central metabolism to increase iron availability.

    Directory of Open Access Journals (Sweden)

    David B Friedman

    2006-08-01

    Full Text Available Staphylococcus aureus pathogenesis is significantly influenced by the iron status of the host. However, the regulatory impact of host iron sources on S. aureus gene expression remains unknown. In this study, we combine multivariable difference gel electrophoresis and mass spectrometry with multivariate statistical analyses to systematically cluster cellular protein response across distinct iron-exposure conditions. Quadruplicate samples were simultaneously analyzed for alterations in protein abundance and/or post-translational modification state in response to environmental (iron chelation, hemin treatment or genetic (Deltafur alterations in bacterial iron exposure. We identified 120 proteins representing several coordinated biochemical pathways that are affected by changes in iron-exposure status. Highlighted in these experiments is the identification of the heme-regulated transport system (HrtAB, a novel transport system which plays a critical role in staphylococcal heme metabolism. Further, we show that regulated overproduction of acidic end-products brought on by iron starvation decreases local pH resulting in the release of iron from the host iron-sequestering protein transferrin. These findings reveal novel strategies used by S. aureus to acquire scarce nutrients in the hostile host environment and begin to define the iron and heme-dependent regulons of S. aureus.

  18. Lysionotin attenuates Staphylococcus aureus pathogenicity by inhibiting α-toxin expression.

    Science.gov (United States)

    Teng, Zihao; Shi, Dongxue; Liu, Huanyu; Shen, Ziying; Zha, Yonghong; Li, Wenhua; Deng, Xuming; Wang, Jianfeng

    2017-07-14

    α-Toxin, one of the best known pore-forming proteins produced by Staphylococcus aureus (S. aureus), is a critical virulence factor in multiple infections. The necessity of α-toxin for S. aureus pathogenicity suggests that this toxin is an important target for the development of a potential treatment strategy. In this study, we showed that lysionotin, a natural compound, can inhibit the hemolytic activity of culture supernatants by S. aureus by reducing α-toxin expression. Using real-time PCR analysis, we showed that transcription of hla (the gene encoding α-toxin) and agr (the locus regulating hla) was significantly inhibited by lysionotin. Lactate dehydrogenase and live/dead assays indicated that lysionotin effectively protected human alveolar epithelial cells against S. aureus, and in vivo studies also demonstrated that lysionotin can protect mice from pneumonia caused by S. aureus. These findings suggest that lysionotin is an efficient inhibitor of α-toxin expression and shows significant protection against S. aureus in vitro and in vivo. This study supports a potential strategy for the treatment of S. aureus infection by inhibiting the expression of virulence factors and indicates that lysionotin may be a potential treatment for S. aureus pneumonia.

  19. Pathogenesis of Staphylococcus aureus abscesses.

    Science.gov (United States)

    Kobayashi, Scott D; Malachowa, Natalia; DeLeo, Frank R

    2015-06-01

    Staphylococcus aureus causes many types of human infections and syndromes-most notably skin and soft tissue infections. Abscesses are a frequent manifestation of S. aureus skin and soft tissue infections and are formed, in part, to contain the nidus of infection. Polymorphonuclear leukocytes (neutrophils) are the primary cellular host defense against S. aureus infections and a major component of S. aureus abscesses. These host cells contain and produce many antimicrobial agents that are effective at killing bacteria, but can also cause non-specific damage to host tissues and contribute to the formation of abscesses. By comparison, S. aureus produces several molecules that also contribute to the formation of abscesses. Such molecules include those that recruit neutrophils, cause host cell lysis, and are involved in the formation of the fibrin capsule surrounding the abscess. Herein, we review our current knowledge of the mechanisms and processes underlying the formation of S. aureus abscesses, including the involvement of polymorphonuclear leukocytes, and provide a brief overview of therapeutic approaches.

  20. Curcumin Reverse Methicillin Resistance in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Su-Hyun Mun

    2014-11-01

    Full Text Available Curcumin, a natural polyphenolic flavonoid extracted from the rhizome of Curcuma longa L., was shown to possess superior potency to resensitize methicillin-resistant Staphylococcus aureus (MRSA to antibiotics. Previous studies have shown the synergistic activity of curcumin with β-lactam and quinolone antibiotics. Further, to understand the anti-MRSA mechanism of curcumin, we investigated the potentiated effect of curcumin by its interaction in diverse conditions. The mechanism of anti-MRSA action of curcumin was analyzed by the viability assay in the presence of detergents, ATPase inhibitors and peptidoglycan (PGN from S. aureus, and the PBP2a protein level was analyzed by western blotting. The morphological changes in the curcumin-treated MRSA strains were investigated by transmission electron microscopy (TEM. We analyzed increased susceptibility to MRSA isolates in the presence of curcumin. The optical densities at 600 nm (OD600 of the suspensions treated with the combinations of curcumin with triton X-100 and Tris were reduced to 63% and 59%, respectively, compared to curcumin without treatment. N,N'-dicyclohexylcarbodiimide (DCCD and sodium azide (NaN3 were reduced to 94% and 55%, respectively. When peptidoglycan (PGN from S. aureus was combined with curcumin, PGN (0–125 μg/mL gradually blocked the antibacterial activity of curcumin (125 μg/mL; however, at a concentration of 125 µg/mL PGN, it did not completely block curcumin. Curcumin has a significant effect on the protein level of PBP2a. The TEM images of MRSA showed damage of the cell wall, disruption of the cytoplasmic contents, broken cell membrane and cell lysis after the treatment of curcumin. These data indicate a remarkable antibacterial effect of curcumin, with membrane permeability enhancers and ATPase inhibitors, and curcumin did not directly bind to PGN on the cell wall. Further, the antimicrobial action of curcumin involved in the PBP2a-mediated resistance mechanism was

  1. Staphylococcus aureus Bacteraemia

    Directory of Open Access Journals (Sweden)

    James Price

    2010-01-01

    Full Text Available Staphylococcus aureus bacteraemia (SAB is commonly complicated by metastatic infection or relapse after treatment. Objectives. The study aim was to determine the role of bacterial, host, and management factors in development of complicated SAB. Methods. A prospectively-conducted observational study gathered data on predisposition, management and outcome of 100 consecutive SAB cases. Antibiotic susceptibilities and genetic lineage of bacterial isolates were determined. Further clinical and microbiological data were gathered on two retrospective series from 1999–2000 (n=57 and 2004 (n=116. Results. In the prospective cases, 27% met our definition of complicated disease. Expressed as RR and 95% CI, complicated disease was associated with diabetes (1.58, 1.00–2.48, injecting-drug use (5.48, 0.88–33.49, community-onset of symptoms (1.4, 1.02–1.92, and symptom duration ≥48 hours prior to starting effective antibiotic therapy (2.10, 1.22–3.61. Uncomplicated disease was associated with the presence of a central line (0.69, 0.55–0.88 and prompt removal of a primary focus (0.71, 0.57–0.90. Neither methicillin resistance nor genetic lineage was associated with complicated disease, but methicillin resistance was associated with higher mortality. Conclusions. This study demonstrates that clinical rather than microbial factors are the major determinants of SAB outcome and underscores the importance of early treatment.

  2. Genetically enhanced cows resist intramammary Staphylococcus aureus infection.

    Science.gov (United States)

    Wall, Robert J; Powell, Anne M; Paape, Max J; Kerr, David E; Bannerman, Douglas D; Pursel, Vernon G; Wells, Kevin D; Talbot, Neil; Hawk, Harold W

    2005-04-01

    Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of dollars annually. To test the feasibility of protecting animals through genetic engineering, transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 micrograms/ml [corrected] in their milk were produced. In vitro assays demonstrated the milk's ability to kill Staphylococcus aureus. Intramammary infusions of S. aureus were administered to three transgenic and ten nontransgenic cows. Increases in milk somatic cells, elevated body temperatures and induced acute phase proteins, each indicative of infection, were observed in all of the nontransgenic cows but in none of the transgenic animals. Protection against S. aureus mastitis appears to be achievable with as little as 3 micrograms/ml [corrected] of lysostaphin in milk. Our results indicate that genetic engineering can provide a viable tool for enhancing resistance to disease and improve the well-being of livestock.

  3. Contribution of coagulases towards Staphylococcus aureus disease and protective immunity.

    Directory of Open Access Journals (Sweden)

    Alice G Cheng

    Full Text Available The bacterial pathogen Staphylococcus aureus seeds abscesses in host tissues to replicate at the center of these lesions, protected from host immune cells via a pseudocapsule. Using histochemical staining, we identified prothrombin and fibrin within abscesses and pseudocapsules. S. aureus secretes two clotting factors, coagulase (Coa and von Willebrand factor binding protein (vWbp. We report here that Coa and vWbp together are required for the formation of abscesses. Coa and vWbp promote the non-proteolytic activation of prothrombin and cleavage of fibrinogen, reactions that are inhibited with specific antibody against each of these molecules. Coa and vWbp specific antibodies confer protection against abscess formation and S. aureus lethal bacteremia, suggesting that coagulases function as protective antigens for a staphylococcal vaccine.

  4. Toxin-mediated gene regulatory mechanism in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Hwang-Soo Joo

    2016-12-01

    Full Text Available The dangerous human pathogen Staphylococcus aureus relies heavily on toxins to cause disease, but toxin production can put a strong burden on the bacteria’s energy balance. Thus, controlling the synthesis of proteins solely needed in times of toxin production represents a way for the bacteria to avoid wasting energy. One hypothetical manner to accomplish this sort of regulation is by gene regulatory functions of the toxins themselves. There have been several reports about gene regulation by toxins in S. aureus, but these were never verified on the molecular level. In our study published in MBio [Joo et al., 7(5. pii: e01579-16], we show that phenol-soluble modulins (PSMs, important peptide toxins of S. aureus, release a repressor from the promoter of the operon encoding the toxin export system, thereby enabling toxin secretion. This study describes the first molecular regulatory mechanism exerted by an S. aureus toxin, setting a paradigmatic example of how S. aureus toxins may influence cell functions to adjust them to times of toxin production.

  5. Evaluation of staphylococcus aureus class C non-specific acid phosphatase (SapS) as a reporter for gene expression and protein secretion in gram-negative and gram-positive bacteria

    CSIR Research Space (South Africa)

    Du Plessis, EM

    2007-11-01

    Full Text Available using a cost-effective plate screen, quantitatively measured by a simple enzyme assay and detected with zymography, its potential use as a reporter system was investigated. The S. aureus acid phosphatase (sapS) gene has been cloned and expressed from its...

  6. Neutrophil-mediated phagocytosis of Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Jos A.G. Van Strijp

    2014-09-01

    Full Text Available For invading staphylococci, phagocytosis an killing bij human neutrophils is the biggest threat. Neutrophils are the only cells that can effectively kill staphylococci by engulfment and subsequent bombardment with proteases, amidases, antimicrobial peptides and proteins in concert with reactive oxygen species that are generated during the metabolic burst.Both complement and antibodies are crucial for effective uptake and neutrophil activation. S. aureus is not an innocent bystander in this process. It actively secretes several proteins to impair every single step in this process from receptor modulation, to complement inhibition to neutrophil lysis to protease, antimicrobial peptide inhibition and resistance to reactive oxygen species. For the design of future novel antimicrobial strategies: therapeutic antibodies, vaccines, novel antibiotics, all this should be taken into account. Still the best way to treat diseases is to help to enhance the natural defence mechanism that are already in place.

  7. Methicillin-resistant Staphylococcus aureus (MRSA)

    Science.gov (United States)

    Methicillin-resistant Staphylococcus aureus; Hospital-acquired MRSA (HA-MRSA); Staph - MRSA; Staphylococcal - MRSA ... Que YA, Moreillon P. Staphylococcus aureus (including ... MJ, eds. Mandell, Douglas, and Bennett's Principles and Practice ...

  8. Staphylococcus aureus Isolates from Kenyan Milk

    African Journals Online (AJOL)

    Milk; Staphylococcus aureus; Antimicrobial Resistance; Multiple resistance. Introduction ... of antibiotic resistant bacterial strains in food in Africa. ..... spread of methicillin resistant staphylococcus aureus. Clin. Infect. Disease. 24S-.74-S79.

  9. The therapeutic effect of Chlorogenic acid against Staphylococcus aureus infection through Sortase A inhibition

    Directory of Open Access Journals (Sweden)

    Lin eWang

    2015-10-01

    Full Text Available The emergence and wide spread of multi-drug resistant Staphylococcus aureus (S. aureus requires the development of new therapeutic agents with alternative modes of action. Anti-virulence strategies are hoped to meet that need. Sortase A (SrtA has attracted great interest as a potential drug target to treat infections caused by S. aureus, as many of the surface proteins displayed by SrtA function as virulence factors by mediating bacterial adhesion to specific organ tissues, invasion of host cells, and evasion of the host-immune responses. It has been suggested that inhibitors of SrtA might be promising candidates for the treatment and/or prevention of S. aureus infections. In this study, we report that Chlorogenic acid (CHA, a natural compound that lacks significant anti–S. aureus activity, inhibit the activity of SrtA in vitro (IC50=33.86±5.55μg/ml and the binding of S. aureus to fibrinogen (Fg. Using molecular dynamics simulations and mutagenesis assays, we further demonstrate that CHA binds to the binding sites of C184 and G192 in the SrtA. In vivo studies demonstrated that CHA prevent mice from S. aureus-induced renal abscess, resulting in a significant survival advantage. These findings indicate that CHA is a promising therapeutic compound against SrtA during S. aureus infections.

  10. Mild Staphylococcus aureus skin infection improves the course of subsequent endogenous S. aureus bacteremia in mice

    NARCIS (Netherlands)

    S. van den Berg (Sanne); C.P. de Vogel (Corné); A.F. van Belkum (Alex); I.A.J.M. Bakker-Woudenberg (Irma)

    2015-01-01

    textabstractStaphylococcus aureus carriers with S. aureus bacteremia may have a reduced mortality risk compared to non-carriers. A role for the immune system is suggested. Here, we study in mice the effect of mild S. aureus skin infection prior to endogenous or exogenous S. aureus bacteremia, and ev

  11. Mild Staphylococcus aureus skin infection improves the course of subsequent endogenous S. aureus bacteremia in mice

    NARCIS (Netherlands)

    S. van den Berg (Sanne); C.P. de Vogel (Corné); A.F. van Belkum (Alex); I.A.J.M. Bakker-Woudenberg (Irma)

    2015-01-01

    textabstractStaphylococcus aureus carriers with S. aureus bacteremia may have a reduced mortality risk compared to non-carriers. A role for the immune system is suggested. Here, we study in mice the effect of mild S. aureus skin infection prior to endogenous or exogenous S. aureus bacteremia, and

  12. Vaccine Protection of Leukopenic Mice against Staphylococcus aureus Bloodstream Infection

    Science.gov (United States)

    Rauch, Sabine; Gough, Portia; Kim, Hwan Keun; Schneewind, Olaf

    2014-01-01

    The risk for Staphylococcus aureus bloodstream infection (BSI) is increased in immunocompromised individuals, including patients with hematologic malignancy and/or chemotherapy. Due to the emergence of antibiotic-resistant strains, designated methicillin-resistant S. aureus (MRSA), staphylococcal BSI in cancer patients is associated with high mortality; however, neither a protective vaccine nor pathogen-specific immunotherapy is currently available. Here, we modeled staphylococcal BSI in leukopenic CD-1 mice that had been treated with cyclophosphamide, a drug for leukemia and lymphoma patients. Cyclophosphamide-treated mice were highly sensitive to S. aureus BSI and developed infectious lesions lacking immune cell infiltrates. Virulence factors of S. aureus that are key for disease establishment in immunocompetent hosts—α-hemolysin (Hla), iron-regulated surface determinants (IsdA and IsdB), coagulase (Coa), and von Willebrand factor binding protein (vWbp)—are dispensable for the pathogenesis of BSI in leukopenic mice. In contrast, sortase A mutants, which cannot assemble surface proteins, display delayed time to death and increased survival in this model. A vaccine with four surface antigens (ClfA, FnBPB, SdrD, and SpAKKAA), which was identified by genetic vaccinology using sortase A mutants, raised antigen-specific immune responses that protected leukopenic mice against staphylococcal BSI. PMID:25183728

  13. 金黄色葡萄球菌ltaS突变株的构建与eLtaS蛋白的表达%Construction of LtaS mutant in Staphylococcus aureus and expression and purification of eLtaS protein

    Institute of Scientific and Technical Information of China (English)

    芦强; 牟春花; 张鑫; 刘玉; 董洁; 高亚萍; 杨光

    2012-01-01

    目的 通过构建金黄色葡萄球菌(SAU)ltaS突变株以及体外重组表达其胞外形式的eLtaS蛋白,研究LtaS以及eLtaS蛋白在SAU中的生物学功能.方法 利用同源重组的方法构建SAU 8325-4来源的ltaS缺失突变菌株,并对其生长以及外毒素水平进行检测;同时,在大肠杆菌中,体外重组表达并纯化eLtaS蛋白,继而观察其对SAU生长的影响.结果在SAU 8325-4中成功构建了ltaS缺失突变菌株,并发现ltaS突变株生长较野生型菌株缓慢;通过表达纯化成功获得高纯度的eLtaS蛋白,其与突变株共培养后对突变株的生长缓慢现象没有回复作用.结论 LtaS蛋白在SAU侵袭机体的过程中发挥着重要的功能,其胞外形式的eLtaS蛋白可能未参与脂磷壁酸(lipoteichoic acid,LTA)的合成.%Objective To investigate the biological function of LtaS and its extracellular form eLtaS in Staphylococcus aureus ( S. Aureus )by constructing the ItaS mutant from S. Aureus 8325-4, expressing and purifying the eLtaS protein. Methods ItaS Depletion mutant was constructed by homologous recombination in S. Aureus 8325-4. The growth curve and the level of its extra-toxins production were measured. The recombinant eLtaS was expressed and purified in vitro by pET-28a vector, and the effect of eLtaS on the growth of S. Aureus was detected. Results The ItaS mutant was successfully constructed in S. Aureus 8325-4. Compared with the wild-type strain, the growth rate of the mutant was inhibited and the level of extra-toxins of the ItaS mutant was decreased. We found that eLtaS could not restore the growth of the ItaS mutant. Conclusion LtaS plays an important role in the growth and pathogenicity of S. Aureus, while the extracellular protein eLtaS may not be involved in the synthesis of lipoteichoic acid.

  14. Distribution of the novel cell wall anchored protein-encoding gene sasX in Staphylococcus aureus strains%新的细胞壁锚定蛋白编码基因sasX在金黄色葡萄球菌中的分布

    Institute of Scientific and Technical Information of China (English)

    杜昕; 宋燕; 田月如; 阮斐怡; 吕元; 李敏

    2011-01-01

    Objective To investigate the distribution and effect on antibiotic resistance of the novel cell wall anchored protein-encoding gene sasX.Methods A total of 300 S.aureus isolates were randomly collected from inpatients with S.aureus infection in Shanghai Huashan hospital in 2004, 2007 and 2010.Meanwhile,170 S.aureus isolates from the nasal swabs of healthy people were collected as part of a population-based community prevalence study.Typing of S.aureus isolates were identified by using multilocus sequence typing (MLST) and S.aureus-specific staphylococcal protein A typing (spa typing).Determination of oxacillin MICs was used to screen MRSA.PCR and sequencing were used to analyze sasX gene.The effect on antibiotic resistance of sasX gene was detect by disc agar diffusion drug sensitive test.Results The major clonal types of the 300 S.aureus isolates collected from inpatients with S.aureus infection were ST239 ( 110,36.7%) and ST5 (122,40.7%).From 2004 to 2010,the percentage of isolates from inpatients with S.aureus infection was increased from 17% to 39%,but sasX was only found in 0.59% of the S.aureus isolates from the nasal swabs of healthy people.The percentage of sasX positive was increased from 47.2% to 83.8% in ST239.The percentage of sasX positive MRSA was increased from 26.4% to 50.8%,but the percentage of sasX positive MSSA was about 10%.Antibiotic resistance of sasX positive strains were higher than that of sasX negative strains.Conclusions SasX gene is mainly detected in nosocomial pathogenic S.aureus and it is a possible virulence factor of S.aureus in hosptal setting.The presence of sasX gene is related to antibiotic resistance.For better understanding the real function of this novel gene,further studies such as expression of the encoded protein should be carried out.%目的 研究细胞壁锚定蛋白编码基因sasX在金黄色葡萄球菌中的分布及对细菌耐药性的影响.方法 随机收集上海华山医院2004、2007

  15. Synthetic peptide inhibitors of DNA replication in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Løbner-Olesen, Anders; Kjelstrup, Susanne

    of clinically important pathogens and is essential for bacterial proliferation. The bacterial replication apparatus fulfill the requirements for a good drug target. The replisome of S. aureus consists of 5 different subunits (2, PolC2, 4, δ and δ`) who’s organization depends on multiple protein......-protein interactions. Centrally in the replisome is the -clamp where to multiple proteins binds through a conserved motif. We have identified the protein-protein interactions in the replisome of S. aureus by use of a bacterial two-hybrid system. A reverse bacterial two-hybrid system (R-BTH) based on Pyr......N (), DnaB and DnaX (). Three peptides identified as inhibitors of DnaN have been purified. Two of these peptides inhibited growth as well as DNA replication in S. aureus. The minimal inhibitory concentration (MIC) of the peptides was approximately 50 g/ml. Overexpression of DnaN reduced the inhibitory...

  16. Staphylococcus aureus Alters Growth Activity, Autolysis, and Antibiotic Tolerance in a Human Host-Adapted Pseudomonas aeruginosa Lineage

    DEFF Research Database (Denmark)

    Frydenlund Michelsen, Charlotte; Christensen, Anne-Mette; Bojer, Martin Saxtorph

    2014-01-01

    Interactions among members of polymicrobial infections or between pathogens and the commensal flora may determine disease outcomes. Pseudomonas aeruginosa and Staphylococcus aureus are important opportunistic human pathogens and are both part of the polymicrobial infection communities in human....... aeruginosa DK2 strains outcompeted S. aureus during coculture on agar plates, we found that later P. aeruginosa DK2 strains showed a commensal-like interaction, where S. aureus was not inhibited by P. aeruginosa and the growth activity of P. aeruginosa was enhanced in the presence of S. aureus. This effect...... is mediated by one or more extracellular S. aureus proteins greater than 10 kDa, which also suppressed P. aeruginosa autolysis and prevented killing by clinically relevant antibiotics through promoting small-colony variant (SCV) formation. The commensal interaction was abolished with S. aureus strains mutated...

  17. Prevention of Healthcare Associated Staphylococcus aureus Infections

    NARCIS (Netherlands)

    L.G.M. Bode (Lonneke)

    2014-01-01

    markdownabstract__Abstract__ S. aureus colonizes the skin and mucosae of a proportion of the human population. Carriers of S. aureus are at increased risk of developing infections with this pathogen. The aim of this thesis was to add to the prevention of healthcare associated S. aureus infections.

  18. Methicillin-resistant Staphylococcus aureus and athletes.

    Science.gov (United States)

    Kirkland, Eugene Brent; Adams, Brian B

    2008-09-01

    Methicillin-resistant Staphylococcus aureus infections have become an increasingly common condition among athletes. Physical contact, shared facilities and equipment, and hygienic practices of athletes all contribute to methicillin-resistant S. aureus transmission among sports participants. This review elucidates the risk factors predisposing to methicillin-resistant S. aureus infection in athletes and provides guidance for treatment and prevention.

  19. Acacetin Protects Mice from Staphylococcus aureus Bloodstream Infection by Inhibiting the Activity of Sortase A.

    Science.gov (United States)

    Bi, Chongwei; Dong, Xiaoyun; Zhong, Xiaobo; Cai, Hongjun; Wang, Dacheng; Wang, Lin

    2016-09-26

    Staphylococcus aureus (S. aureus) is a major cause of infection in hospitals and communities. Widespread dissemination of multi-drug resistant S. aureus is a serious threat to the health of humans and animals. An anti-virulence strategy has been widely considered as an alternative therapeutic approach. Inhibitors of virulence factors are able to treat S. aureus infections without influencing the growth or viability of bacteria and rarely lead to bacterial resistance. Sortase A (SrtA) is a membrane-associated cysteine transpeptidase that catalyzes up to 25 surface proteins that covalently bind to cell wall peptidoglycans. In S. aureus, most of these surface proteins have been identified as important virulence factors that are vital in bacterial pathogenesis. In the present study, we show that acacetin, a natural flavonoid compound, inhibits the activity of SrtA in S. aureus (IC50 = 36.46 ± 4.69 μg/mL, 128 μM) which affects the assembly of protein A (SpA) to cell walls and reduces the binding of S. aureus to fibrinogen (Fg). The mechanism of the interaction between acacetin and SrtA were preliminarily discussed using molecular dynamics simulations. The results suggested that acacetin adopted a compact conformation binding at the pocket of the SrtA via residues Arg-139 and Lys-140. By performing an animal infection model, we demonstrated that acacetin was able to protect mice from renal abscess formation induced by S. aureus and significantly increased survival rates. Taken together, these findings suggest that acacetin may be a promising candidate for the development of anti-S. aureus drugs.

  20. Characterization of immune response in Staphylococcus aureus chronically infected bovine mammary glands during active involution.

    Science.gov (United States)

    Andreotti, Carolina S; Baravalle, Celina; Sacco, Sofía C; Lovato, Melisa; Pereyra, Elizabet A L; Renna, María S; Ortega, Hugo H; Calvinho, Luis F; Dallard, Bibiana E

    2017-10-01

    The aim of this study was to characterize the immune response in Staphylococcus aureus chronically infected bovine mammary glands during active involution. Twenty-one Holstein non-pregnant cows in late lactation either uninfected or with chronic naturally acquired S. aureus intramammary infections (IMI) were included in this study. Cows were slaughtered at 7, 14 and 21 d after cessation of milking and samples for immunohistochemical analysis were taken. Protein expression of toll-like receptor 2 (TLR2) and TLR4 was significantly higher in S. aureus-infected quarters than in uninfected controls at the three involution stages studied. Protein expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1α and IL-17 was significantly affected by IMI; being higher in S. aureus-infected than uninfected quarters during all evaluated stages. In S. aureus-infected and uninfected quarters protein expression of lactoferrin increased from day 7-14 of involution, decreasing significantly to day 21 in mammary quarters with chronic infections. The number of monocytes-macrophages was significantly higher in S. aureus-infected than in uninfected control quarters at 7 and 21 d of involution. The number of T lymphocytes was significantly higher in S. aureus-infected than in uninfected quarters at 7 and 14 d of involution while the number of B lymphocytes was significantly higher in S. aureus-infected than in uninfected quarters during all evaluated stages, showing a progressive increase as involution advanced. These results demonstrated a sustained and exacerbated innate and adaptive immune response during chronic S. aureus IMI, playing a critical role in the infection control during active involution. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Aureolib - a proteome signature library: towards an understanding of staphylococcus aureus pathophysiology.

    Directory of Open Access Journals (Sweden)

    Stephan Fuchs

    Full Text Available Gel-based proteomics is a powerful approach to study the physiology of Staphylococcus aureus under various growth restricting conditions. We analyzed 679 protein spots from a reference 2-dimensional gel of cytosolic proteins of S. aureus COL by mass spectrometry resulting in 521 different proteins. 4,692 time dependent protein synthesis profiles were generated by exposing S. aureus to nine infection-related stress and starvation stimuli (H2O2, diamide, paraquat, NO, fermentation, nitrate respiration, heat shock, puromycin, mupirocin. These expression profiles are stored in an online resource called Aureolib (http://www.aureolib.de. Moreover, information on target genes of 75 regulators and regulatory elements were included in the database. Cross-comparisons of this extensive data collection of protein synthesis profiles using the tools implemented in Aureolib lead to the identification of stress and starvation specific marker proteins. Altogether, 226 protein synthesis profiles showed induction ratios of 2.5-fold or higher under at least one of the tested conditions with 157 protein synthesis profiles specifically induced in response to a single stimulus. The respective proteins might serve as marker proteins for the corresponding stimulus. By contrast, proteins whose synthesis was increased or repressed in response to more than four stimuli are rather exceptional. The only protein that was induced by six stimuli is the universal stress protein SACOL1759. Most strikingly, cluster analyses of synthesis profiles of proteins differentially synthesized under at least one condition revealed only in rare cases a grouping that correlated with known regulon structures. The most prominent examples are the GapR, Rex, and CtsR regulon. In contrast, protein synthesis profiles of proteins belonging to the CodY and σ(B regulon are widely distributed. In summary, Aureolib is by far the most comprehensive protein expression database for S. aureus and provides

  2. Staphylococcus aureus produces membrane-derived vesicles that induce host cell death.

    Directory of Open Access Journals (Sweden)

    Mamata Gurung

    Full Text Available Gram-negative bacteria produce outer membrane vesicles that play a role in the delivery of virulence factors to host cells. However, little is known about the membrane-derived vesicles (MVs produced by gram-positive bacteria. The present study examined the production of MVs from Staphylococcus aureus and investigated the delivery of MVs to host cells and subsequent cytotoxicity. Four S. aureus strains tested, two type strains and two clinical isolates, produced spherical nanovesicles during in vitro culture. MVs were also produced during in vivo infection of a clinical S. aureus isolate in a mouse pneumonia model. Proteomic analysis showed that 143 different proteins were identified in the S. aureus-derived MVs. S. aureus MVs were interacted with the plasma membrane of host cells via a cholesterol-rich membrane microdomain and then delivered their component protein A to host cells within 30 min. Intact S. aureus MVs induced apoptosis of HEp-2 cells in a dose-dependent manner, whereas lysed MVs neither delivered their component into the cytosol of host cells nor induced cytotoxicity. In conclusion, this study is the first report that S. aureus MVs are an important vehicle for delivery of bacterial effector molecules to host cells.

  3. Rapid detection of viral antibodies based on multifunctional Staphylococcus aureus nanobioprobes.

    Science.gov (United States)

    Qiao, Jinjuan; Li, Yunpeng; Wei, Cuihua; Yang, Hang; Yu, Junping; Wei, Hongping

    2016-12-01

    Biosynthesis of nanoparticles inside S. aureus cells has enhanced the sensitivity of immunoassays based on the S. aureus nanoparticles. However, the current methods are limited to antigen detection by conjugating IgG antibodies on S. aureus nanoparticles. In this study, a simple way to conjugate antigens to the S. aureus nanobioparticles was developed by utilizing a cell wall binding domain (CBD) from a bacteriophage lysin PlyV12. Based on this novel design, simple agglutination tests of the IgG antibodies of Ebola virus (EBOV) nucleoprotein (NP) and Middle East Respiratory Virus (MERS) NP in rabbit sera were successfully developed by conjugating the S. aureus nanobioparticles with two fusion proteins EBOV NP- CBD and MERS NP-CBD, respectively. The conjugation was done easily by just mixing the fusion proteins with the S. aureus nanoparticles. The detection time was within 20 min without any special equipment or expertise. As far as we know, this is the first time to realize the detection of viral antibodies based on S. aureus nanoparticles.

  4. Proteome-wide antigen discovery of novel protective vaccine candidates against Staphylococcus aureus infection

    DEFF Research Database (Denmark)

    Rasmussen, Karina Juhl; Mattsson, Andreas Holm; Pilely, Katrine;

    2016-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a rapidly growing problem, especially in hospitals where MRSA cause increased morbidity and mortality and a significant rise in health expenditures. As many strains of MRSA are resistant to other antimicrobials in addition to methicillin......-five different S. aureus proteins were identified, recombinantly expressed, and tested for protection in a lethal sepsis mouse model using S. aureus strain MRSA252 as the challenge organism. We found that 13 of the 35 recombinant peptides yielded significant protection and that 12 of these antigens were highly...

  5. Dermatology case: segmental lichen aureus

    OpenAIRE

    Fernandes, I.; S. Carvalho; Machado, S.; Alves,R.; Selores, M.

    2012-01-01

    ABSTRACT The authors describe a clinical case of a six-year-old boy with history of a segmental brownish maculopapular skin eruption on his left thoracic and lumbar wall, since the last four months. Based on clinical and histological findings he was diagnosed with segmental lichen aureus.

  6. Stress Responses in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Frees, Dorte; Ingmer, Hanne

    2016-01-01

    Staphylococcus aures are prominent members of the normal flora of humans and animals, but are also a major cause of mild and severe infections. To persist and disseminate in the human host, and to survive in environmental settings, such as hospitals, S. aureus have developed a plethora of cellula...

  7. Binary typing of staphylococcus aureus

    NARCIS (Netherlands)

    W.B. van Leeuwen (Willem)

    2002-01-01

    textabstractThis thesis describes the development. application and validation of straindifferentiating DNA probes for the characterization of Staphylococcus aureus strains in a system. that yields a binary output. By comparing the differential hybridization of these DNA probes to staphylococcal geno

  8. Treatment of Staphylococcus aureus Infections.

    Science.gov (United States)

    David, Michael Z; Daum, Robert S

    2017-09-13

    Staphylococcus aureus, although generally identified as a commensal, is also a common cause of human bacterial infections, including of the skin and other soft tissues, bones, bloodstream, and respiratory tract. The history of S. aureus treatment is marked by the development of resistance to each new class of antistaphylococcal antimicrobial drugs, including the penicillins, sulfonamides, tetracyclines, glycopeptides, and others, complicating therapy. S. aureus isolates identified in the 1960s were sometimes resistant to methicillin, a ß-lactam antimicrobial active initially against a majority S. aureus strains. These MRSA isolates, resistant to nearly all ß-lactam antimicrobials, were first largely confined to the health care environment and the patients who attended it. However, in the mid-1990s, new strains, known as community-associated (CA-) MRSA strains, emerged. CA-MRSA organisms, compared with health care-associated (HA-) MRSA strain types, are more often susceptible to multiple classes of non ß-lactam antimicrobials. While infections caused by methicillin-susceptible S. aureus (MSSA) strains are usually treated with drugs in the ß-lactam class, such as cephalosporins, oxacillin or nafcillin, MRSA infections are treated with drugs in other antimicrobial classes. The glycopeptide drug vancomycin, and in some countries teicoplanin, is the most common drug used to treat severe MRSA infections. There are now other classes of antimicrobials available to treat staphylococcal infections, including several that have been approved after 2009. The antimicrobial management of invasive and noninvasive S. aureus infections in the ambulatory and in-patient settings is the topic of this review. Also discussed are common adverse effects of antistaphylococcal antimicrobial agents, advantages of one agent over another for specific clinical syndromes, and the use of adjunctive therapies such as surgery and intravenous immunoglobulin. We have detailed considerations in the

  9. An introduction to staphylococcus aureus, and techniques for identifying and quantifying s. aureus adhesins in relation to adhesion to biomaterials: review

    Directory of Open Access Journals (Sweden)

    Harris L. G.

    2002-12-01

    Full Text Available The ability of Staphylococcus aureus to adhere to the extracellular matrix and plasma proteins deposited on biomaterials is a significant factor in the pathogenesis of orthopaedic-device related infections. S. aureus possesses many adhesion proteins on its surface, but it is not known how they interact with each other to form stable interactions with the substrate. A novel method was developed for extracting adhesins from the S. aureus cell wall, which could then be further analysed. The protocol involves using a FastPrep instrument to mechanically disrupt the cell walls resulting in native cell walls. Ionically and covalently bound proteins were then solubilised using sodium dodecyl sulphate (SDS and lysostaphin, respectively. Western blot analysis of covalently bound proteins using anti-protein A and anti-clumping factor A sera showed that S. aureus produces most surface proteins in early growth, and less in post-exponential and stationary growth. Immuno-gold labelling of protein A, and clumping factor A was observed all over the bacteria and showed no distinct surface distribution pattern. However, this labelling showed expression of surface associated proteins varied in a growth-phase dependent and cell-density dependent manner.

  10. Proteomic Characterization of Lytic Bacteriophages of Staphylococcus aureus Isolated from Sewage Affluent of India

    Science.gov (United States)

    Sangha, Kamalpreet Kaur; Kumar, B. V. Sunil; Agrawal, Ravi Kant; Verma, Ramneek

    2014-01-01

    Staphylococcus aureus is a Gram-positive bacterium that causes a variety of diseases, including bovine mastitis, which has severe economic consequences. Standard antibiotic treatment results in selection of resistant strains, leading to need for an alternative treatment such as bacteriophage therapy. Present study describes isolation and characterization of a staphylococcal phage from sewage samples. S. aureus isolates obtained from microbial type culture collection (MTCC), Chandigarh, India, were used to screen staphylococcal phages. A phage designated as ΦMSP was isolated from sewage samples by soft agar overlay method. It produced clear plaques on tryptone soya agar overlaid with S. aureus. Transmission electron microscopy revealed that the phage had an icosahedral symmetry. It had 5 major proteins and possessed a peptidoglycan hydrolase corresponding to 70 kDa. ΦMSP infection induced 26 proteins to be uniquely expressed in S. aureus. This phage can be proposed as a candidate phage to treat staphylococcal infections. PMID:27355013

  11. Response of Staphylococcus Aureus to a Spaceflight Analogue

    Science.gov (United States)

    Castro, S. L.; Ott, C. M.

    2010-01-01

    demonstrated that the low-shear response could be lost in as few as 2.5 hours. These changes in phenotypic properties prompted investigation into variations occurring at the genetic level. Microarray analysis of low-shear cultured S. aureus revealed the differential regulation of genes involved in metabolism, stress response, and phosphate transfer. Additional genetic analysis with quantitative real-time PCR revealed alterations in the expression of Hfq, the conserved RNA chaperone protein involved in global gene regulation. Hfq has been connected to the regulation of a spaceflight microgravity response in S. typhimurium. These findings in S. aureus suggest an evolutionary conserved response to spaceflight conditions among structurally-diverse microorganisms. Furthermore, the reduction in pigmentation, hemolysin secretion, and survival against oxidative stress and immunologically active whole blood demonstrate an overall decrease in the virulence factors of S. aureus in response to spaceflight-like conditions.

  12. Response of Staphylococcus Aureus to a Spaceflight Analogue

    Science.gov (United States)

    Castro, S. L.; Ott, C. M.

    2010-01-01

    demonstrated that the low-shear response could be lost in as few as 2.5 hours. These changes in phenotypic properties prompted investigation into variations occurring at the genetic level. Microarray analysis of low-shear cultured S. aureus revealed the differential regulation of genes involved in metabolism, stress response, and phosphate transfer. Additional genetic analysis with quantitative real-time PCR revealed alterations in the expression of Hfq, the conserved RNA chaperone protein involved in global gene regulation. Hfq has been connected to the regulation of a spaceflight microgravity response in S. typhimurium. These findings in S. aureus suggest an evolutionary conserved response to spaceflight conditions among structurally-diverse microorganisms. Furthermore, the reduction in pigmentation, hemolysin secretion, and survival against oxidative stress and immunologically active whole blood demonstrate an overall decrease in the virulence factors of S. aureus in response to spaceflight-like conditions.

  13. Rhesus macaques (Macaca mulatta are natural hosts of specific Staphylococcus aureus lineages.

    Directory of Open Access Journals (Sweden)

    Sanne van den Berg

    Full Text Available Currently, there is no animal model known that mimics natural nasal colonization by Staphylococcus aureus in humans. We investigated whether rhesus macaques are natural nasal carriers of S. aureus. Nasal swabs were taken from 731 macaques. S. aureus isolates were typed by pulsed-field gel electrophoresis (PFGE, spa repeat sequencing and multi-locus sequence typing (MLST, and compared with human strains. Furthermore, the isolates were characterized by several PCRs. Thirty-nine percent of 731 macaques were positive for S. aureus. In general, the macaque S. aureus isolates differed from human strains as they formed separate PFGE clusters, 50% of the isolates were untypeable by agr genotyping, 17 new spa types were identified, which all belonged to new sequence types (STs. Furthermore, 66% of macaque isolates were negative for all superantigen genes. To determine S. aureus nasal colonization, three nasal swabs from 48 duo-housed macaques were taken during a 5 month period. In addition, sera were analyzed for immunoglobulin G and A levels directed against 40 staphylococcal proteins using a bead-based flow cytometry technique. Nineteen percent of the animals were negative for S. aureus, and 17% were three times positive. S. aureus strains were easily exchanged between macaques. The antibody response was less pronounced in macaques compared to humans, and nasal carrier status was not associated with differences in serum anti-staphylococcal antibody levels. In conclusion, rhesus macaques are natural hosts of S. aureus, carrying host-specific lineages. Our data indicate that rhesus macaques are useful as an autologous model for studying S. aureus nasal colonization and infection prevention.

  14. Induction of Staphylococcus aureus-specific IgA and agglutination potency in milk of cows by mucosal immunization.

    Science.gov (United States)

    Tempelmans Plat-Sinnige, Marjan J; Verkaik, Nelianne J; van Wamel, Willem J B; de Groot, Nanda; Acton, Dennis S; van Belkum, Alex

    2009-06-19

    Lactating cows were immunized with inactivated Staphylococcus aureus strains and concentrated culture supernatants. Application of a repeated mucosal immunization scheme resulted in significant levels of S. aureus-specific IgA in milk of dairy cows. Average IgA titers against whole cell S. aureus increased during the first 10 weeks of immunization after which a plateau level was reached and maintained during lactation. Immune whey agglutinated both bovine and human S. aureus strains including methicillin-resistant S. aureus (MRSA) strains and recognized extracted S. aureus proteins on Western blot. ELISAs to quantify milk IgA reactive with a number of S. aureus virulence proteins (e.g. enterotoxins, microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) and immune modulating proteins) and cell wall components, demonstrated the polyclonality of the IgA. Correlations observed between agglutination and specific IgA titers for whey and for purified IgA suggested functionality of the induced antibodies. Milk from immunized cows may provide a way of producing potentially therapeutic polyclonal antibodies against S. aureus colonization and infection.

  15. Involvement of superoxide and myeloperoxidase in oxygen-dependent killing of Staphylococcus aureus by neutrophils.

    OpenAIRE

    Hampton, M B; A. J. Kettle; Winterbourn, C C

    1996-01-01

    We have used a quantitative assay that measures independent rate constants for phagocytosis and killing of Staphylococcus aureus to investigate the involvement of superoxide and myeloperoxidase in bacterial killing by human neutrophils. To inhibit superoxide-dependent processes, superoxide dismutase was cross-linked to immunoglobulin G and the conjugate was attached to the surface of S. aureus via protein A in its cell wall. Myeloperoxidase was inhibited with azide, and myeloperoxidase-defici...

  16. Antibacterial synergy between rosmarinic acid and antibiotics against methicillin-resistant Staphylococcus aureus

    Science.gov (United States)

    Ekambaram, Sanmuga Priya; Perumal, Senthamil Selvan; Balakrishnan, Ajay; Marappan, Nathiya; Gajendran, Sabari Srinivasan; Viswanathan, Vinodhini

    2016-01-01

    Aim/Background: Medicinal plants have ability to resist microorganisms by synthesizing secondary metabolites such as phenols. Rosmarinic acid (RA) is a phenylpropanoid widely distributed in plants and well known as therapeutic and cosmetic agent. Methicillin-resistant Staphylococcus aureus (MRSA) which is resistant to all kinds of β-lactams, threatens even most potent antibiotics. To improve the efficiency of antibiotics against multi-drug resistant bacteria and to reduce the antibiotic dose, the antibacterial activity and the synergistic effect of RA with standard antibiotics against S. aureus and MRSA was investigated. Materials and Methods: Antibacterial activity of RA against S. aureus and a clinical isolate of MRSA was evaluated by agar well diffusion method. Minimum inhibitory concentration (MIC) of RA was determined by broth dilution method. Synergism of RA with various antibiotics against S. aureus and MRSA was studied by broth checkerboard method and time-kill kinetic assay. Effect of RA on microbial surface components recognizing adhesive matrix molecules (MSCRAMM’s) of S. aureus and MRSA was studied using sodium dodecyl sulfate - polyacrylamide gel electrophoresis. Results: MIC of RA was found to be 0.8 and 10 mg/ml against S. aureus and MRSA, respectively. RA was synergistic with vancomycin, ofloxacin, and amoxicillin against S. aureus and only with vancomycin against MRSA. The time-kill analysis revealed that synergistic combinations were a more effective than individual antibiotics. MSCRAMM’s protein expression of S. aureus and MRSA was markedly suppressed by RA + vancomycin combination rather than RA alone. Conclusion: The synergistic effects of RA with antibiotics were observed against S. aureus and MRSA. RA showed inhibitory effect on the surface proteins MSCRAMM’s. Even though RA was shown to exhibit a synergistic effect with antibiotics, the MIC was found to be higher. Thus, further studies on increasing the efficacy of RA can develop it

  17. A pig model of acute Staphylococcus aureus induced pyemia

    DEFF Research Database (Denmark)

    Nielsen, O. L.; Iburg, T.; Aalbæk, B.;

    2009-01-01

    Background: Sepsis caused by Staphylococcus aureus constitutes an important cause of morbidity and mortality in humans, and the incidence of this disease-entity is increasing. In this paper we describe the initial microbial dynamics and lesions in pigs experimentally infected with S. aureus......, with the aim of mimicking human sepsis and pyemia. Methods: The study was conducted in anaesthetized and intravenously inoculated pigs, and was based on bacteriological examination of blood and testing of blood for IL-6 and C-reactive protein. Following killing of the animals and necropsy bacteriological...... was not detected in the blood and C-reactive protein did not increase, probably because of the short time course of the study. Conclusion: This study demonstrates the successful induction of acute pyemia (microabscesses), and forms a basis for future experiments that should include inoculation with strains of S...

  18. A pig model of acute Staphylococcus aureus induced pyemia

    DEFF Research Database (Denmark)

    Nielsen, O. L.; Iburg, T.; Aalbæk, B.

    2009-01-01

    Background: Sepsis caused by Staphylococcus aureus constitutes an important cause of morbidity and mortality in humans, and the incidence of this disease-entity is increasing. In this paper we describe the initial microbial dynamics and lesions in pigs experimentally infected with S. aureus......, with the aim of mimicking human sepsis and pyemia. Methods: The study was conducted in anaesthetized and intravenously inoculated pigs, and was based on bacteriological examination of blood and testing of blood for IL-6 and C-reactive protein. Following killing of the animals and necropsy bacteriological...... was not detected in the blood and C-reactive protein did not increase, probably because of the short time course of the study. Conclusion: This study demonstrates the successful induction of acute pyemia (microabscesses), and forms a basis for future experiments that should include inoculation with strains of S...

  19. Assessing the potential for raw meat to influence human colonization with Staphylococcus aureus.

    Science.gov (United States)

    Carrel, Margaret; Zhao, Chang; Thapaliya, Dipendra; Bitterman, Patrick; Kates, Ashley E; Hanson, Blake M; Smith, Tara C

    2017-09-07

    The role of household meat handling and consumption in the transfer of Staphylococcus aureus (S. aureus) from livestock to consumers is not well understood. Examining the similarity of S. aureus colonizing humans and S. aureus in meat from the stores in which those individuals shop can provide insight into the role of meat in human S. aureus colonization. S. aureus isolates were collected from individuals in rural and urban communities in Iowa (n = 3347) and contemporaneously from meat products in stores where participants report purchasing meat (n = 913). The staphylococcal protein A (spa) gene was sequenced for all isolates to determine a spa type. Morisita indices and Permutational Multivariate Analysis of Variance Using Distance Matrices (PERMANOVA) were used to determine the relationship between spa type composition among human samples and meat samples. spa type composition was significantly different between households and meat sampled from their associated grocery stores. spa types found in meat were not significantly different regardless of the store or county in which they were sampled. spa types in people also exhibit high similarity regardless of residential location in urban or rural counties. Such findings suggest meat is not an important source of S. aureus colonization in shoppers.

  20. Analysis of the features of 45 identified CRISPR loci in 32 Staphylococcus aureus.

    Science.gov (United States)

    Yang, Siyu; Liu, Jing; Shao, Fuye; Wang, Pengfei; Duan, Guangcai; Yang, Haiyan

    2015-08-28

    Staphylococcus aureus (S. aureus) is a common pathogen that can cause serious infections, even death. Because of the horizontal gene transfer (HGT) of antibiotic resistance genes, the drug resistant condition is becoming increasingly prevalent. Recently, an adaptive immunity system, named clustered regularly interspaced short palindromic repeats (CRISPR), was discovered and demonstrated to confer a defense against foreign invading elements that may carry the antibiotic resistance genes. In this study, we reveal the features of 45 identified CRISPR loci and the CRISPR associated gene (Cas) in 32 S. aureus strains from CRISPR database. Five spacers of S. aureus 08BA02176 and MSHR1132 were homologous with foreign genetic sequences from phages or plasmids, even containing a spacer sequence identical to part of some phages' genomes containing lukPV gene that encodes the PVL toxin. Many S. aureus strains with the same CRISPR type shared the same MLST type. CRISPR loci that had 3 or more similar protein loci mostly belonged to the same CRISPR type. We came to the conclusion that the CRISPR/Cas of strains 08BA02176 and MSHR1132 were inherited from a common ancestor or recombined from Staphylococcus lugdunensis. CRISPR loci can be mobilized and can transfer among different but closely related species, and the same types of MLST strains exhibit a higher affinity to the same types of CRISPR loci. Bacteriophages may be the predominant challenge facing S. aureus. The CRISPR/Cas structure may limit the transmission of bacterial virulence among S. aureus.

  1. Stress Responses in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Frees, Dorte; Ingmer, Hanne

    2016-01-01

    Staphylococcus aures are prominent members of the normal flora of humans and animals, but are also a major cause of mild and severe infections. To persist and disseminate in the human host, and to survive in environmental settings, such as hospitals, S. aureus have developed a plethora of cellular...... of the specific and general S. aureusstress responses, with a special emphasis on how stress responses contribute to virulence and antibiotic resistance in this important human pathogen....

  2. SDS Interferes with SaeS Signaling of Staphylococcus aureus Independently of SaePQ

    OpenAIRE

    Makgotlho, Phuti E.; Gabriella Marincola; Daniel Schäfer; Qian Liu; Taeok Bae; Tobias Geiger; Elizabeth Wasserman; Christiane Wolz; Wilma Ziebuhr; Bhanu Sinha

    2016-01-01

    The Staphylococcus aureus regulatory saePQRS system controls the expression of numerous virulence factors, including extracellular adherence protein (Eap), which amongst others facilitates invasion of host cells. The saePQRS operon codes for 4 proteins: the histidine kinase SaeS, the response regulator SaeR, the lipoprotein SaeP and the transmembrane protein SaeQ. S. aureus strain Newman has a single amino acid substitution in the transmembrane domain of SaeS (L18P) which results in constitut...

  3. A dual antibacterial mechanism involved in membrane disruption and DNA binding of 2R,3R-dihydromyricetin from pine needles of Cedrus deodara against Staphylococcus aureus.

    Science.gov (United States)

    Wu, Yanping; Bai, Jinrong; Zhong, Kai; Huang, Yina; Gao, Hong

    2017-03-01

    The antibacterial activity and mechanism of 2R,3R-dihydromyricetin (DMY) against Staphylococcus aureus were investigated. The minimum inhibitory concentration of DMY against S. aureus was 0.125mg/ml, and the growth inhibitory assay also revealed that DMY showed a potent antibacterial activity against S. aureus. Massive nucleotide leakage and flow cytometric analysis demonstrated that DMY disrupted the membrane integrity of S. aureus. Morphological changes and membrane hyperpolarization of S. aureus cells treated with DMY further suggested that DMY destroyed cell membrane. Meanwhile, DMY probably interacted with membrane lipids and proteins, causing a significant reduction in membrane fluidity and changes in conformation of membrane protein. Moreover, DMY could interact with S. aureus DNA through the groove binding mode. Overall, the results suggested that DMY could be applied as a candidate for the development of new food preservatives as it achieved bactericidal activity by damaging cell membrane and binding to intracellular DNA.

  4. Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis

    Science.gov (United States)

    2011-01-01

    Staphylococcus aureus is a major cause of mastitis in ruminants. In ewe mastitis, symptoms range from subclinical to gangrenous mastitis. S. aureus factors or host-factors contributing to the different outcomes are not completely elucidated. In this study, experimental mastitis was induced on primiparous ewes using two S. aureus strains, isolated from gangrenous (strain O11) or subclinical (strain O46) mastitis. Strains induced drastically distinct clinical symptoms when tested in ewe and mice experimental mastitis. Notably, they reproduced mild (O46) or severe (O11) mastitis in ewes. Ewe sera were used to identify staphylococcal immunoreactive proteins commonly or differentially produced during infections of variable severity and to define core and accessory seroproteomes. Such SERological Proteome Analysis (SERPA) allowed the identification of 89 immunoreactive proteins, of which only 52 (58.4%) were previously identified as immunogenic proteins in other staphylococcal infections. Among the 89 proteins identified, 74 appear to constitute the core seroproteome. Among the 15 remaining proteins defining the accessory seroproteome, 12 were specific for strain O11, 3 were specific for O46. Distribution of one protein specific for each mastitis severity was investigated in ten other strains isolated from subclinical or clinical mastitis. We report here for the first time the identification of staphylococcal immunogenic proteins common or specific to S. aureus strains responsible for mild or severe mastitis. These findings open avenues in S. aureus mastitis studies as some of these proteins, expressed in vivo, are likely to account for the success of S. aureus as a pathogen of the ruminant mammary gland. PMID:21324116

  5. Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis

    Directory of Open Access Journals (Sweden)

    Le Maréchal Caroline

    2011-02-01

    Full Text Available Abstract Staphylococcus aureus is a major cause of mastitis in ruminants. In ewe mastitis, symptoms range from subclinical to gangrenous mastitis. S. aureus factors or host-factors contributing to the different outcomes are not completely elucidated. In this study, experimental mastitis was induced on primiparous ewes using two S. aureus strains, isolated from gangrenous (strain O11 or subclinical (strain O46 mastitis. Strains induced drastically distinct clinical symptoms when tested in ewe and mice experimental mastitis. Notably, they reproduced mild (O46 or severe (O11 mastitis in ewes. Ewe sera were used to identify staphylococcal immunoreactive proteins commonly or differentially produced during infections of variable severity and to define core and accessory seroproteomes. Such SERological Proteome Analysis (SERPA allowed the identification of 89 immunoreactive proteins, of which only 52 (58.4% were previously identified as immunogenic proteins in other staphylococcal infections. Among the 89 proteins identified, 74 appear to constitute the core seroproteome. Among the 15 remaining proteins defining the accessory seroproteome, 12 were specific for strain O11, 3 were specific for O46. Distribution of one protein specific for each mastitis severity was investigated in ten other strains isolated from subclinical or clinical mastitis. We report here for the first time the identification of staphylococcal immunogenic proteins common or specific to S. aureus strains responsible for mild or severe mastitis. These findings open avenues in S. aureus mastitis studies as some of these proteins, expressed in vivo, are likely to account for the success of S. aureus as a pathogen of the ruminant mammary gland.

  6. Population structure of Staphylococcus aureus from remote African Babongo Pygmies.

    Directory of Open Access Journals (Sweden)

    Frieder Schaumburg

    Full Text Available BACKGROUND: Pandemic community-acquired methicillin-resistant Staphylococcus aureus isolates (CA-MRSA predominantly encode the Panton-Valentine leukocidin (PVL, which can be associated with severe infections. Reports from non-indigenous Sub-Saharan African populations revealed a high prevalence of PVL-positive isolates. The objective of our study was to investigate the S. aureus carriage among a remote indigenous African population and to determine the molecular characteristics of the isolates, particularly those that were PVL-positive. METHODOLOGY/PRINCIPAL FINDINGS: Nasal S. aureus carriage and risk factors of colonization were systematically assessed in remote Gabonese Babongo Pygmies. Susceptibility to antibiotics, possession of toxin-encoding genes (i.e., PVL, enterotoxins, and exfoliative toxins, S. aureus protein A (spa types and multi-locus sequence types (MLST were determined for each isolate. The carriage rate was 33%. No MRSA was detected, 61.8% of the isolates were susceptible to penicillin. Genes encoding PVL (55.9%, enterotoxin B (20.6%, exfoliative toxin D (11.7% and the epidermal cell differentiation inhibitor B (11.7% were highly prevalent. Thirteen spa types were detected and were associated with 10 STs predominated by ST15, ST30, ST72, ST80, and ST88. CONCLUSIONS: The high prevalence of PVL-positive isolates among Babongo Pygmies demands our attention as PVL can be associated with necrotinzing infection and may increase the risk of severe infections in remote Pygmy populations. Many S. aureus isolates from Babongo Pygmies and pandemic CA-MRSA-clones have a common genetic background. Surveillance is needed to control the development of resistance to antibiotic drugs and to assess the impact of the high prevalence of PVL in indigenous populations.

  7. Heme Recognition By a Staphylococcus Aureus IsdE

    Energy Technology Data Exchange (ETDEWEB)

    Grigg, J.C.; Vermeiren, C.L.; Heinrichs, D.E.; Murphy, M.E.P.

    2009-06-03

    Staphylococcus aureus is a Gram-positive bacterial pathogen and a leading cause of hospital acquired infections. Because the free iron concentration in the human body is too low to support growth, S. aureus must acquire iron from host sources. Heme iron is the most prevalent iron reservoir in the human body and a predominant source of iron for S. aureus. The iron-regulated surface determinant (Isd) system removes heme from host heme proteins and transfers it to IsdE, the cognate substrate-binding lipoprotein of an ATP-binding cassette transporter, for import and subsequent degradation. Herein, we report the crystal structure of the soluble portion of the IsdE lipoprotein in complex with heme. The structure reveals a bi-lobed topology formed by an N- and C-terminal domain bridged by a single {alpha}-helix. The structure places IsdE as a member of the helical backbone metal receptor superfamily. A six-coordinate heme molecule is bound in the groove established at the domain interface, and the heme iron is coordinated in a novel fashion for heme transporters by Met{sup 78} and His{sup 229}. Both heme propionate groups are secured by H-bonds to IsdE main chain and side chain groups. Of these residues, His{sup 299} is essential for IsdE-mediated heme uptake by S. aureus when growth on heme as a sole iron source is measured. Multiple sequence alignments of homologues from several other Gram-positive bacteria, including the human pathogens pyogenes, Bacillus anthracis, and Listeria monocytogenes, suggest that these other systems function equivalently to S. aureus IsdE with respect to heme binding and transport.

  8. Methicillin resistant S. aureus in human and bovine mastitis.

    Science.gov (United States)

    Holmes, Mark A; Zadoks, Ruth N

    2011-12-01

    Staphylococcus aureus is a ubiquitous organism that causes a variety of diseases including mastitis in cattle and humans. High-level resistance of S. aureus to β-lactams conferred by a mecA gene encoding a modified penicillin binding protein (PBP2a) was first observed in the early 1960's. These methicillin resistant S. aureus (MRSA) have been responsible for both hospital acquired infections (HA-MRSA) and, more recently, community acquired MRSA (CA-MRSA). A small number of human MRSA mastitis cases and outbreaks in maternity or neonatal units have been reported which are generally the result of CA-MRSA. The establishment of the sequence type 398 (ST398) in farm animals, primarily pigs, in the early 2000's has provided a reservoir of infection for humans and dairy cattle, particularly in continental Europe, described as livestock-associated MRSA (LA-MRSA). Prior to the emergence of ST398 there were sporadic reports of MRSA in bovine milk and cases of mastitis, often caused by strains from human associated lineages. Subsequently, there have been several reports describing bovine udder infections caused by ST-398 MRSA. Recently, another group of LA-MRSA strains was discovered in humans and dairy cattle in Europe. This group carries a divergent mecA gene and includes a number of S. aureus lineages (CC130, ST425, and CC1943) that were hitherto thought to be bovine-specific but are now also found as carriage or clinical isolates in humans. The emergence of MRSA in dairy cattle may be associated with contact with other host species, as in the case of ST398, or with the exchange of genetic material between S. aureus and coagulase negative Staphylococcus species, which are the most common species associated with bovine intramammary infections and commonly carry antimicrobial resistance determinants.

  9. Catalase-negative, methicillin-resistant Staphylococcus aureus as a cause of septicemia Staphylococcus aureus catalase-negativo resistente a meticilina como causa de septicemia

    Directory of Open Access Journals (Sweden)

    Ana Lúcia Innaco de Carvalho

    2003-01-01

    Full Text Available A catalase-negative methicillin-resistant Staphylococcus aureus (MRSA was isolated from blood, venous catheter spike and bone marrow collected from an HIV-positive man with lobar pneumonia and sepsis after ten days of hospitalization. The isolate was resistant to oxacillin (positive for penicillin-binding protein 2', ceftriaxone, clindamycin and clarithromycin, and susceptible to vancomycin. This is the first case of septicemia due to a catalase-negative S. aureus reported in Brazil, and, to our knowledge, it is the first case of catalase-negative MRSA reported in the literature. We believe that the patient acquired the S. aureus infection within the hospital environment since it was isolated ten days after hospitalization, it was isolated in a venous catheter spike, and the antibiotic resistance profile is similar to other S. aureus isolates recovered from infections in our hospital.Em um paciente HIV-positivo, com pneumonia lobar e septicemia, foi isolada, após dez dias de internação, uma cepa de Staphylococcus aureus catalase-negativa, resistente a meticilina/oxacilina (MRSA, de culturas de sangue, cateter venoso central e medula óssea. A cepa era resistente a oxacilina (PBP 2' positivo, ceftriaxona, clindamicina e claritromicina, e sensível a vancomicina. Este é o primeiro caso, reportado no Brasil, de uma septicemia por S. aureus catalase-negativo e, em nosso conhecimento, o primeiro caso de um S. aureus catalase-negativo resistente a meticilina. Nós acreditamos que o paciente tenha adquirido a infecção no ambiente hospitalar, uma vez que esta cepa foi isolada aos dez dias de internação, foi isolada em cateter venoso central e o perfil de sensibilidade aos antimicrobianos é semelhante ao dos S. aureus de infecções nosocomiais que ocorrem em nosso hospital.

  10. Identification of pathogenic factors potentially involved in Staphylococcus aureus keratitis using proteomics.

    Science.gov (United States)

    Khan, Shamila; Cole, Nerida; Hume, Emma B H; Garthwaite, Linda L; Nguyen-Khuong, Terry; Walsh, Bradley J; Willcox, Mark D P

    2016-10-01

    Staphylococcus is a leading cause of microbial keratitis, characterized by destruction of the cornea by bacterial exoproteins and host-associated factors. The aim of this study was to compare extracellular and cell-associated proteins produced by two different isolates of S. aureus, a virulent clinical isolate (Staph 38) and a laboratory strain (Staphylococcus aureus 8325-4) of weaker virulence in the mouse keratitis model. Proteins were analyzed using 2D polyacrylamide gel electrophoresis and identified by subsequent mass spectrometry. Activity of staphylococcal adhesins was assessed by allowing strains to bind to various proteins adsorbed onto polymethylmethacrylate squares. Thirteen proteins in the extracellular fraction and eight proteins in the cell-associated fractions after bacterial growth were produced in increased amounts in the clinical isolate Staph 38. Four of these proteins were S. aureus virulence factor adhesins, fibronectin binding protein A, staphopain, glyceraldehyde-3-phosphate dehydrogenase 2 and extracellular adherence protein. The clinical isolate Staph 38 adhered to a greater extent to all mammalian proteins tested, indicating the potential of the adhesins to be active on its surface. Other proteins with increased expression in Staph 38 included potential moonlighting proteins and proteins involved in transcription or translation. This is the first demonstration of the proteome of S. aureus isolates from keratitis. These results indicate that the virulent clinical isolate produces more potentially important virulence factors compared to the less virulent laboratory strain and these may be associated with the ability of a S. aureus strain to cause more severe keratitis.

  11. Evasion of Neutrophil Killing by Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Will A. McGuinness

    2016-03-01

    Full Text Available Staphylococcus aureus causes many types of infections, ranging from self-resolving skin infections to severe or fatal pneumonia. Human innate immune cells, called polymorphonuclear leukocytes (PMNs or neutrophils, are essential for defense against S. aureus infections. Neutrophils are the most prominent cell type of the innate immune system and are capable of producing non-specific antimicrobial molecules that are effective at eliminating bacteria. Although significant progress has been made over the past few decades, our knowledge of S. aureus-host innate immune system interactions is incomplete. Most notably, S. aureus has the capacity to produce numerous molecules that are directed to protect the bacterium from neutrophils. Here we review in brief the role played by neutrophils in defense against S. aureus infection, and correspondingly, highlight selected S. aureus molecules that target key neutrophil functions.

  12. Tracing and inhibiting growth of Staphylococcus aureus in barbecue cheese production after product recall.

    Science.gov (United States)

    Johler, S; Zurfluh, K; Stephan, R

    2016-05-01

    Staphylococcal food poisoning is one of the most prevalent causes of foodborne intoxication worldwide. It is caused by ingestion of enterotoxins formed by Staphylococcus aureus during growth in the food matrix. Following a recall of barbecue cheese due to the detection of staphylococcal enterotoxins in Switzerland in July 2015, we analyzed the production process of the respective dairy. Although most cheese-making processes involve acidification to inhibit the growth of pathogenic bacteria, barbecue cheese has to maintain a pH >6.0 to prevent undesired melting of the cheese. In addition, the dairy decided to retain the traditional manual production process of the barbecue cheese. In this study, therefore, we aimed to (1) trace Staph. aureus along the barbecue cheese production process, and (2) develop a sustainable strategy to inhibit growth of Staph. aureus and decrease the risk of staphylococcal food poisoning without changing the traditional production process. To this end, we traced Staph. aureus in a step-wise blinded process analysis on 4 different production days using spa (Staphylococcus protein A gene) typing, DNA microarray profiling, and pulsed-field gel electrophoresis analysis. We subsequently selected a new starter culture and used a model cheese production including a challenge test assay to assess its antagonistic effect on Staph. aureus growth, as well as its sensory and technological implications. We detected Staph. aureus in 30% (37/124) of the collected samples taken from the barbecue cheese production at the dairy. This included detection of Staph. aureus in the final product on all 4 production days, either after enrichment or using quantitative detection. We traced 2 enterotoxigenic Staph. aureus strains (t073/CC45 and t282/CC45) colonizing the nasal cavity and the forearms of the cheesemakers to the final product. In the challenge test assay, we were able to show that the new starter culture inhibited growth of Staph. aureus while meeting

  13. Selenium Deficiency Facilitates Inflammation Following S. aureus Infection by Regulating TLR2-Related Pathways in the Mouse Mammary Gland.

    Science.gov (United States)

    Gao, Xuejiao; Zhang, Zecai; Li, Ying; Shen, Peng; Hu, Xiaoyu; Cao, Yongguo; Zhang, Naisheng

    2016-08-01

    Selenium (Se) is an essential micronutrient affecting various aspects of health. Se deficiency has been associated with inflammation and immune responses. Mastitis poses a serious problem for humans and animals in the postpartum period. Staphylococcus aureus (S. aureus) is the most common infectious bacterial pathogen associated with mastitis. The present study sought to determine the effects and underlying mechanism of dietary Se on S. aureus-induced inflammation using a model of mouse mastitis. ELISA and Western blotting were performed to detect protein levels. Quantitative PCR (qPCR) was performed to detect messenger RNA (mRNA) levels. The histopathological changes indicated that Se deficiency resulted in increased inflammatory lesions in S. aureus mastitis, whereas Se deficiency did not induce inflammatory lesions in the mammary gland. Myeloperoxidase (MPO) activity was increased in Se-deficient mice with S. aureus mastitis. Analysis of cytokine mRNA and protein showed that Se deficiency leads to increased TNF-α, IL-1β, and IL-6 production in S. aureus mastitis. In addition, Se deficiency enhanced the mRNA and protein expressions of toll-like receptor 2 (TLR2), which were originally upregulated by S. aureus in the mammary gland tissues and human embryonic kidney 293 (HEK293)-mTLR2 cells. When Se-deficient mice were infected with S. aureus, the phosphorylation of IκB, nuclear factor-κB (NF-κB), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 was greatly increased. The results indicate that Se deficiency could intensify the inflammatory reaction in S. aureus mastitis. This work contributes to the exploration of new methods of preventing or treating of S. aureus mastitis and other infectious diseases.

  14. The adherens junctions control susceptibility to Staphylococcus aureus α-toxin.

    Science.gov (United States)

    Popov, Lauren M; Marceau, Caleb D; Starkl, Philipp M; Lumb, Jennifer H; Shah, Jimit; Guerrera, Diego; Cooper, Rachel L; Merakou, Christina; Bouley, Donna M; Meng, Wenxiang; Kiyonari, Hiroshi; Takeichi, Masatoshi; Galli, Stephen J; Bagnoli, Fabio; Citi, Sandra; Carette, Jan E; Amieva, Manuel R

    2015-11-17

    Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7(-/-) mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections.

  15. Staphylococcus aureus α-toxin-dependent induction of host cell death by membrane-derived vesicles.

    Directory of Open Access Journals (Sweden)

    Bernard Thay

    Full Text Available Staphylococcus aureus causes a wide spectrum of infections in humans, ranging from superficial cutaneous infections, infections in the circum-oral region, to life-threatening bacteremia. It was recently demonstrated that Gram-positive organisms such as S. aureus liberate membrane-derived vesicles (MVs, which analogously to outer membrane vesicles (OMVs of Gram-negative bacteria can play a role in delivering virulence factors to host cells. In the present study we have shown that cholesterol-dependent fusion of S. aureus MVs with the plasma membrane represents a route for delivery of a key virulence factor, α-toxin (α-hemolysin; Hla to human cells. Most S. aureus strains produce this 33-kDa pore-forming protein, which can lyse a wide range of human cells, and induce apoptosis in T-lymphocytes. Our results revealed a tight association of biologically active α-toxin with membrane-derived vesicles isolated from S. aureus strain 8325-4. Concomitantly, α-toxin contributed to HeLa cell cytotoxicity of MVs, and was the main vesicle-associated protein responsible for erythrocyte lysis. In contrast, MVs obtained from an isogenic hla mutant were significantly attenuated with regards to both causing lysis of erythrocytes and death of HeLa cells. This is to our knowledge the first recognition of an S. aureus MV-associated factor contributing to host cell cytotoxicity.

  16. [Quantitative specific detection of Staphylococcus aureus based on recombinant lysostaphin and ATP bioluminescence].

    Science.gov (United States)

    Li, Yuyuan; Mi, Zhiqiang; An, Xiaoping; Zhou, Yusen; Tong, Yigang

    2014-08-01

    Quantitative specific detection of Staphylococcus aureus is based on recombinant lysostaphin and ATP bioluminescence. To produce recombinant lysostaphin, the lysostaphin gene was chemically synthesized and inserted it into prokaryotic expression vector pQE30, and the resulting expression plasmid pQE30-Lys was transformed into E. coli M15 for expressing lysostaphin with IPTG induction. The recombinant protein was purified by Ni(2+)-NTA affinity chromatography. Staphylococcus aureus was detected by the recombinant lysostaphin with ATP bioluminescence, and plate count method. The results of the two methods were compared. The recombinant lysostaphin was successfully expressed, and a method of quantitative specific detection of S. aureus has been established, which showed a significant linear correlation with the colony counting. The detection method developed has good perspective to quantify S. aureus.

  17. Global changes in Staphylococcus aureus gene expression during human prosthetic joint infection

    DEFF Research Database (Denmark)

    Xu, Yijuan; Nielsen, Per Halkjær; Nielsen, Jeppe Lund

    2016-01-01

    sequencing (RNA-seq) was used for transcriptome profile of joint fluid obtained from a patient undergoing surgery due to acute S. aureus prosthetic joint infection. The S. aureus gene expression in the infection was compared with exponential culture of a S. aureus isolate obtained from the same sample using...... of known resistant genes. The patient showed no signs of improvement during 2-days treatment with antibiotics (different β-lactams and gentamicin) prior to the surgery. The RNA-seq data indicated that the strategy employed by S. aureus to survive and proliferate in the host during antibiotic treatment......, hemolysins, and immune evasion proteins. A number of chaperones and stress related genes were overexpressed indicating a stress response. Furthermore, the RNA-seq data provided clues of the potential major nutrient sources for the pathogen in vivo. Several amino acid degradation pathways were highly...

  18. Skrining Staphylococcus aureus dengan Resistansi Berperantara MecA dari Sediaan Usap Hidung pada Dokter Muda di Instalasi Perawatan Intensif Rumah Sakit Umum Pusat Haji Adam Malik Medan

    OpenAIRE

    Krishnamoorthy, Mungunthanii

    2015-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) merupakan isolat S.aureus yang resistan terhadap semua antibiotika ß-laktam termasuk penicillin. Gen MecA yang mengkode protein pengikat penicillin 2a (PBP 2a) menentukan sifat resistansi tersebut. MRSA merupakan penyebab infeksi yang signifikan seperti infeksi kulit dan jaringan lunak (SSTI), bakteremia, endokarditis, pneumonia, osteomyelitis dan artritis infektif. Tujuan penelitian ini adalah untuk mengetahui jumlah S.aureus dengan resistan...

  19. Postoperative Staphylococcus aureus infections in Medicare beneficiaries.

    Directory of Open Access Journals (Sweden)

    Moaven Razavi

    Full Text Available Staphylococcus aureus (S. aureus infections are important because of their increasing frequency, resistance to antibiotics, and high associated rates of disabilities and deaths. We examined the incidence and correlates of S. aureus infections following 219,958 major surgical procedures in a 5% random sample of fee-for-service Medicare beneficiaries from 2004-2007. Of these surgical patients, 0.3% had S. aureus infections during the hospitalizations when index surgical procedures were performed; and 1.7% and 2.3%, respectively, were hospitalized with infections within 60 days or 180 days following admissions for index surgeries. S. aureus infections occurred within 180 days in 1.9% of patients following coronary artery bypass graft surgery, 2.3% following hip surgery, and 5.9% following gastric or esophageal surgery. Of patients first hospitalized with any major infection reported during the first 180 days after index surgery, 15% of infections were due to S. aureus, 18% to other documented organisms, and no specific organism was reported on claim forms in 67%. Patient-level predictors of S. aureus infections included transfer from skilled nursing facilities or chronic hospitals and comorbid conditions (e.g., diabetes, congestive heart failure, chronic obstructive pulmonary disease, and chronic renal disease. In a logarithmic regression, elective index admissions with S. aureus infection stayed 130% longer than comparable patients without that infection. Within 180 days of the index surgery, 23.9% of patients with S. aureus infection and 10.6% of patients without this infection had died. In a multivariate logistic regression of death within 180 days of admission for the index surgery with adjustment for demographics, co-morbidities, and other risks, S. aureus was associated with a 42% excess risk of death. Due to incomplete documentation of organisms in Medicare claims, these statistics may underestimate the magnitude of S. aureus infection

  20. Cyclic peptide inhibitors of the β-sliding clamp in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Kjelstrup, Susanne; Hansen, Paula Melo Paulon; Thomsen, Line Elnif;

    2013-01-01

    Interaction between pairs of Staphylococcus aureus replication proteins was detected in an Escherichia coli based two-hybrid analysis. A reverse two-hybrid system was constructed for selection of compounds that hindered interaction between interacting protein pairs. A number of cyclic peptides...

  1. Binding of Efb from Staphylococcus aureus to fibrinogen blocks neutrophil adherence

    Science.gov (United States)

    In addition to its pivotal role in hemostasis, fibrinogen (Fg) and provisional fibrin matrices play important roles in inflammation and regulate innate immune responses by interacting with leukocytes. Efb (the extracellular fibrinogen-binding protein) is a secreted Staphylococcus aureus protein that...

  2. CHROMOSOMAL MAPPING IN STRAINS OF STAPHYLOCOCCUS AUREUS,

    Science.gov (United States)

    STAPHYLOCOCCUS AUREUS , CHROMOSOMES), (*CHROMOSOMES, MAPPING), NITROSO COMPOUNDS, GUANIDINES, GENETICS, MUTATIONS, DRUGS, TOLERANCES(PHYSIOLOGY), TEST METHODS, DEOXYRIBONUCLEIC ACIDS, INHIBITION, RESISTANCE(BIOLOGY).

  3. Mastite com lesões sistêmicas por Staphylococus aureus subesp. aureus em coelhos Mastitis with systemic lesions due to Staphylococus aureus subesp. aureus in rabbits

    OpenAIRE

    Sandra Davi Traverso; Leonardo da Cunha; Joaquim César Teixeira Fernandes; Alexandre Paulino Loretti; Adriana Rhoden; Elsio Wunder Jr.; David Driemeier

    2003-01-01

    Em uma criação composta por 1800 coelhos, 33% das matrizes apresentaram mastite e lesões cutâneas crostosas e purulentas. Estes animais apresentavam-se entre 10 a- 12 meses de idade e em segunda parição. Quinze coelhos afetados foram sacrificados e necropsiados. Na necropsia, além das lesões cutâneas haviam microabscessos em diversos órgãos. Das amostras coletadas isolou-se Staphylococcus aureus subesp. aureus. S. aureus subesp. aureus também foi isolado de "swab" nasal coletado do tratador e...

  4. S. aureus colonization at ICU admission as a risk factor for developing S. aureus ICU pneumonia

    NARCIS (Netherlands)

    Paling, Fleur P; Wolkewitz, Martin; Bode, Lonneke G M; Klein Klouwenberg, Peter M C; Ong, David S Y; Depuydt, Pieter; de Bus, Liesbet; Sifakis, Frangiscos; Bonten, Marc J M; Kluijtmans, Jan

    OBJECTIVE: To quantify the incidence of intensive care unit (ICU) acquired pneumonia caused by Staphylococcus aureus (S. aureus) and its association with S. aureus colonization at ICU admission. METHODS: This was a post-hoc analysis of two cohort studies in critically ill patients. The primary

  5. Mild Staphylococcus aureus Skin Infection Improves the Course of Subsequent Endogenous S. aureus Bacteremia in Mice.

    Science.gov (United States)

    van den Berg, Sanne; de Vogel, Corné P; van Belkum, Alex; Bakker-Woudenberg, Irma A J M

    2015-01-01

    Staphylococcus aureus carriers with S. aureus bacteremia may have a reduced mortality risk compared to non-carriers. A role for the immune system is suggested. Here, we study in mice the effect of mild S. aureus skin infection prior to endogenous or exogenous S. aureus bacteremia, and evaluate protection in relation to anti-staphylococcal antibody levels. Skin infections once or twice by a clinical S. aureus isolate (isolate P) or S. aureus strain 8325-4 were induced in mice free of S. aureus and anti-staphylococcal antibodies. Five weeks later, immunoglobulin G (IgG) levels in blood against 25 S. aureus antigens were determined, and LD50 or LD100 bacteremia caused by S. aureus isolate P was induced. S. aureus skin infections led to elevated levels of anti-staphylococcal IgG in blood. One skin infection improved the course of subsequent severe endogenous bacteremia only. A second skin infection further improved animal survival rate, which was associated with increased pre-bacteremia IgG levels against Efb, IsaA, LukD, LukE, Nuc, PrsA and WTA. In conclusion, S. aureus isolate P skin infection in mice reduces the severity of subsequent endogenous S. aureus bacteremia only. Although cellular immune effects cannot be rules out, anti-staphylococcal IgG against specified antigens may contribute to this effect.

  6. Mild Staphylococcus aureus Skin Infection Improves the Course of Subsequent Endogenous S. aureus Bacteremia in Mice.

    Directory of Open Access Journals (Sweden)

    Sanne van den Berg

    Full Text Available Staphylococcus aureus carriers with S. aureus bacteremia may have a reduced mortality risk compared to non-carriers. A role for the immune system is suggested. Here, we study in mice the effect of mild S. aureus skin infection prior to endogenous or exogenous S. aureus bacteremia, and evaluate protection in relation to anti-staphylococcal antibody levels. Skin infections once or twice by a clinical S. aureus isolate (isolate P or S. aureus strain 8325-4 were induced in mice free of S. aureus and anti-staphylococcal antibodies. Five weeks later, immunoglobulin G (IgG levels in blood against 25 S. aureus antigens were determined, and LD50 or LD100 bacteremia caused by S. aureus isolate P was induced. S. aureus skin infections led to elevated levels of anti-staphylococcal IgG in blood. One skin infection improved the course of subsequent severe endogenous bacteremia only. A second skin infection further improved animal survival rate, which was associated with increased pre-bacteremia IgG levels against Efb, IsaA, LukD, LukE, Nuc, PrsA and WTA. In conclusion, S. aureus isolate P skin infection in mice reduces the severity of subsequent endogenous S. aureus bacteremia only. Although cellular immune effects cannot be rules out, anti-staphylococcal IgG against specified antigens may contribute to this effect.

  7. Active immunization with an octa-valent Staphylococcus aureus antigen mixture in models of S. aureus bacteremia and skin infection in mice.

    Directory of Open Access Journals (Sweden)

    Sanne van den Berg

    Full Text Available Proteomic studies with different Staphylococcus aureus isolates have shown that the cell surface-exposed and secreted proteins IsaA, LytM, Nuc, the propeptide of Atl (pro-Atl and four phenol-soluble modulins α (PSMα are invariantly produced by this pathogen. Therefore the present study was aimed at investigating whether these proteins can be used for active immunization against S. aureus infection in mouse models of bacteremia and skin infection. To this end, recombinant His-tagged fusions of IsaA, LytM, Nuc and pro-Atl were isolated from Lactococcus lactis or Escherichia coli, while the PSMα1-4 peptides were chemically synthesized. Importantly, patients colonized by S. aureus showed significant immunoglobulin G (IgG responses against all eight antigens. BALB/cBYJ mice were immunized subcutaneously with a mixture of the antigens at day one (5 μg each, and boosted twice (25 μg of each antigen with 28 days interval. This resulted in high IgG responses against all antigens although the response against pro-Atl was around one log lower compared to the other antigens. Compared to placebo-immunized mice, immunization with the octa-valent antigen mixture did not reduce the S. aureus isolate P load in blood, lungs, spleen, liver, and kidneys in a bacteremia model in which the animals were challenged for 14 days with a primary load of 3 × 10(5 CFU. Discomfort scores and animal survival rates over 14 days did not differ between immunized mice and placebo-immunized mice upon bacteremia with S. aureus USA300 (6 × 10(5 CFU. In addition, this immunization did not reduce the S. aureus isolate P load in mice with skin infection. These results show that the target antigens are immunogenic in both humans and mice, but in the used animal models do not result in protection against S. aureus infection.

  8. Active Immunization with an Octa-Valent Staphylococcus aureus Antigen Mixture in Models of S. aureus Bacteremia and Skin Infection in Mice

    Science.gov (United States)

    van den Berg, Sanne; Koedijk, Dennis G. A. M.; Back, Jaap Willem; Neef, Jolanda; Dreisbach, Annette; van Dijl, Jan Maarten; Bakker-Woudenberg, Irma A. J. M.; Buist, Girbe

    2015-01-01

    Proteomic studies with different Staphylococcus aureus isolates have shown that the cell surface-exposed and secreted proteins IsaA, LytM, Nuc, the propeptide of Atl (pro-Atl) and four phenol-soluble modulins α (PSMα) are invariantly produced by this pathogen. Therefore the present study was aimed at investigating whether these proteins can be used for active immunization against S. aureus infection in mouse models of bacteremia and skin infection. To this end, recombinant His-tagged fusions of IsaA, LytM, Nuc and pro-Atl were isolated from Lactococcus lactis or Escherichia coli, while the PSMα1-4 peptides were chemically synthesized. Importantly, patients colonized by S. aureus showed significant immunoglobulin G (IgG) responses against all eight antigens. BALB/cBYJ mice were immunized subcutaneously with a mixture of the antigens at day one (5 μg each), and boosted twice (25 μg of each antigen) with 28 days interval. This resulted in high IgG responses against all antigens although the response against pro-Atl was around one log lower compared to the other antigens. Compared to placebo-immunized mice, immunization with the octa-valent antigen mixture did not reduce the S. aureus isolate P load in blood, lungs, spleen, liver, and kidneys in a bacteremia model in which the animals were challenged for 14 days with a primary load of 3 × 105 CFU. Discomfort scores and animal survival rates over 14 days did not differ between immunized mice and placebo-immunized mice upon bacteremia with S. aureus USA300 (6 × 105 CFU). In addition, this immunization did not reduce the S. aureus isolate P load in mice with skin infection. These results show that the target antigens are immunogenic in both humans and mice, but in the used animal models do not result in protection against S. aureus infection. PMID:25710376

  9. Modulation of Drug Resistance in Staphylococcus aureus with Coumarin Derivatives

    Directory of Open Access Journals (Sweden)

    Rodrigo Santos Aquino de Araújo

    2016-01-01

    Full Text Available Semisynthetic and commercial coumarins were investigated for their antibacterial and adjuvant properties with antibiotic agents against norfloxacin, erythromycin, and tetracycline resistant Staphylococcus aureus as based on efflux mechanisms. The coumarins and certain commercial antibiotics had their Minimum Inhibitory Concentrations determined by broth microdilution assay against resistant S. aureus strains which overexpress efflux pump proteins. For evaluation of the modulatory activity, the antibiotics MICs were determined in the presence of the coumarin derivatives at subinhibitory concentration. Although the coumarins did not display relevant antibacterial activity (MIC ≥ 128 µg/mL, they did modulate the antibiotics activities. Various coumarins, especially the alkylated derivatives in combination with antibiotics at subinhibitory concentrations, modulated antibiotic activity, reducing the MIC for tetracycline and norfloxacin by 2 to 8 times. Polar Surface Area (PSA studies were performed and the fact that the presence of apolar groups is an important factor for the modulatory activity of coumarins was corroborated. Docking on the Penicillin-Binding Protein from MRSA identified that 18 is a potential ligand presenting low Ebinding. The results indicate that coumarin derivatives modulated antibiotic resistance and may be used as potential antibiotic adjuvants, acting by bacterial efflux pump inhibition in S. aureus.

  10. Inhibitory effects of antibiofilm compound 1 against Staphylococcus aureus biofilms.

    Science.gov (United States)

    Shrestha, Looniva; Kayama, Shizuo; Sasaki, Michiko; Kato, Fuminori; Hisatsune, Junzo; Tsuruda, Keiko; Koizumi, Kazuhisa; Tatsukawa, Nobuyuki; Yu, Liansheng; Takeda, Kei; Sugai, Motoyuki

    2016-03-01

    A novel benzimidazole molecule that was identified in a small-molecule screen and is known as antibiofilm compound 1 (ABC-1) has been found to prevent bacterial biofilm formation by multiple bacterial pathogens, including Staphylococcus aureus, without affecting bacterial growth. Here, the biofilm inhibiting ability of 156 μM ABC-1 was tested in various biofilm-forming strains of S. aureus. It was demonstrated that ABC-1 inhibits biofilm formation by these strains at micromolar concentrations regardless of the strains' dependence on Polysaccharide Intercellular Adhesin (PIA), cell wall-associated protein dependent or cell wall- associated extracellular DNA (eDNA). Of note, ABC-1 treatment primarily inhibited Protein A (SpA) expression in all strains tested. spa gene disruption showed decreased biofilm formation; however, the mutants still produced more biofilm than ABC-1 treated strains, implying that ABC-1 affects not only SpA but also other factors. Indeed, ABC-1 also attenuated the accumulation of PIA and eDNA on cell surface. Our results suggest that ABC-1 has pleotropic effects on several biofilm components and thus inhibits biofilm formation by S. aureus.

  11. Biochemical characters and antibiotic susceptibility of Staphylococcus aureus isolates

    Institute of Scientific and Technical Information of China (English)

    Subhankari Prasad Chakraborty; Santanu Kar Mahapatra; Somenath Roy

    2011-01-01

    Objective: To observe the biochemical characters and antibiotic susceptibility of isolated Staphylococcus aureus (S. auerus) strains against some conventional and traditional antibiotics.Methods:Bacterial culture was done in Mueller-Hinton broth at 37 ℃. Characters of these strains were determined by traditional biochemical tests such as hydrolysis test of gelatin, urea, galactose, starch and protein, and fermentation of lactose and sucrose. Antibiotic susceptibility were carried out by minimum inhibitory concentration test, minium bactericidal concentration test, disc agar diffusion test and brain heart infusion oxacillin screening agar. Results: From this study, it was observed that 100% S. aureus isolates showed positive results in gelatin, urea and galactose hydrolysis test, 50% isolates were positive in starch hydrolysis test, 35% in protein hydrolysis test, 100% isolates in lactose fermenting test, but no isolate was positive in sucrose fermenting test. Antibiotic susceptibility testing suggested that 20% of isolates were resistant to kanamycin and 46.67% were resistant to oxacillin. Conclusions: These findings show that all these isolates have gelatin, urea, galactose hydrolysis and lactose fermenting activity. 20% of these isolates were resistant to kanamycin and 46.67% were resistant to oxacillin. Thirty post operative pathogenic isolated S. aureus strains were used in this study.

  12. Characterisation of Staphylococcus aureus bacteraemia at Tygerberg hospital

    NARCIS (Netherlands)

    Orth, H.; Dreyer, Z.S.; Makgotlho, E.; Oosthuysen, W.; Sinha, B.; Wasserman, E.

    2013-01-01

    To elucidate the local epidemiology of Staphylococcus aureus bacteraemia, we characterised blood culture isolates using molecular methods and prospectively collected clinical data to determine the occurrence of community-acquired, methicillinresistant S. aureus (MRSA). Consecutive S. aureus blood cu

  13. Characterisation of Staphylococcus aureus bacteraemia at Tygerberg hospital

    NARCIS (Netherlands)

    Orth, H.; Dreyer, Z.S.; Makgotlho, E.; Oosthuysen, W.; Sinha, B.; Wasserman, E.

    2013-01-01

    To elucidate the local epidemiology of Staphylococcus aureus bacteraemia, we characterised blood culture isolates using molecular methods and prospectively collected clinical data to determine the occurrence of community-acquired, methicillinresistant S. aureus (MRSA). Consecutive S. aureus blood cu

  14. An Aromatic Hydroxyamide Attenuates Multiresistant Staphylococcus aureus Toxin Expression.

    Science.gov (United States)

    Vomacka, Jan; Korotkov, Vadim S; Bauer, Bianca; Weinandy, Franziska; Kunzmann, Martin H; Krysiak, Joanna; Baron, Oliver; Böttcher, Thomas; Lorenz-Baath, Katrin; Sieber, Stephan A

    2016-01-26

    Methicillin-resistant Staphylococcus aureus (MRSA) causes severe infections with only few effective antibiotic therapies currently available. To approach this challenge, chemical entities with a novel and resistance-free mode of action are desperately needed. Here, we introduce a new hydroxyamide compound that effectively reduces the expression of devastating toxins in various S. aureus and MRSA strains. The molecular mechanism was investigated by transcriptome analysis as well as by affinity-based protein profiling. Down-regulation of several pathogenesis associated genes suggested the inhibition of a central virulence-related pathway. Mass spectrometry-based chemical proteomics revealed putative molecular targets. Systemic treatment with the hydroxyamide showed significant reduction of abscess sizes in a MRSA mouse skin infection model. The absence of resistance development in vitro further underlines the finding that targeting virulence could lead to prolonged therapeutic options in comparison to antibiotics that directly address bacterial survival.

  15. Antibacterial Activity of Shikimic Acid from Pine Needles of Cedrus deodara against Staphylococcus aureus through Damage to Cell Membrane.

    Science.gov (United States)

    Bai, Jinrong; Wu, Yanping; Liu, Xiaoyan; Zhong, Kai; Huang, Yina; Gao, Hong

    2015-11-13

    Shikimic acid (SA) has been reported to possess antibacterial activity against Staphylococcus aureus, whereas the mode of action of SA is still elusive. In this study, the antibacterial activity and mechanism of SA toward S. aureus by cell membrane damage was investigated. After SA treatment, massive K+ and nucleotide leakage from S. aureus, and a significant change in the membrane potential was observed, suggesting SA may act on the membrane by destroying the cell membrane permeability. Through transmission electron microscopic observations we further confirmed that SA can disrupt the cell membrane and membrane integrity. Meanwhile, SA was found to be capable of reducing the membrane fluidity of the S. aureus cell. Moreover, the fluorescence experiments indicated that SA could quench fluorescence of Phe residues of the membrane proteins, thus demonstrating that SA can bind to S. aureus membrane proteins. Therefore, these results showed the antibacterial activity of SA against S. aureus could be caused by the interactions of SA with S. aureus membrane proteins and lipids, resulting in causing cell membrane dysfunction and bacterial damage or even death. This study reveals the potential use of SA as an antibacterial agent.

  16. Evaluation of Two New Chromogenic Media, CHROMagar MRSA and S. aureus ID, for Identifying Staphylococcus aureus and Screening Methicillin-Resistant S. aureus

    OpenAIRE

    Hedin, Göran; Fang,Hong

    2005-01-01

    Thirty-nine methicillin-resistant Staphylococcus aureus (MRSA) isolates with diverse genetic backgrounds and two reference strains were correctly identified as S. aureus on CHROMagar MRSA and S. aureus ID media. Growth inhibition on CHROMagar MRSA was noted. A combination of cefoxitin disk and S. aureus ID was found suitable for rapid MRSA screening.

  17. Protein

    Science.gov (United States)

    ... Food Service Resources Additional Resources About FAQ Contact Protein Protein is found throughout the body—in muscle, ... the heart and respiratory system, and death. All Protein Isn’t Alike Protein is built from building ...

  18. Evaluation of S. aureus ID, a New Chromogenic Agar Medium for Detection of Staphylococcus aureus

    OpenAIRE

    Perry, John D.; Rennison, Claire; Butterworth, Lynne A.; Hopley, Andrew L. J.; Gould, F. Kate

    2003-01-01

    S. aureus ID (bioMérieux, La Balme Les Grottes, France) is a new chromogenic agar medium designed to enable the isolation of staphylococci and the specific identification of Staphylococcus aureus. S. aureus produces green colonies on this medium due to production of α-glucosidase. To evaluate this medium, a total of 350 wound swabs were cultured onto S. aureus ID, CHROMagar Staph. aureus, and conventional media routinely used in our laboratory. After 18 to 20 h of incubation, 96.8% of strains...

  19. Metabolic activity, urease production, antibiotic resistance and virulence in dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus

    Science.gov (United States)

    Vandecandelaere, Ilse; Van Nieuwerburgh, Filip; Deforce, Dieter

    2017-01-01

    In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other’s behavior, but additional studies are required necessary to elucidate the exact

  20. Metabolic activity, urease production, antibiotic resistance and virulence in dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus.

    Science.gov (United States)

    Vandecandelaere, Ilse; Van Nieuwerburgh, Filip; Deforce, Dieter; Coenye, Tom

    2017-01-01

    In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact

  1. C-jun N-terminal Kinase-mediated Signaling Is Essential for Staphylococcus Aureus-induced U937 Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Jia-he Wang; Bo Yu; Hui-yan Niu; Hui Li; Yi Zhang; Xin Wang; Ping He

    2009-01-01

    Objective To investigate the effect of SP600125, a specific c-jun N-terminal protein kinase (JNK) inhibitor, on Staphylococcus aureus (S. aureus)-induced U937 cell death and the underlying mechanism. Methods The human monocytic U937 cells were treated with S. aureus at different time with or without SP600125. Cell apoptosis was analyzed by flow cytometry. JNK, Bax, and caspase-3 activities were detected by Western blotting. Results S. aureus induced apoptosis in cultured U937 cells in a time-dependent manner. Expression of Bax and phospho-JNK significantly increased in S. aureus-treated U937 cells, and the level of activated caspase-3 also increased in a time-dependent manner. Inhibition of JNK with SP600125 significantly inhibited S. aureus-induced apoptosis in U937 cells. Conclusions S. aureus can induce apoptosis in U937 cells by phosphorylation of JNK and activation of Bax and caspase-3. SP600125 protects U937 cells from apoptosis induced by S. aureus via inhibiting the activity of JNK.

  2. Montanide ISA 71 VG is Advantageous to Freund's Adjuvant in Immunization Against S. aureus Infection of Mice.

    Science.gov (United States)

    Klimka, A; Michels, L; Glowalla, E; Tosetti, B; Krönke, M; Krut, O

    2015-05-01

    The enormous capacity of Staphylococcus aureus to acquire antibiotic resistance makes it a permanent task to search for and to develop new anti-infectives. One of the possible approaches is the early active immunization of risk patients and animal stocks to prevent S. aureus infections. Based on a S. aureus proteome screen with S. aureus-specific human antiserum, we have previously identified several anchorless cell wall proteins to be used as novel vaccine candidates. To develop an efficient anti-S. aureus vaccine, the supplemented adjuvants Montanide(™) ISA 71 VG and ISA 206 were compared to Freund's adjuvant in terms of handling, induction of cytokine profile, triggering antigen-specific immunoglobulin production of different IgG subclasses and provision of increased survival rates in our S. aureus sepsis mouse model. Immunization with ISA 71 VG in comparison with Freund's adjuvant induced slightly delayed but comparably strong increase of antigen-specific antibody titres and conferred protective effect against S. aureus challenge. In contrast using ISA 206 as adjuvant, significantly lower IgG titres and consequently, no protective effect against S. aureus infection were observed. Handling and tolerability of the Montanide is superior to Freund's adjuvant. Montanide(™) ISA 71 VG can serve as an effective adjuvant replacement for Freund's adjuvant in research with a prospective usage in animal and human vaccines against bacterial pathogens. © 2015 John Wiley & Sons Ltd.

  3. Active Immunization with an Octa-Valent Staphylococcus aureus Antigen Mixture in Models of S. aureus Bacteremia and Skin Infection in Mice

    NARCIS (Netherlands)

    van den Berg, Sanne; Koedijk, Dennis G. A. M.; Back, Jaap Willem; Neef, Jolanda; Dreisbach, Annette; van Dijl, Jan Maarten; Bakker-Woudenberg, Irma A. J. M.; Buist, Girbe

    2015-01-01

    Proteomic studies with different Staphylococcus aureus isolates have shown that the cell surface-exposed and secreted proteins IsaA, LytM, Nuc, the propeptide of Atl (pro-Atl) and four phenol-soluble modulins alpha (PSM alpha) are invariantly produced by this pathogen. Therefore the present study wa

  4. Active immunization with an octa-valent Staphylococcus aureus antigen mixture in models of S. aureus bacteremia and skin infection in mice

    NARCIS (Netherlands)

    Van Den Berg, S. (Sanne); Koedijk, D.G.A.M. (Dennis G. A. M.); Back, J.W. (Jaap Willem); J. Neef (Jolanda); A. Dreisbach (Annette); J.M. Dijl (Jan Maarten); I.A.J.M. Bakker-Woudenberg (Irma); G. Buist (Girbe)

    2015-01-01

    textabstractProteomic studies with different Staphylococcus aureus isolates have shown that the cell surface-exposed and secreted proteins IsaA, LytM, Nuc, the propeptide of Atl (pro-Atl) and four phenol-soluble modulins α (PSMα) are invariantly produced by this pathogen. Therefore the present study

  5. Immunogenicity of toxins during Staphylococcus aureus infection

    NARCIS (Netherlands)

    N.J. Verkaik (Nelianne); O. Dauwalder (Olivier); K. Antri (Kenza); I. Boubekri (Ilhem); C.P. de Vogel (Corné); C. Badiou (Cédric); M. Bes (Michèle); F. Vandenesch (François); M. Tazir (Mohammed); H. Hooijkaas (Herbert); H.A. Verbrugh (Henri); A.F. van Belkum (Alex); J. Etienne (Jerome); G. Lina (Gérard); N. Ramdani-Bouguessa (Nadjia); W.J.B. van Wamel (Willem)

    2010-01-01

    textabstractAB - BACKGROUND: Toxins are important Staphylococcus aureus virulence factors, but little is known about their immunogenicity during infection. Here, additional insight is generated. METHODS: Serum samples from 206 S. aureus-infected patients and 201 hospital-admitted control subjects we

  6. Chromones from an ascomycete,Chaetomium aureus

    Institute of Scientific and Technical Information of China (English)

    Li Mei Li; Qiang Zou; Guo You Li

    2010-01-01

    A novel chromone,named chaetoaurin (1),along with six known chromone derivatives (2-7),was isolated from the ethyl acetate extract of a solid-state fermented culture of Chaetomium aureus.Their structures were elucidated by extensive spectral analysis.All of these compounds were reported from C.aureus for the first time.

  7. Staphylococcus aureus and hand eczema severity

    DEFF Research Database (Denmark)

    Haslund, P; Bangsgaard, N; Jarløv, J O

    2009-01-01

    BACKGROUND: The role of bacterial infections in hand eczema (HE) remains to be assessed. OBJECTIVES: To determine the prevalence of Staphylococcus aureus in patients with HE compared with controls, and to relate presence of S. aureus, subtypes and toxin production to severity of HE. METHODS......: Bacterial swabs were taken at three different visits from the hand and nose in 50 patients with HE and 50 controls. Staphylococcus aureus was subtyped by spa typing and assigned to clonal complexes (CCs), and isolates were tested for exotoxin-producing S. aureus strains. The Hand Eczema Severity Index...... was used for severity assessment. RESULTS: Staphylococcus aureus was found on the hands in 24 patients with HE and four controls (P hands...

  8. Transfer of Antibiotic Resistance in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Haaber, Jakob; Penadés, José R; Ingmer, Hanne

    2017-01-01

    Staphylococcus aureus is a serious human pathogen with remarkable adaptive powers. Antibiotic-resistant clones rapidly emerge mainly by acquisition of antibiotic-resistance genes from other S. aureus strains or even from other genera. Transfer is mediated by a diverse complement of mobile genetic...... of plasmids that can be transferred by conjugation and the efficiency with which transduction occurs. Here, we review the main routes of antibiotic resistance gene transfer in S. aureus in the context of its biology as a human commensal and a life-threatening pathogen. Staphylococcus aureus cells...... are effective in exchanging mobile genetic elements, including antibiotic-resistance genes.During colonization or infection of host organisms, the exchange appears to be particularly effective.Bacteriophage-mediated transfer involves both transduction and autotransduction, which may enable lysogenic S. aureus...

  9. Rapid detection and semi-quantification of IgG-accessible Staphylococcus aureus surface-associated antigens using a multiplex competitive Luminex assay

    NARCIS (Netherlands)

    Hansenova Manaskova, S.; Bikker, F.J.; Veerman, E.C.I.; van Belkum, A.; van Wamel, W.J.B.

    2013-01-01

    The surface characterization of Staphylococcus aureus is currently labor intensive and time consuming. Therefore, we developed a novel method for the rapid yet comprehensive characterization of S. aureus cell-surface-associated proteins and carbohydrates, based on a competitive Luminex assay. In thi

  10. Regulation of Expression of Oxacillin-Inducible Methionine Sulfoxide Reductases in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Kyle R. Baum

    2015-01-01

    Full Text Available Cell wall-active antibiotics cause induction of a locus that leads to elevated synthesis of two methionine sulfoxide reductases (MsrA1 and MsrB in Staphylococcus aureus. To understand the regulation of this locus, reporter strains were constructed by integrating a DNA fragment consisting of the msrA1/msrB promoter in front of a promoterless lacZ gene in the chromosome of wild-type and MsrA1-, MsrB-, MsrA1/MsrB-, and SigB-deficient methicillin-sensitive S. aureus strain SH1000 and methicillin-resistant S. aureus strain COL. These reporter strains were cultured in TSB and the cellular levels of β-galactosidase activity in these cultures were assayed during different growth phases. β-galactosidase activity assays demonstrated that the lack of MsrA1, MsrB, and SigB upregulated the msrA1/msrB promoter in S. aureus strain SH1000. In S. aureus strain COL, the highest level of β-galactosidase activity was observed under the conditions when both MsrA1 and MsrB proteins were absent. The data suggest that the msrA1/msrB locus, in part, is negatively regulated by MsrA1, MsrB, and SigB in S. aureus.

  11. Pseudomonas aeruginosa Alters Staphylococcus aureus Sensitivity to Vancomycin in a Biofilm Model of Cystic Fibrosis Infection.

    Science.gov (United States)

    Orazi, Giulia; O'Toole, George A

    2017-07-18

    The airways of cystic fibrosis (CF) patients have thick mucus, which fosters chronic, polymicrobial infections. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent respiratory pathogens in CF patients. In this study, we tested whether P. aeruginosa influences the susceptibility of S. aureus to frontline antibiotics used to treat CF lung infections. Using our in vitro coculture model, we observed that addition of P. aeruginosa supernatants to S. aureus biofilms grown either on epithelial cells or on plastic significantly decreased the susceptibility of S. aureus to vancomycin. Mutant analyses showed that 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), a component of the P. aeruginosa Pseudomonas quinolone signal (PQS) system, protects S. aureus from the antimicrobial activity of vancomycin. Similarly, the siderophores pyoverdine and pyochelin also contribute to the ability of P. aeruginosa to protect S. aureus from vancomycin, as did growth under anoxia. Under our experimental conditions, HQNO, P. aeruginosa supernatant, and growth under anoxia decreased S. aureus growth, likely explaining why this cell wall-targeting antibiotic is less effective. P. aeruginosa supernatant did not confer additional protection to slow-growing S. aureus small colony variants. Importantly, P. aeruginosa supernatant protects S. aureus from other inhibitors of cell wall synthesis as well as protein synthesis-targeting antibiotics in an HQNO- and siderophore-dependent manner. We propose a model whereby P. aeruginosa causes S. aureus to shift to fermentative growth when these organisms are grown in coculture, leading to reduction in S. aureus growth and decreased susceptibility to antibiotics targeting cell wall and protein synthesis.IMPORTANCE Cystic fibrosis (CF) lung infections are chronic and difficult to eradicate. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent respiratory pathogens in CF patients and are

  12. Laboratory Mice Are Frequently Colonized with Staphylococcus aureus and Mount a Systemic Immune Response-Note of Caution for In vivo Infection Experiments.

    Science.gov (United States)

    Schulz, Daniel; Grumann, Dorothee; Trübe, Patricia; Pritchett-Corning, Kathleen; Johnson, Sarah; Reppschläger, Kevin; Gumz, Janine; Sundaramoorthy, Nandakumar; Michalik, Stephan; Berg, Sabine; van den Brandt, Jens; Fister, Richard; Monecke, Stefan; Uy, Benedict; Schmidt, Frank; Bröker, Barbara M; Wiles, Siouxsie; Holtfreter, Silva

    2017-01-01

    Whether mice are an appropriate model for S. aureus infection and vaccination studies is a matter of debate, because they are not considered as natural hosts of S. aureus. We previously identified a mouse-adapted S. aureus strain, which caused infections in laboratory mice. This raised the question whether laboratory mice are commonly colonized with S. aureus and whether this might impact on infection experiments. Publicly available health reports from commercial vendors revealed that S. aureus colonization is rather frequent, with rates as high as 21% among specific-pathogen-free mice. In animal facilities, S. aureus was readily transmitted from parents to offspring, which became persistently colonized. Among 99 murine S. aureus isolates from Charles River Laboratories half belonged to the lineage CC88 (54.5%), followed by CC15, CC5, CC188, and CC8. A comparison of human and murine S. aureus isolates revealed features of host adaptation. In detail, murine strains lacked hlb-converting phages and superantigen-encoding mobile genetic elements, and were frequently ampicillin-sensitive. Moreover, murine CC88 isolates coagulated mouse plasma faster than human CC88 isolates. Importantly, S. aureus colonization clearly primed the murine immune system, inducing a systemic IgG response specific for numerous S. aureus proteins, including several vaccine candidates. Phospholipase C emerged as a promising test antigen for monitoring S. aureus colonization in laboratory mice. In conclusion, laboratory mice are natural hosts of S. aureus and therefore, could provide better infection models than previously assumed. Pre-exposure to the bacteria is a possible confounder in S. aureus infection and vaccination studies and should be monitored.

  13. Nanoscale Plasma Coating Inhibits Formation of Staphylococcus aureus Biofilm.

    Science.gov (United States)

    Xu, Yuanxi; Jones, John E; Yu, Haiqing; Yu, Qingsong; Christensen, Gordon D; Chen, Meng; Sun, Hongmin

    2015-12-01

    Staphylococcus aureus commonly infects medical implants or devices, with devastating consequences for the patient. The infection begins with bacterial attachment to the device, followed by bacterial multiplication over the surface of the device, generating an adherent sheet of bacteria known as a biofilm. Biofilms resist antimicrobial therapy and promote persistent infection, making management difficult to futile. Infections might be prevented by engineering the surface of the device to discourage bacterial attachment and multiplication; however, progress in this area has been limited. We have developed a novel nanoscale plasma coating technology to inhibit the formation of Staphylococcus aureus biofilms. We used monomeric trimethylsilane (TMS) and oxygen to coat the surfaces of silicone rubber, a material often used in the fabrication of implantable medical devices. By quantitative and qualitative analysis, the TMS/O2 coating significantly decreased the in vitro formation of S. aureus biofilms; it also significantly decreased in vivo biofilm formation in a mouse model of foreign-body infection. Further analysis demonstrated TMS/O2 coating significantly changed the protein adsorption, which could lead to reduced bacterial adhesion and biofilm formation. These results suggest that TMS/O2 coating can be used to effectively prevent medical implant-related infections.

  14. Heterogeneity of host TLR2 stimulation by Staphylocoocus aureus isolates.

    Directory of Open Access Journals (Sweden)

    Dina Hilmi

    Full Text Available High lipoprotein expression and potent activation of host Toll-like receptor-2 (TLR2 are characteristic features of the staphylococcal species. Expression of TLR2 in the host is important for clearance of Staphylococcus aureus infection and host survival. Thus, we hypothesized that bacterial regulation of its intrinsic TLR2-stimulatory capacity could represent a means for immune evasion or host adaptation. We, therefore, compared clinical S. aureus isolates in regards to their TLR2 activation potential and assessed the bacterial factors that modulate TLR2-mediated recognition. S. aureus isolates displayed considerable variability in TLR2-activity with low to absent TLR2-activity in 64% of the isolates tested (68/106. Notably, strain-specific TLR2-activity was independent of the strain origin, e.g. no differences were found between strains isolated from respiratory specimen from cystic fibrosis patients or those isolated from invasive disease specimen. TLR2-activity correlated with protein A expression but not with the agr status. Capsule expression and small colony variant formation had a negative impact on TLR2-activity but any disruption of cell wall integrity enhanced TLR2 activation. Altogether, heterogeneity in host TLR2-activity reflects differences in metabolic activity and cell wall synthesis and/or remodeling.

  15. Rot is a key regulator of Staphylococcus aureus biofilm formation

    Science.gov (United States)

    Mootz, Joe M.; Benson, Meredith A.; Heim, Cortney E.; Crosby, Heidi A.; Kavanaugh, Jeffrey S.; Dunman, Paul M.; Kielian, Tammy; Torres, Victor J.; Horswill, Alexander R.

    2015-01-01

    AUTHOR SUMMARY Staphylococcus aureus is a significant cause of chronic biofilm infections on medical implants. We investigated the biofilm regulatory cascade and discovered that the repressor of toxins (Rot) is part of this pathway. A USA300 community-associated methicillin-resistant S. aureus (CA-MRSA) strain deficient in Rot was unable to form a biofilm using multiple different assays, and we found rot mutants in other strain lineages were also biofilm deficient. By performing a global analysis of transcripts and protein production controlled by Rot, we observed that all the secreted protease genes were upregulated in a rot mutant, and we hypothesized that this regulation could be responsible for the biofilm phenotype. To investigate this question, we determined that Rot bound to the protease promoters, and we observed that activity levels of these enzymes, in particular the cysteine proteases, were increased in a rot mutant. By inactivating these proteases, biofilm capacity was restored to the mutant, demonstrating they are responsible for the biofilm negative phenotype. Finally, we tested the rot mutant in a mouse catheter model of biofilm infection and observed a significant reduction in biofilm burden. Thus S. aureus uses the transcription factor Rot to repress secreted protease levels in order to build a biofilm. PMID:25612137

  16. Reversal of methicillin resistance in Staphylococcus aureus by thioridazine

    DEFF Research Database (Denmark)

    Klitgaard, Janne K; Skov, Marianne N; Kallipolitis, Birgitte H;

    2008-01-01

    Objectives Thioridazine has been shown to reverse oxacillin resistance in methicillin-resistant Staphylococcus aureus (MRSA) in vitro. The aim of this study was to investigate whether thioridazine alone or in combination with oxacillin affects the transcription of the methicillin resistance gene...... mecA and the protein level of the encoded protein PBP2a. Methods Viability of MRSA was determined in liquid media in the presence of oxacillin or thioridazine alone or in combination. Transcription of mecA was analysed by primer extension, and the protein level of PBP2a was analysed by western...... of thioridazine in the presence of a fixed amount of oxacillin. Furthermore, the protein level of PBP2a was reduced when bacteria were treated with the combination of oxacillin and thioridazine. The two drugs also affected the mRNA level of the beta-lactamase gene, blaZ. Conclusions The present study indicates...

  17. Antibacterial Activity of Metabolites Products of Vibrio Alginolyticus Isolated from Sponge Haliclona sp. Against Staphylococcus Aureus

    Science.gov (United States)

    Nursyam, Happy

    2017-01-01

    The objective of this study was to investigate the antibacterial activity of primary and secondary metabolites from Vibrio alginoliticus isolated from sponge Haliclona sp. against Staphylococcus aureus. A descriptive method was used in this research. The antibacterial activity was analysed by paper disk method. The results showed that the primary metabolites produced by Vibrio alginoliticus that is in symbiosis with sponge Haliclona sp. were able to effectively inhibit Staphylococcus aureus growth with an inhibition zone diameter of 12.9 mm, while the secondary metabolites of 9.9 mm. Electrophoresis analysis of the primary metabolites showed that there were 11 protein bands which were not found in secondary metabolites. Protein bands with low molecular weights presumably had an inhibiting effect on the growth of Staphylococcus aureus.

  18. Staphylococcus aureus Manipulates Innate Immunity through Own and Host-Expressed Proteases

    Science.gov (United States)

    Pietrocola, Giampiero; Nobile, Giulia; Rindi, Simonetta; Speziale, Pietro

    2017-01-01

    Neutrophils, complement system and skin collectively represent the main elements of the innate immune system, the first line of defense of the host against many common microorganisms. Bacterial pathogens have evolved strategies to counteract all these defense activities. Specifically, Staphylococcus aureus, a major human pathogen, secretes a variety of immune evasion molecules including proteases, which cleave components of the innate immune system or disrupt the integrity of extracellular matrix and intercellular connections of tissues. Additionally, S. aureus secretes proteins that can activate host zymogens which, in turn, target specific defense components. Secreted proteins can also inhibit the anti-bacterial function of neutrophils or complement system proteases, potentiating S. aureus chances of survival. Here, we review the current understanding of these proteases and modulators of host proteases in the functioning of innate immunity and describe the importance of these mechanisms in the pathology of staphylococcal diseases. PMID:28529927

  19. Staphylococcus aureus resistente a vancomicina.

    Directory of Open Access Journals (Sweden)

    Carlos Andrés Rodríguez

    2005-12-01

    Full Text Available Objetivo. Revisar la evolución y mecanismos moleculares de la resistencia de Staphylococcus aureus a vancomicina. Fuente de los datos. Se consultó la base de datos MEDLINE y se seleccionaron artículos tipo reportes de caso, estudios bioquímicos, de microscopía electrónica y biología molecular pertinentes. Síntesis. Después de casi 40 años de eficacia ininterrumpida de la vancomicina, en 1997 se reportaron los primeros casos de fracaso terapéutico debido a cepas de Staphylococcus aureus con resistencia intermedia, denominadas VISA (concentración inhibitoria mínima, CIM, 8 a 16 ?g/ml, así como a cepas con resistencia heterogénea hVISA (CIM global = 4 ?g/ml, pero con subpoblaciones VISA, en las cuales la resistencia está mediada por engrosamiento de la pared celular y disminución de su entrecruzamiento, lo que afecta la llegada del antibiótico al blanco principal, los monómeros del peptidoglicano en la membrana plasmática. En 2002 se aisló la primera de las 3 cepas reportadas hasta la fecha con resistencia total al antibiótico, denominadas VRSA (CIM>32 ?g/ml, en las que se encontró el transposón Tn1546 proveniente de Enterococcus spp, responsable del reemplazo de la terminación D-Ala-D-Ala por D-Ala-Dlactato en los precursores de la pared celular con pérdida de la afinidad por el glicopéptido. Conclusiones. La resistencia a vancomicina es una realidad en S. aureus, mediada en el caso de VISA por alteraciones en la pared celular que atrapan el antibiótico antes de llegar al sitio de acción, y en el caso de VRSA, por transferencia desde Enterococcus spp. de genes que llevan a la modificación del blanco molecular.

  20. Applying Convergent Immunity to Innovative Vaccines Targeting Staphylococcus aureus

    Science.gov (United States)

    Yeaman, Michael R.; Filler, Scott G.; Schmidt, Clint S.; Ibrahim, Ashraf S.; Edwards, John E.; Hennessey, John P.

    2014-01-01

    Recent perspectives forecast a new paradigm for future “third generation” vaccines based on commonalities found in diverse pathogens or convergent immune defenses to such pathogens. For Staphylococcus aureus, recurring infections and a limited success of vaccines containing S. aureus antigens imply that native antigens induce immune responses insufficient for optimal efficacy. These perspectives exemplify the need to apply novel vaccine strategies to high-priority pathogens. One such approach can be termed convergent immunity, where antigens from non-target organisms that contain epitope homologs found in the target organism are applied in vaccines. This approach aims to evoke atypical immune defenses via synergistic processes that (1) afford protective efficacy; (2) target an epitope from one organism that contributes to protective immunity against another; (3) cross-protect against multiple pathogens occupying a common anatomic or immunological niche; and/or (4) overcome immune subversion or avoidance strategies of target pathogens. Thus, convergent immunity has a potential to promote protective efficacy not usually elicited by native antigens from a target pathogen. Variations of this concept have been mainstays in the history of viral and bacterial vaccine development. A more far-reaching example is the pre-clinical evidence that specific fungal antigens can induce cross-kingdom protection against bacterial pathogens. This trans-kingdom protection has been demonstrated in pre-clinical studies of the recombinant Candida albicans agglutinin-like sequence 3 protein (rAls3) where it was shown that a vaccine containing rAls3 provides homologous protection against C. albicans, heterologous protection against several other Candida species, and convergent protection against several strains of S. aureus. Convergent immunity reflects an intriguing new approach to designing and developing vaccine antigens and is considered here in the context of vaccines to target S

  1. Applying Convergent Immunity to Innovative Vaccines Targeting Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Michael R Yeaman

    2014-09-01

    Full Text Available Recent perspectives forecast a new paradigm for future 3rd generation vaccines based on commonalities found in diverse pathogens or convergent immune defenses to such pathogens. For Staphylococcus aureus, recurring infections and a limited success of vaccines containing S. aureus antigens imply that native antigens induce immune responses insufficient for optimal efficacy. These perspectives exemplify the need to apply novel vaccine strategies to high priority pathogens. One such approach can be termed convergent immunity, where antigens from non-target organisms that contain epitope homologues found in the target organism are applied in vaccines. This approach aims to evoke atypical immune defenses via synergistic processes that 1 afford protective efficacy; 2 target an epitope from one organism that contributes to protective immunity against another; 3 cross-protect against multiple pathogens occupying a common anatomic or immunologic niche; and/or 4 overcome immune subversion or avoidance strategies of target pathogens. Thus, convergent immunity has a potential to promote protective efficacy not usually elicited by native antigens from a target pathogen. Variations of this concept have been mainstays in the history of viral and bacterial vaccine development. A more far-reaching example is the pre–clinical evidence that specific fungal antigens can induce cross-kingdom protection against bacterial pathogens. This trans-kingdom protection has been demonstrated in preclinical studies of the recombinant Candida albicans agglutinin-like sequence 3 protein (rAls3 where it was shown that a vaccine containing rAls3 provides homologous protection against C. albicans, heterologous protection against several other Candida species, and convergent protection against several strains of S. aureus. Convergent immunity reflects an intriguing new approach to designing and developing vaccine antigens and is considered here in the context of vaccines to target

  2. A type IV modification-dependent restriction enzyme SauUSI from Staphylococcus aureus subsp. aureus USA300.

    Science.gov (United States)

    Xu, Shuang-Yong; Corvaglia, Anna R; Chan, Siu-Hong; Zheng, Yu; Linder, Patrick

    2011-07-01

    A gene encoding a putative DNA helicase from Staphylococcus aureus USA300 was cloned and expressed in Escherichia coli. The protein was purified to over 90% purity by chromatography. The purified enzyme, SauUSI, predominantly cleaves modified DNA containing 5mC and 5-hydroxymethylcytosine. Cleavage of 5mC-modified plasmids indicated that the sites S5mCNGS (S = C or G) are preferentially digested. The endonuclease activity requires the presence of adenosine triphosphate (ATP) or dATP whereas the non-hydrolyzable γ-S-ATP does not support activity. SauUSI activity was inhibited by ethylenediaminetetraacetic acid. It is most active in Mg(++) buffers. No companion methylase gene was found near the SauUSI restriction gene. The absence of a cognate methylase and cleavage of modified DNA indicate that SauUSI belongs to type IV restriction endonucleases, a group that includes EcoK McrBC and Mrr. SauUSI belongs to a family of highly similar homologs found in other sequenced S. aureus, S. epidermidis and S. carnosus genomes. More distant SauUSI orthologs can be found in over 150 sequenced bacterial/archaea genomes. Finally, we demonstrated the biological function of the type IV REase in restricting 5mC-modified plasmid DNA by transformation into clinical S. aureus strain SA564, and in restricting phage λ infection when the endonuclease is expressed in E. coli.

  3. Genotyping of Staphylococcus aureus in bovine mastitis and correlation to phenotypic characteristics.

    Science.gov (United States)

    Artursson, Karin; Söderlund, Robert; Liu, Lihong; Monecke, Stefan; Schelin, Jenny

    2016-09-25

    Reducing the prevalence of mastitis caused by Staphylococcus aureus (S. aureus) is essential to improve animal health and reduce economic losses for farmers. The clinical outcome of acute mastitis and risk of progression to persistent mastitis can, at least to some extent, be related to genetic variants of the strain causing the infection. In the present study we have used microarrays to investigate the presence of virulence genes in S. aureus isolates from dairy cows with acute clinical mastitis (n=70) and correlated the findings to other genotypic and phenotypic characteristics. Among the most commonly found virulence factors were genes encoding several hemolysin types, leukocidins D and lukM/lukF-P83, clumping factors A and B, fibrinogen binding protein and fibronectin-binding protein A. Some virulence factors e.g. fibronectin-binding protein B and Staphylococcus aureus surface protein G were less common. Genes coding for several staphylococcal enterotoxins and toxic shock syndrome toxin-1 (TSST-1) were commonly found, especially in one major pulsotype. No beta-lactamase genes were found in any common pulsotype, while present in some rare pulsotypes, indicated to be of human origin. Production of TSST-1, enterotoxins, hemolysins and beta-lactamase could all be positively correlated to presence of the corresponding genes. This study reveals a number of genotypic differences and similarities among common and rare pulsotypes of S. aureus from cases of mastitis in Sweden. The results could help the design of diagnostic tools to guide on-farm interventions according to the expected impact on udder health from a specific S. aureus genotype. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Antibacterial activity of staphylococcus aureus hydrolyzed from brewer's spent grains protein%啤酒糟蛋白水解物对金黄色葡萄球菌抑菌能力研究

    Institute of Scientific and Technical Information of China (English)

    宗绪岩; 李丽; 罗惠波; 刘长江

    2012-01-01

    Four proteases were used in different hydrolysis time and hydrolysis's antibacterial ability was detected. The results showed: hydrolysis has better inhibition ability of Staphylococcus aureus with Flavourzyme in 180 min. Peptides was separated and tested with pol-yamide column chromatography, ion exchange column chromatogra-phy, and gel column chromatography. Molecular weight is about 1 877. 67. The minimal inhibitory concentration for Escherichia coli was 2%.%选择4种蛋白酶制备出不同时间水解的啤酒糟蛋白水解物,分别检测其对金黄色葡萄球菌的抑茵能力.结果表明:采用Flavourzyme水解180 min的水解物具有较好的抑制金黄色葡萄球菌的能力;采用聚酰胺柱层析、离子交换柱层析方法及凝胶柱层析方法对水解物进行分离测定,得到分子量约为1 877.67的啤酒糟多肽样品,其对于金黄色葡萄球菌的最小抑菌浓度为2%.

  5. The effect of metal ions on Staphylococcus aureus revealed by biochemical and mass spectrometric analyses.

    Science.gov (United States)

    Chudobova, Dagmar; Dostalova, Simona; Ruttkay-Nedecky, Branislav; Guran, Roman; Rodrigo, Miguel Angel Merlos; Tmejova, Katerina; Krizkova, Sona; Zitka, Ondrej; Adam, Vojtech; Kizek, Rene

    2015-01-01

    In this study, we focused on the effect of heavy metal ions in resistant strains of gram-positive bacteria Staphylococcus aureus using biochemical methods and mass spectrometry. Five nitrate solutions of heavy metals (Ag(+), Cu(2+), Cd(2+), Zn(2+) and Pb(2+)) were used to create S. aureus resistant strains. Biochemical changes of resistant strains in comparison with the non-resistant control strain of S. aureus were observed by microbiological (measuring - growth curves and inhibition zones) and spectrophotometric methods (antioxidant activity and alaninaminotransferase, aspartateaminotransferase, alkaline phosphatase, γ-glutamyltransferase activities). Mass spectrometry was employed for the qualitative analysis of the samples (changes in S. aureus protein composition) and for the identification of the strains database MALDI Biotyper was employed. Alterations, in terms of biochemical properties and protein composition, were observed in resistant strains compared to non-resistant control strain. Our results describe the possible option for the analysis of S. aureus resistant strains and may thus serve as a support for monitoring of changes in genetic information caused by the forming of resistance to heavy metals.

  6. Epidemic Increase in Methicillin-resistant Staphylococcus aureus in Copenhagen

    DEFF Research Database (Denmark)

    Westh, Henrik; Boye, Kit; Bartels, Mette Damkjær;

    2006-01-01

    in 2004. All isolates have been spa-typed and epidemiologic information collected. RESULTS: The number of MRSA cases has a doubling time of about six months. The epidemic has been caused by many different MRSA types and 31 staphylococcus protein A genotypes (spa types). MRSA has caused several hospital......INTRODUCTION: We have found an epidemic increase in methicillin-resistant Staphylococcus aureus (MRSA) in Copenhagen. The increase has a complex background and involves hospitals, nursing homes and persons nursed in their own home. MATERIAL AND METHODS: We found 33 MRSA patients in 2003 and 121...

  7. Synthesis and function of phospholipids in Staphylococcus aureus.

    Science.gov (United States)

    Kuhn, Sebastian; Slavetinsky, Christoph J; Peschel, Andreas

    2015-02-01

    Phospholipids are the major components of bacterial membranes, and changes in phospholipid composition affect important cellular processes such as metabolism, stress response, antimicrobial resistance, and virulence. The most prominent phospholipids in Staphylococcus aureus are phosphatidylglycerol, lysyl-phosphatidylglycerol, and cardiolipin, whose biosynthesis is mediated by a complex protein machinery. Phospholipid composition of the staphylococcal membrane has to be continuously adjusted to changing external conditions, which is achieved by a series of transcriptional and biochemical regulatory mechanisms. This mini-review outlines the current state of knowledge concerning synthesis, regulation, and function of the major staphylococcal phospholipids.

  8. Expression and crystallization of DsbA from Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Heras, B., E-mail: b.heras@imb.uq.edu.au; Kurz, M.; Jarrott, R.; Byriel, K. A.; Jones, A. [Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane QLD 4072 (Australia); Thöny-Meyer, L. [EMPA, Abteilung Biokompatible Werkstoffe, Lerchenfeldstrasse 5, CH-9014 St Gallen (Switzerland); Martin, J. L., E-mail: b.heras@imb.uq.edu.au [Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane QLD 4072 (Australia)

    2007-11-01

    Free-interface diffusion crystallization chips were used to identify crystallization conditions for S. aureus DsbA, representing the first Gram-positive DsbA to be crystallized. Native and selenomethionine-derivative crystals diffracted to 2.1 and 2.4 Å resolution, respectively. Bacterial Dsb proteins catalyse the in vivo formation of disulfide bonds, a critical step in the stability and activity of many proteins. Most studies on Dsb proteins have focused on Gram-negative bacteria and thus the process of oxidative folding in Gram-positive bacteria is poorly understood. To help elucidate this process in Gram-positive bacteria, DsbA from Staphylococcus aureus (SaDsbA) has been focused on. Here, the expression, purification, crystallization and preliminary diffraction analysis of SaDsbA are reported. SaDsbA crystals diffract to a resolution limit of 2.1 Å and belong to the hexagonal space group P6{sub 5} or P6{sub 1}, with unit-cell parameters a = b = 72.1, c = 92.1 Å and one molecule in the asymmetric unit (64% solvent content)

  9. Mastite com lesões sistêmicas por Staphylococus aureus subesp. aureus em coelhos

    OpenAIRE

    Traverso Sandra Davi; Cunha Leonardo da; Fernandes Joaquim César Teixeira; Loretti Alexandre Paulino; Rhoden Adriana; Wunder Jr. Elsio; Driemeier David

    2003-01-01

    Em uma criação composta por 1800 coelhos, 33% das matrizes apresentaram mastite e lesões cutâneas crostosas e purulentas. Estes animais apresentavam-se entre 10 a- 12 meses de idade e em segunda parição. Quinze coelhos afetados foram sacrificados e necropsiados. Na necropsia, além das lesões cutâneas haviam microabscessos em diversos órgãos. Das amostras coletadas isolou-se Staphylococcus aureus subesp. aureus. S. aureus subesp. aureus também foi isolado de "swab" nasal coletado do tratador e...

  10. Staphylococcus aureus Survives with a Minimal Peptidoglycan Synthesis Machine but Sacrifices Virulence and Antibiotic Resistance.

    Directory of Open Access Journals (Sweden)

    Patricia Reed

    2015-05-01

    Full Text Available Many important cellular processes are performed by molecular machines, composed of multiple proteins that physically interact to execute biological functions. An example is the bacterial peptidoglycan (PG synthesis machine, responsible for the synthesis of the main component of the cell wall and the target of many contemporary antibiotics. One approach for the identification of essential components of a cellular machine involves the determination of its minimal protein composition. Staphylococcus aureus is a Gram-positive pathogen, renowned for its resistance to many commonly used antibiotics and prevalence in hospitals. Its genome encodes a low number of proteins with PG synthesis activity (9 proteins, when compared to other model organisms, and is therefore a good model for the study of a minimal PG synthesis machine. We deleted seven of the nine genes encoding PG synthesis enzymes from the S. aureus genome without affecting normal growth or cell morphology, generating a strain capable of PG biosynthesis catalyzed only by two penicillin-binding proteins, PBP1 and the bi-functional PBP2. However, multiple PBPs are important in clinically relevant environments, as bacteria with a minimal PG synthesis machinery became highly susceptible to cell wall-targeting antibiotics, host lytic enzymes and displayed impaired virulence in a Drosophila infection model which is dependent on the presence of specific peptidoglycan receptor proteins, namely PGRP-SA. The fact that S. aureus can grow and divide with only two active PG synthesizing enzymes shows that most of these enzymes are redundant in vitro and identifies the minimal PG synthesis machinery of S. aureus. However a complex molecular machine is important in environments other than in vitro growth as the expendable PG synthesis enzymes play an important role in the pathogenicity and antibiotic resistance of S. aureus.

  11. Streptokinase Treatment Reverses Biofilm-Associated Antibiotic Resistance in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Jørgensen, Nis Pedersen; Zobek, Natalia; Dreier, Cindy;

    2016-01-01

    Biofilms formed by Staphylococcus aureus is a serious complication to the use of medical implants. A central part of the pathogenesis relies on S. aureus’ ability to adhere to host extracellular matrix proteins, which adsorb to medical implants and stimulate biofilm formation. Being coagulase...

  12. The olive compound 4-hydroxytyrosol inactivates Staphyloccoccus aureus bacteria and Staphylococcal enterotoxin A (SEA)

    Science.gov (United States)

    The foodborne pathogen Staphylococcus aureus produces the virulent staphylococcal enterotoxin A (SEA), a single chain protein which consists of 233 amino acid residues with a molecular weight of 27,078 Da. SEA is a superantigen that is reported to contribute to animal (mastitis) and human (emesis, ...

  13. Rapid increase of genetically diverse methicillin-resistant Staphylococcus aureus, Copenhagen, Denmark

    DEFF Research Database (Denmark)

    Bartels, Mette Damkjaer; Boye, Kit; Rhod Larsen, Anders;

    2007-01-01

    by pulsed-field gel electrophoresis, Staphylococcus protein A (spa) typing, multilocus sequence typing, staphylococcal chromosome cassette (SCC) mec typing, and detection of Panton-Valentine leukocidin (PVL) genes. Seventy-one percent of cases were community-onset MRSA (CO-MRSA); of these, 36% had......In Copenhagen, methicillin-resistant Staphylococcus aureus (MRSA) accounted for

  14. Brief report: biomarkers of aortic vascular prosthetic graft infection in a porcine model with Staphylococcus aureus

    DEFF Research Database (Denmark)

    Langerhuus, S. N.; Tønnesen, E. K.; Jensen, K. H.;

    2010-01-01

    Aortic vascular prosthetic graft infection (AVPGI) with Staphylococcus aureus is a feared post-operative complication. This study was conducted to evaluate the clinical signs and potential biomarkers of infection in a porcine AVPGI model. The biomarkers evaluated were: C-reactive protein (CRP...

  15. Rapid Increase of Genetically Diverse Methicillin-Resistant Staphylococcus aureus, Copenhagen, Denmark

    DEFF Research Database (Denmark)

    Bartels, Mette Damkjær; Boye, Kit; Larsen, Anders Rhod;

    2007-01-01

    by pulsed-field gel electrophoresis, Staphylococcus protein A (spa) typing, multilocus sequence typing, staphylococcal chromosome cassette (SCC) mec typing, and detection of Panton-Valentine leukocidin (PVL) genes. Seventy-one percent of cases were community-onset MRSA (CO-MRSA); of these, 36% had......In Copenhagen, methicillin-resistant Staphylococcus aureus (MRSA) accounted for

  16. Identification of Secreted Exoproteome Fingerprints of Highly-Virulent and Non-Virulent Staphylococcus aureus Strains

    NARCIS (Netherlands)

    Bonar, Emilia; Wojcik, Iwona; Jankowska, Urszula; Kedracka-Krok, Sylwia; Bukowski, Michal; Polakowska, Klaudia; Lis, Marcin W; Kosecka-Strojek, Maja; Sabat, Artur J; Dubin, Grzegorz; Friedrich, Alexander W; Miedzobrodzki, Jacek; Dubin, Adam; Wladyka, Benedykt

    2016-01-01

    Staphylococcus aureus is a commensal inhabitant of skin and mucous membranes in nose vestibule but also an important opportunistic pathogen of humans and livestock. The extracellular proteome as a whole constitutes its major virulence determinant; however, the involvement of particular proteins is s

  17. Identification of Secreted Exoproteome Fingerprints of Highly-Virulent and Non-Virulent Staphylococcus aureus Strains

    NARCIS (Netherlands)

    Bonar, Emilia; Wojcik, Iwona; Jankowska, Urszula; Kedracka-Krok, Sylwia; Bukowski, Michal; Polakowska, Klaudia; Lis, Marcin W; Kosecka-Strojek, Maja; Sabat, Artur J; Dubin, Grzegorz; Friedrich, Alexander W; Miedzobrodzki, Jacek; Dubin, Adam; Wladyka, Benedykt

    2016-01-01

    Staphylococcus aureus is a commensal inhabitant of skin and mucous membranes in nose vestibule but also an important opportunistic pathogen of humans and livestock. The extracellular proteome as a whole constitutes its major virulence determinant; however, the involvement of particular proteins is

  18. Global analysis of the impact of linezolid onto virulence factor production in S. aureus USA300.

    Science.gov (United States)

    Bonn, Florian; Pané-Farré, Jan; Schlüter, Rabea; Schaffer, Marc; Fuchs, Stephan; Bernhardt, Jörg; Riedel, Katharina; Otto, Andreas; Völker, Uwe; van Dijl, Jan Maarten; Hecker, Michael; Mäder, Ulrike; Becher, Dörte

    2016-05-01

    The translation inhibitor linezolid is an antibiotic of last resort against Gram-positive pathogens including methicillin resistant strains of the nosocomial pathogen Staphylococcus aureus. Linezolid is reported to inhibit production of extracellular virulence factors, but the molecular cause is unknown. To elucidate the physiological response of S. aureus to linezolid in general and the inhibition of virulence factor synthesis in particular a holistic study was performed. Linezolid was added to exponentially growing S. aureus cells and the linezolid stress response was analyzed with transcriptomics and quantitative proteomics methods. In addition, scanning and transmission electron microscopy experiments as well as fluorescence microscopy analyses of the cellular DNA and membrane were performed. As previously observed in studies on other translation inhibitors, S. aureus adapts its protein biosynthesis machinery to the reduced translation efficiency. For example the synthesis of ribosomal proteins was induced. Also unexpected results like a decline in the amount of extracellular and membrane proteins were obtained. In addition, cell shape and size changed after linezolid stress and cell division was diminished. Finally, the chromosome was condensed after linezolid stress and lost contact to the membrane. These morphological changes cannot be explained by established theories. A new hypothesis is discussed, which suggests that the reduced amount of membrane and extracellular proteins and observed defects in cell division are due to the disintegration of transertion complexes by linezolid.

  19. Epidemiology of Methicillin-Resistant Staphylococcus aureus

    National Research Council Canada - National Science Library

    Helen W. Boucher; G. Ralph Corey

    2008-01-01

    The frequency of methicillin-resistant Staphylococcus aureus (MRSA) infections continues to grow in hospital-associated settings and, more recently, in community settings in the United States and globally...

  20. minimising antibiotic resistance to staphylococcus aureus in ...

    African Journals Online (AJOL)

    2002-11-11

    Nov 11, 2002 ... changes in the epidemiology and antibiotic resistance of S. aureus. Strategies aimed at ... There has been much interest in the media, national and ..... may resort to self-medication with consumption of inadequate doses of ...

  1. Piperine Plays an Anti-Inflammatory Role in Staphylococcus aureus Endometritis by Inhibiting Activation of NF-κB and MAPK Pathways in Mice

    Directory of Open Access Journals (Sweden)

    Wen-jun Zhai

    2016-01-01

    Full Text Available Endometritis is commonly caused by pathogenic microorganisms, including Staphylococcus aureus (S. aureus. Piperine, which is a natural medicine, has shown a variety of biological activities. To explore the effect and mechanism of piperine on S. aureus endometritis, a mouse model of S. aureus endometritis was successfully established in the present study. Histopathological changes were observed with H&E staining, cytokines were analyzed by ELISA, mRNA was analyzed by qPCR, and proteins were detected by western blot. The results showed that piperine could significantly alleviate inflammatory injury in S. aureus endometritis. The qPCR and ELISA results showed that piperine effectively reduced the S. aureus-induced overexpression of TNF-α, IL-1β, and IL-6 but increased the expression of IL-10. The S. aureus-induced inflammation was related to TLR-2 and TLR-4 because the results showed that their expression was increased in S. aureus infection but then decreased with piperine treatment. To further confirm that piperine caused an anti-inflammatory response by targeting NF-κB and MAPKs, the expression of I-κB, p65, p38, ERK, and JNK was measured. The phosphorylation of I-κB, p65, p38, ERK, and JNK was inhibited by piperine in a dose-dependent manner. All of the results indicated that piperine may be a potential anti-inflammatory drug both in endometritis and in other S. aureus-induced diseases.

  2. Deficiency of the Complement Component 3 but Not Factor B Aggravates Staphylococcus aureus Septic Arthritis in Mice.

    Science.gov (United States)

    Na, Manli; Jarneborn, Anders; Ali, Abukar; Welin, Amanda; Magnusson, Malin; Stokowska, Anna; Pekna, Marcela; Jin, Tao

    2016-04-01

    The complement system plays an essential role in the innate immune response and protection against bacterial infections. However, detailed knowledge regarding the role of complement in Staphylococcus aureus septic arthritis is still largely missing. In this study, we elucidated the roles of selected complement proteins in S. aureus septic arthritis. Mice lacking the complement component 3 (C3(-/-)), complement factor B (fB(-/-)), and receptor for C3-derived anaphylatoxin C3a (C3aR(-/-)) and wild-type (WT) control mice were intravenously or intra-articularly inoculated with S. aureus strain Newman. The clinical course of septic arthritis, as well as histopathological and radiological changes in joints, was assessed. After intravenous inoculation, arthritis severity and frequency were significantly higher in C3(-/-)mice than in WT controls, whereas fB(-/-)mice displayed intermediate arthritis severity and frequency. This was in accordance with both histopathological and radiological findings. C3, but not fB, deficiency was associated with greater weight loss, more frequent kidney abscesses, and higher bacterial burden in kidneys. S. aureus opsonized with C3(-/-)sera displayed decreased uptake by mouse peritoneal macrophages compared with bacteria opsonized with WT or fB(-/-)sera. C3aR deficiency had no effect on the course of hematogenous S. aureus septic arthritis. We conclude that C3 deficiency increases susceptibility to hematogenous S. aureus septic arthritis and impairs host bacterial clearance, conceivably due to diminished opsonization and phagocytosis of S. aureus.

  3. Vinculin and Rab5 complex is required [correction of requited]for uptake of Staphylococcus aureus and interleukin-6 expression.

    Directory of Open Access Journals (Sweden)

    Makoto Hagiwara

    Full Text Available Vinculin, a 116-kDa membrane cytoskeletal protein, is an important molecule for cell adhesion; however, little is known about its other cellular functions. Here, we demonstrated that vinculin binds to Rab5 and is required for Staphylococcus aureus (S. aureus uptake in cells. Viunculin directly bound to Rab5 and enhanced the activation of S. aureus uptake. Over-expression of active vinculin mutants enhanced S. aureus uptake, whereas over-expression of an inactive vinculin mutant decreased S. aureus uptake. Vinculin bound to Rab5 at the N-terminal region (1-258 of vinculin. Vinculin and Rab5 were involved in the S. aureus-induced phosphorylation of MAP kinases (p38, Erk, and JNK and IL-6 expression. Finally, vinculin and Rab5 knockdown reduced infection of S. aureus, phosphorylation of MAPKs and IL-6 expression in murine lungs. Our results suggest that vinculin binds to Rab5 and that these two molecules cooperatively enhance bacterial infection and the inflammatory response.

  4. Homology modeling and validation of SAS2271 transcriptional regulator of AraC family in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Money Gupta

    2013-02-01

    Full Text Available Objective: To predict three dimensional structure of AraC Family transcription regulator protein in staphylococcus aureus involved in causing virulence. Methods: Evolutionary dynamics of S. aureus reveled that several mutations preceding virulence lead to truncated proteins that plays an important role in virulence. The Structural templates are identified using homology search and then homology modelling is used to get the 3-D structure of the protein. Results: The 3-D structure of SAS2271 transcriptional factor of AraC family in MSSA476 strain of S. aureus was modelled and validated using the Ramachandran plot. Conclusions: The knowledge of 3-D structure of the protein will be helpful in identifying its biochemical function along with its regulatory mechanism in causing virulence.

  5. A global Staphylococcus aureus proteome resource applied to the in vivo characterization of host-pathogen interactions.

    Science.gov (United States)

    Michalik, Stephan; Depke, Maren; Murr, Annette; Gesell Salazar, Manuela; Kusebauch, Ulrike; Sun, Zhi; Meyer, Tanja C; Surmann, Kristin; Pförtner, Henrike; Hildebrandt, Petra; Weiss, Stefan; Palma Medina, Laura Marcela; Gutjahr, Melanie; Hammer, Elke; Becher, Dörte; Pribyl, Thomas; Hammerschmidt, Sven; Deutsch, Eric W; Bader, Samuel L; Hecker, Michael; Moritz, Robert L; Mäder, Ulrike; Völker, Uwe; Schmidt, Frank

    2017-09-08

    Data-independent acquisition mass spectrometry promises higher performance in terms of quantification and reproducibility compared to data-dependent acquisition mass spectrometry methods. To enable high-accuracy quantification of Staphylococcus aureus proteins, we have developed a global ion library for data-independent acquisition approaches employing high-resolution time of flight or Orbitrap instruments for this human pathogen. We applied this ion library resource to investigate the time-resolved adaptation of S. aureus to the intracellular niche in human bronchial epithelial cells and in a murine pneumonia model. In epithelial cells, abundance changes for more than 400 S. aureus proteins were quantified, revealing, e.g., the precise temporal regulation of the SigB-dependent stress response and differential regulation of translation, fermentation, and amino acid biosynthesis. Using an in vivo murine pneumonia model, our data-independent acquisition quantification analysis revealed for the first time the in vivo proteome adaptation of S. aureus. From approximately 2.15 × 10(5)  S. aureus cells, 578 proteins were identified. Increased abundance of proteins required for oxidative stress response, amino acid biosynthesis, and fermentation together with decreased abundance of ribosomal proteins and nucleotide reductase NrdEF was observed in post-infection samples compared to the pre-infection state.

  6. Occurrence of methicillin-resistant Staphylococcus aureus in surface waters near industrial hog operation spray fields.

    Science.gov (United States)

    Hatcher, S M; Myers, K W; Heaney, C D; Larsen, J; Hall, D; Miller, M B; Stewart, J R

    2016-09-15

    Industrial hog operations (IHOs) have been identified as a source of antibiotic-resistant Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). However, few studies have investigated the presence of antibiotic-resistant S. aureus in the environment near IHOs, specifically surface waters proximal to spray fields where IHO liquid lagoon waste is sprayed. Surface water samples (n=179) were collected over the course of approximately one year from nine locations in southeastern North Carolina and analyzed for the presence of presumptive MRSA using CHROMagar MRSA media. Culture-based, biochemical, and molecular tests, as well as matrix-assisted laser desorption/ionization-time of flight mass spectrometry were used to confirm that isolates that grew on CHROMagar MRSA media were S. aureus. Confirmed S. aureus isolates were then tested for susceptibility to 16 antibiotics and screened for molecular markers of MRSA (mecA, mecC) and livestock adaptation (absence of scn). A total of 12 confirmed MRSA were detected in 9 distinct water samples. Nine of 12 MRSA isolates were also multidrug-resistant (MDRSA [i.e., resistant to ≥3 antibiotic classes]). All MRSA were scn-positive and most (11/12) belonged to a staphylococcal protein A (spa) type t008, which is commonly associated with humans. Additionally, 12 confirmed S. aureus that were methicillin-susceptible (MSSA) were recovered, 7 of which belonged to spa type t021 and were scn-negative (a marker of livestock-adaptation). This study demonstrated the presence of MSSA, MRSA, and MDRSA in surface waters adjacent to IHO lagoon waste spray fields in southeastern North Carolina. To our knowledge, this is the first report of waterborne S. aureus from surface waters proximal to IHOs.

  7. Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus

    Science.gov (United States)

    Irazoqui, Javier E.; Troemel, Emily R.; Feinbaum, Rhonda L.; Luhachack, Lyly G.; Cezairliyan, Brent O.; Ausubel, Frederick M.

    2010-01-01

    The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR) protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs). Because our data suggest that neither the P. aeruginosa nor the S. aureus–triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C. elegans, with

  8. MerTK Does Not Mediate Phagocytosis of Staphylococcus aureus but Attenuates Inflammation Induced by Staphylococcal Lipoteichoic Acid Through Blocking NF-κB Activation.

    Science.gov (United States)

    Zhang, Bing; Wu, Huimei; Fang, Lei; Ding, Peishan; Xu, Ke; Yang, Qingbin; Liu, Rongyu

    2017-05-20

    Mer receptor tyrosine kinase (MerTK) expressed in macrophages is essential for phagocytosis of apoptotic cells. Here, we investigate whether MerTK is involved in the phagocytosis of Staphylococcus aureus (S. aureus) and regulation of staphylococcal lipoteichoic acid (LTA)-induced inflammatory response in macrophages. We found that stimulating RAW264.7 macrophages with S. aureus activated multiple signaling pathways including toll-like receptor 2 (TLR2), scavenger receptor A (SR-A), and MerTK. Meanwhile, S. aureus stimulation also induced activation of proteins focal adhesion kinase (FAK) and Rac1, which are related to phagocytosis. Pretreatment with a specific Mer-blocking antibody significantly inhibited S. aureus-induced phosphorylation of MerTK, while it had no effect on S. aureus-induced activation of FAK and Rac1. Moreover, by confocal laser microscope, we observed that the antibody blockade of MerTK had little impact on the phagocytosis of S. aureus by RAW264.7 macrophages. Additionally, pretreatment with this antibody further promoted LTA-induced phosphorylation of nuclear factor κB (NF-κB) p65 subunit and production of pro-inflammatory cytokines, such as TNF-α, IL-6, IL-1β, and macrophage inflammatory protein-2 (MIP-2). Collectively, these results suggest that MerTK does not play an essential role in the phagocytosis of S. aureus but attenuates inflammation induced by staphylococcal LTA through blocking NF-κB activation.

  9. 新的细胞壁锚定蛋白SasX对金黄葡萄球菌生物膜形成和毒力的影响%The novel surface-anchored protein SasX promotes biofilm formation and is a virulence factor of Staphylococcus aureus

    Institute of Scientific and Technical Information of China (English)

    杜昕; 宋燕; 胡锦辉; 阮斐怡; 吕元; 李敏

    2011-01-01

    Objective To determine whether the novel surface-anchored protein SasX mediates biofilm formation of Staphylococcus aureus and to investigate the function of SasX in the virulence of S.aureus.Methods Methicillin-resistant Staphylococcus aureus(MRSA) ST-239 HS sasX gene mutant (HS770 △ sasX) and complement [ HS770 △sasX(pRBsasX) ] were gotten by gene knock out and complement methods.Semiquantitative biofilm assay was used for detection of the biofilm formation of wild type and mutant.By using abscess model in mice,we investigate the function of SasX in the virulence of S.aureus.Results The sasX gene mutant strain was gotten successfully by using pKOR1 plasmid.HS770 △sasX with a very clear reduction of biofilm formation compared to wild type and complement(P<0.05),there was no significant difference of biofilm formation between wild type and complement(P>0.05).Primary attachment assays demonstrated that comparing to wild type,there was significant reduction of initial accumulative phases of biofilm development in HS770△sasX(P<0.05),but there was no difference between wild type and complement(P>0.05).Both sasX wild type and mutant could cause abscess in the skin of mice,no abscess were forming in the PBS control group.The biggest abscess size was found on the second day after injection.The size of abscess was smaller with extension of the time.But on the same time,the wild type group produced significantly larger abscesses compared to mutant group( P<0.05 ).Conclusion It could improve the effect of gene knocking out by using pKOR1 plasmid.SasX promotes biofilm formation by influencing on the initial accumulative phases of biofilm development,SasX is a marker of invasive infection of S.aureus,it is very important for S.aureus persistence in the hospital setting.%目的 研究新的细胞壁锚定蛋白SasX对金黄葡萄球菌生物膜形成及毒力的影响.方法 采用基因敲除及基因互补技术获得耐甲

  10. Carotenoid Formation by Staphylococcus aureus

    Science.gov (United States)

    Hammond, Ray K.; White, David C.

    1970-01-01

    The carotenoid pigments of Staphylococcus aureus U-71 were identified as phytoene; ζ-carotene; δ-carotene; phytofluenol; a phytofluenol-like carotenoid, rubixanthin; and three rubixanthin-like carotenoids after extraction, saponification, chromatographic separation, and determination of their absorption spectra. There was no evidence of carotenoid esters or glycoside ethers in the extract before saponification. During the aerobic growth cycle the total carotenoids increased from 45 to 1,000 nmoles per g (dry weight), with the greatest increases in the polar, hydroxylated carotenoids. During the anaerobic growth cycle, the total carotenoids increased from 20 nmoles per g (dry weight) to 80 nmoles per g (dry weight), and only traces of the polar carotenoids were formed. Light had no effect on carotenoid synthesis. About 0.14% of the mevalonate-2-14C added to the culture was incorporated into the carotenoids during each bacterial doubling. The total carotenoids did not lose radioactivity when grown in the absence of 14C for 2.5 bacterial doublings. The total carotenoids did not lose radioactivity when grown in the absence of 14C for 2.5 bacterial doublings. The incorporation and turnover of 14C indicated the carotenes were sequentially desaturated and hydroxylated to form the polar carotenoids. PMID:5423369

  11. Extracellular DNA facilitates the formation of functional amyloids in Staphylococcus aureus biofilms.

    Science.gov (United States)

    Schwartz, Kelly; Ganesan, Mahesh; Payne, David E; Solomon, Michael J; Boles, Blaise R

    2016-01-01

    Persistent staphylococcal infections often involve surface-associated communities called biofilms. Staphylococcus aureus biofilm development is mediated by the co-ordinated production of the biofilm matrix, which can be composed of polysaccharides, extracellular DNA (eDNA) and proteins including amyloid fibers. The nature of the interactions between matrix components, and how these interactions contribute to the formation of matrix, remain unclear. Here we show that the presence of eDNA in S. aureus biofilms promotes the formation of amyloid fibers. Conditions or mutants that do not generate eDNA result in lack of amyloids during biofilm growth despite the amyloidogeneic subunits, phenol soluble modulin peptides, being produced. In vitro studies revealed that the presence of DNA promotes amyloid formation by PSM peptides. Thus, this work exposes a previously unacknowledged interaction between biofilm matrix components that furthers our understanding of functional amyloid formation and S. aureus biofilm biology.

  12. Choose wisely: Network, ontology and annotation resources for the analysis of Staphylococcus aureus omics data.

    Science.gov (United States)

    Broadbent, J A; Sampson, D L; Broszczak, D A; Upton, Z; Huygens, F

    2015-05-01

    Staphylococcus aureus (S. aureus) is a prominent human and livestock pathogen investigated widely using omic technologies. Critically, due to availability, low visibility or scattered resources, robust network and statistical contextualisation of the resulting data is generally under-represented. Here, we present novel meta-analyses of freely-accessible molecular network and gene ontology annotation information resources for S. aureus omics data interpretation. Furthermore, through the application of the gene ontology annotation resources we demonstrate their value and ability (or lack-there-of) to summarise and statistically interpret the emergent properties of gene expression and protein abundance changes using publically available data. This analysis provides simple metrics for network selection and demonstrates the availability and impact that gene ontology annotation selection can have on the contextualisation of bacterial omics data. Copyright © 2015 Elsevier GmbH. All rights reserved.

  13. Madurahydroxylactone, an Inhibitor of Staphylococcus aureus FtsZ from Nonomuraea sp. AN100570.

    Science.gov (United States)

    Kim, Bo-Min; Choi, Ha Young; Kim, Geon-Woo; Zheng, Chang-Ji; Kim, Young-Ho; Kim, Won-Gon

    2017-09-15

    FtsZ, a bacterial cell-division protein, is an attractive antibacterial target. In the screening for an inhibitor of Staphylococcus aureus FtsZ, madurahydroxylactone (1) and its related derivatives 2-5 were isolated from Nonomuraea sp. AN1000570. Compound 1 inhibited S. aureus FtsZ with an IC50 of 53.4 µM and showed a potent antibacterial activity against S. aureus and MRSA with an MIC of 1 µg/mL, while 2-5 were weak or inactive. Importantly, 1 induced cell elongation in the cell division phenotype assay, while 2-5 didn't. It indicates that 1 exhibits its potent antibacterial activity via inhibition of FtsZ, and the hydroxyl group and the hydroxylactone ring of 1 are critical for the activity. Thus, madurahydroxylactone is a new type inhibitor of FtsZ.

  14. Structure of the 70S ribosome from human pathogen Staphylococcus aureus.

    Science.gov (United States)

    Khusainov, Iskander; Vicens, Quentin; Bochler, Anthony; Grosse, François; Myasnikov, Alexander; Ménétret, Jean-François; Chicher, Johana; Marzi, Stefano; Romby, Pascale; Yusupova, Gulnara; Yusupov, Marat; Hashem, Yaser

    2016-12-01

    Comparative structural studies of ribosomes from various organisms keep offering exciting insights on how species-specific or environment-related structural features of ribosomes may impact translation specificity and its regulation. Although the importance of such features may be less obvious within more closely related organisms, their existence could account for vital yet species-specific mechanisms of translation regulation that would involve stalling, cell survival and antibiotic resistance. Here, we present the first full 70S ribosome structure from Staphylococcus aureus, a Gram-positive pathogenic bacterium, solved by cryo-electron microscopy. Comparative analysis with other known bacterial ribosomes pinpoints several unique features specific to S. aureus around a conserved core, at both the protein and the RNA levels. Our work provides the structural basis for the many studies aiming at understanding translation regulation in S. aureus and for designing drugs against this often multi-resistant pathogen.

  15. Proteomic analyses of iron-responsive, Clp-dependent changes in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Farrand, Allison J; Friedman, David B; Reniere, Michelle L;

    2015-01-01

    Staphylococcus aureus is a frequent human pathogen that is capable of causing a wide range of life-threatening infections. A promising antibacterial target is the Clp proteolytic system, which performs the vital function of maintaining protein turnover within the cell. This system primarily impacts...... the bacterial response to various stresses by degrading specific proteins but can also regulate a number of physiological processes through protein degradation. A critical stress to which S. aureus must adapt during infection of a vertebrate host is nutrient iron limitation. We have previously shown...... of mutants inactivated for components of the Clp protease, including ClpP, ClpC and ClpX, in high- and low-iron conditions. Our results reveal numerous proteins altered in abundance in the clp mutants and provide new insights into the staphylococcal proteolytic network during nutrient iron limitation....

  16. SarA and not sigmaB is essential for biofilm development by Staphylococcus aureus.

    Science.gov (United States)

    Valle, Jaione; Toledo-Arana, Alejandro; Berasain, Carmen; Ghigo, Jean-Marc; Amorena, Beatriz; Penadés, José R; Lasa, Iñigo

    2003-05-01

    Staphylococcus aureus biofilm formation is associated with the production of the polysaccharide intercellular adhesin (PIA/PNAG), the product of the ica operon. The staphylococcal accessory regulator, SarA, is a central regulatory element that controls the production of S. aureus virulence factors. By screening a library of Tn917 insertions in a clinical S. aureus strain, we identified SarA as being essential for biofilm development. Non-polar mutations of sarA in four genetically unrelated S. aureus strains decreased PIA/PNAG production and completely impaired biofilm development, both in steady state and flow conditions via an agr-independent mechanism. Accordingly, real-time PCR showed that the mutation in the sarA gene resulted in downregulation of the ica operon transcription. We also demonstrated that complete deletion of sigmaB did not affect PIA/PNAG production and biofilm formation, although it slightly decreased ica operon transcription. Furthermore, the sarA-sigmaB double mutant showed a significant decrease of ica expression but an increase of PIA/PNAG production and biofilm formation compared to the sarA single mutant. We propose that SarA activates S. aureus development of biofilm by both enhancing the ica operon transcription and suppressing the transcription of either a protein involved in the turnover of PIA/PNAG or a repressor of its synthesis, whose expression would be sigmaB-dependent.

  17. Staphylococcus aureus Nuclease Is an SaeRS-Dependent Virulence Factor

    Science.gov (United States)

    Olson, Michael E.; Nygaard, Tyler K.; Ackermann, Laynez; Watkins, Robert L.; Zurek, Oliwia W.; Pallister, Kyler B.; Griffith, Shannon; Kiedrowski, Megan R.; Flack, Caralyn E.; Kavanaugh, Jeffrey S.; Kreiswirth, Barry N.

    2013-01-01

    Several prominent bacterial pathogens secrete nuclease (Nuc) enzymes that have an important role in combating the host immune response. Early studies of Staphylococcus aureus Nuc attributed its regulation to the agr quorum-sensing system. However, recent microarray data have indicated that nuc is under the control of the SaeRS two-component system, which is a major regulator of S. aureus virulence determinants. Here we report that the nuc gene is directly controlled by the SaeRS two-component system through reporter fusion, immunoblotting, Nuc activity measurements, promoter mapping, and binding studies, and additionally, we were unable identify a notable regulatory link to the agr system. The observed SaeRS-dependent regulation was conserved across a wide spectrum of representative S. aureus isolates. Moreover, with community-associated methicillin-resistant S. aureus (CA MRSA) in a mouse model of peritonitis, we observed in vivo expression of Nuc activity in an SaeRS-dependent manner and determined that Nuc is a virulence factor that is important for in vivo survival, confirming the enzyme's role as a contributor to invasive disease. Finally, natural polymorphisms were identified in the SaeRS proteins, one of which was linked to Nuc regulation in a CA MRSA USA300 endocarditis isolate. Altogether, our findings demonstrate that Nuc is an important S. aureus virulence factor and part of the SaeRS regulon. PMID:23381999

  18. Staphylococcus aureus nuclease is an SaeRS-dependent virulence factor.

    Science.gov (United States)

    Olson, Michael E; Nygaard, Tyler K; Ackermann, Laynez; Watkins, Robert L; Zurek, Oliwia W; Pallister, Kyler B; Griffith, Shannon; Kiedrowski, Megan R; Flack, Caralyn E; Kavanaugh, Jeffrey S; Kreiswirth, Barry N; Horswill, Alexander R; Voyich, Jovanka M

    2013-04-01

    Several prominent bacterial pathogens secrete nuclease (Nuc) enzymes that have an important role in combating the host immune response. Early studies of Staphylococcus aureus Nuc attributed its regulation to the agr quorum-sensing system. However, recent microarray data have indicated that nuc is under the control of the SaeRS two-component system, which is a major regulator of S. aureus virulence determinants. Here we report that the nuc gene is directly controlled by the SaeRS two-component system through reporter fusion, immunoblotting, Nuc activity measurements, promoter mapping, and binding studies, and additionally, we were unable identify a notable regulatory link to the agr system. The observed SaeRS-dependent regulation was conserved across a wide spectrum of representative S. aureus isolates. Moreover, with community-associated methicillin-resistant S. aureus (CA MRSA) in a mouse model of peritonitis, we observed in vivo expression of Nuc activity in an SaeRS-dependent manner and determined that Nuc is a virulence factor that is important for in vivo survival, confirming the enzyme's role as a contributor to invasive disease. Finally, natural polymorphisms were identified in the SaeRS proteins, one of which was linked to Nuc regulation in a CA MRSA USA300 endocarditis isolate. Altogether, our findings demonstrate that Nuc is an important S. aureus virulence factor and part of the SaeRS regulon.

  19. IsdB-dependent hemoglobin binding is required for acquisition of heme by Staphylococcus aureus.

    Science.gov (United States)

    Pishchany, Gleb; Sheldon, Jessica R; Dickson, Claire F; Alam, Md Tauqeer; Read, Timothy D; Gell, David A; Heinrichs, David E; Skaar, Eric P

    2014-06-01

    Staphylococcus aureus is a Gram-positive pathogen responsible for tremendous morbidity and mortality. As with most bacteria, S. aureus requires iron to cause disease, and it can acquire iron from host hemoglobin. The current model for staphylococcal hemoglobin-iron acquisition proposes that S. aureus binds hemoglobin through the surface-exposed hemoglobin receptor IsdB. IsdB removes heme from bound hemoglobin and transfers this cofactor to other proteins of the Isd system, which import and degrade heme to release iron in the cytoplasm. Here we demonstrate that the individual components of the Isd system are required for growth on low nanomolar concentrations of hemoglobin as a sole source of iron. An in-depth study of hemoglobin binding by IsdB revealed key residues that are required for hemoglobin binding. Further, we show that these residues are necessary for heme extraction from hemoglobin and growth on hemoglobin as a sole iron source. These processes are found to contribute to the pathogenicity of S. aureus in a murine model of infection. Together these results build on the model for Isd-mediated hemoglobin binding and heme-iron acquisition during the pathogenesis of S. aureus infection.

  20. Bacterial findings in optimised sampling and characterisation of S. aureus in chronic rhinosinusitis.

    Science.gov (United States)

    Thunberg, Ulrica; Söderquist, Bo; Hugosson, Svante

    2017-01-01

    The bacterial spectrum in chronic rhinosinusitis (CRS) is clinically relevant. This study aimed to compare two sampling techniques and to characterise Staphylococcus aureus isolated from CRS patients. Bacterial specimens were collected from the nares and maxillary sinus in 42 CRS patients and from the nares in 57 healthy controls. Maxillary sinus sampling was performed in two ways in each patient: with a cotton-tipped aluminium swab through the enlarged sinus ostium, and with a protected brush. S. aureus was characterised by DNA-sequencing of the repeat region of the S. aureus protein A gene, spa typing. The protected brush technique was superior to the cotton-tipped aluminium swab in reducing contamination rate. However, the two sampling methods were consistent in terms of clinically relevant bacterial findings, and the easy-to-handle cotton-tipped swab can still be recommended when culturing the maxillary sinus. Patients showed a significantly higher presence of S. aureus in the nares compared with healthy controls, and healthy controls showed a significantly higher presence of coagulase-negative staphylococci in the nares compared with patients. The spa types were identical for the nares and maxillary sinus in all patients except one. The sampling techniques showed equivalent results, indicating a low risk of unnecessary antibiotic treatment when using the easy-to-handle cotton-tipped aluminium swab. The high rate of identical spa types of S. aureus isolated from the nares and maxillary sinus of CRS patients might indicate colonisation of the maxillary sinus from the nares.

  1. Evaluation of the presence of the bap gene in Staphylococcus aureus isolates recovered from human and animals species.

    Science.gov (United States)

    Vautor, E; Abadie, G; Pont, A; Thiery, R

    2008-03-18

    The implication of biofilm in chronic bacterial infection in many species has triggered an increasing interest in the characterization of genes involved in biofilm formation. The bap gene is a newly identified gene that encodes the biofilm-associated protein, BAP, which is involved in biofilm formation in Staphylococcus aureus. So far the bap gene has only been found in a small proportion of S. aureus strains from bovine mastitis in Spain. In order to study the presence of the bap gene in S. aureus isolates obtained from other species and various locations, a collection of 262 isolates was tested by PCR, using published primers and dot-blot. The results indicated that none of the isolates carried the bap gene suggesting that the prevalence of this gene among S. aureus isolates should be very low.

  2. Haem Recognition By a Staphylococcus Aureus NEAT Domain

    Energy Technology Data Exchange (ETDEWEB)

    Grigg, J.C.; Vermeiren, C.; Heinrichs, D.E.; Murphy, M.E.P.

    2009-06-01

    Successful pathogenic organisms have developed mechanisms to thrive under extreme levels of iron restriction. Haem-iron represents the largest iron reservoir in the human body and is a significant source of iron for some bacterial pathogens. NEAT (NEAr Transporter) domains are found exclusively in a family of cell surface proteins in Gram-positive bacteria. Many NEAT domain-containing proteins, including IsdA in Staphylococcus aureus, are implicated in haem binding. Here, we show that overexpression of IsdA in S. aureus enhances growth and an inactivation mutant of IsdA has a growth defect, compared with wild type, when grown in media containing haem as the sole iron source. Furthermore, the haem-binding property of IsdA is contained within the NEAT domain. Crystal structures of the apo-IsdA NEAT domain and in complex with haem were solved and reveal a clathrin adapter-like beta-sandwich fold with a large hydrophobic haem-binding pocket. Haem is bound with the propionate groups directed at the molecular surface and the iron is co-ordinated solely by Tyr(166). The phenol groups of Tyr(166) and Tyr(170) form an H-bond that may function in regulating haem binding and release. An analysis of IsdA structure-sequence alignments indicate that conservation of Tyr(166) is a predictor of haem binding by NEAT domains.

  3. Coordinated Molecular Cross-Talk between Staphylococcus aureus, Endothelial Cells and Platelets in Bloodstream Infection

    Directory of Open Access Journals (Sweden)

    Carolina D. Garciarena

    2015-12-01

    Full Text Available Staphylococcus aureus is an opportunistic pathogen often carried asymptomatically on the human body. Upon entry to the otherwise sterile environment of the cardiovascular system, S. aureus can lead to serious complications resulting in organ failure and death. The success of S. aureus as a pathogen in the bloodstream is due to its ability to express a wide array of cell wall proteins on its surface that recognise host receptors, extracellular matrix proteins and plasma proteins. Endothelial cells and platelets are important cells in the cardiovascular system and are a major target of bloodstream infection. Endothelial cells form the inner lining of a blood vessel and provide an antithrombotic barrier between the vessel wall and blood. Platelets on the other hand travel throughout the cardiovascular system and respond by aggregating around the site of injury and initiating clot formation. Activation of either of these cells leads to functional dysregulation in the cardiovascular system. In this review, we will illustrate how S. aureus establish intimate interactions with both endothelial cells and platelets leading to cardiovascular dysregulation.

  4. Antibiofilm Effect of Octenidine Hydrochloride on Staphylococcus aureus, MRSA and VRSA

    Directory of Open Access Journals (Sweden)

    Mary Anne Roshni Amalaradjou

    2014-05-01

    Full Text Available Millions of indwelling devices are implanted in patients every year, and staphylococci (S. aureus, MRSA and vancomycin-resistant S. aureus (VRSA are responsible for a majority of infections associated with these devices, thereby leading to treatment failures. Once established, staphylococcal biofilms become resistant to antimicrobial treatment and host response, thereby serving as the etiological agent for recurrent infections. This study investigated the efficacy of octenidine hydrochloride (OH for inhibiting biofilm synthesis and inactivating fully-formed staphylococcal biofilm on different matrices in the presence and absence of serum protein. Polystyrene plates and stainless steel coupons inoculated with S. aureus, MRSA or VRSA were treated with OH (zero, 0.5, one, 2 mM at 37 °C for the prevention of biofilm formation. Additionally, the antibiofilm effect of OH (zero, 2.5, five, 10 mM on fully-formed staphylococcal biofilms on polystyrene plates, stainless steel coupons and urinary catheters was investigated. OH was effective in rapidly inactivating planktonic and biofilm cells of S. aureus, MRSA and VRSA on polystyrene plates, stainless steel coupons and urinary catheters in the presence and absence of serum proteins. The use of two and 10 mM OH completely inactivated S. aureus planktonic cells and biofilm (>6.0 log reduction on all matrices tested immediately upon exposure. Further, confocal imaging revealed the presence of dead cells and loss in biofilm architecture in the OH-treated samples when compared to intact live biofilm in the control. Results suggest that OH could be applied as an effective antimicrobial to control biofilms of S. aureus, MRSA and VRSA on appropriate hospital surfaces and indwelling devices.

  5. Antibiofilm Effect of Octenidine Hydrochloride on Staphylococcus aureus, MRSA and VRSA.

    Science.gov (United States)

    Amalaradjou, Mary Anne Roshni; Venkitanarayanan, Kumar

    2014-05-06

    Millions of indwelling devices are implanted in patients every year, and staphylococci (S. aureus, MRSA and vancomycin-resistant S. aureus (VRSA)) are responsible for a majority of infections associated with these devices, thereby leading to treatment failures. Once established, staphylococcal biofilms become resistant to antimicrobial treatment and host response, thereby serving as the etiological agent for recurrent infections. This study investigated the efficacy of octenidine hydrochloride (OH) for inhibiting biofilm synthesis and inactivating fully-formed staphylococcal biofilm on different matrices in the presence and absence of serum protein. Polystyrene plates and stainless steel coupons inoculated with S. aureus, MRSA or VRSA were treated with OH (zero, 0.5, one, 2 mM) at 37 °C for the prevention of biofilm formation. Additionally, the antibiofilm effect of OH (zero, 2.5, five, 10 mM) on fully-formed staphylococcal biofilms on polystyrene plates, stainless steel coupons and urinary catheters was investigated. OH was effective in rapidly inactivating planktonic and biofilm cells of S. aureus, MRSA and VRSA on polystyrene plates, stainless steel coupons and urinary catheters in the presence and absence of serum proteins. The use of two and 10 mM OH completely inactivated S. aureus planktonic cells and biofilm (>6.0 log reduction) on all matrices tested immediately upon exposure. Further, confocal imaging revealed the presence of dead cells and loss in biofilm architecture in the OH-treated samples when compared to intact live biofilm in the control. Results suggest that OH could be applied as an effective antimicrobial to control biofilms of S. aureus, MRSA and VRSA on appropriate hospital surfaces and indwelling devices.

  6. Vancomycin modifies the expression of the agr system in multidrug-resistant Staphylococcus aureus clinical isolates

    Directory of Open Access Journals (Sweden)

    Vicenta eCázares-Domínguez

    2015-05-01

    Full Text Available Staphylococcus aureus is an opportunistic pathogen that colonizes human hosts and causes a wide variety of diseases. Two interacting regulatory systems called agr (accessory gene regulator and sar (staphylococcal accessory regulator are involved in the regulation of virulence factors. The aim of this study was to evaluate the effect of vancomycin on hld and spa gene expression during the exponential and post-exponential growth phases in multidrug resistant (MDR S. aureus. Methods. Antibiotic susceptibility was evaluated by the standard microdilution method. The phylogenetic profile was obtained by pulsed-field gel electrophoresis. Polymorphisms of agr and SCCmec were analyzed by multiplex polymerase chain reaction. The expression levels of hld and spa were analyzed by reverse transcription-PCR. An enzyme-linked immunosorbent assay assay was performed to detect protein A, and biofilm formation was analyzed via crystal violet staining. Results. In total, 60.60% (20/33 of S. aureus clinical isolates were MDR. Half (10/20 of the MDR S. aureus isolates were distributed in subcluster 10, with > 90% similarity among them. In the isolates of this subcluster, a high prevalence (100% for the agrII and the cassette SCCmec II polymorphisms was found. Our data showed significant increases in hld expression during the post-exponential phase in the presence and absence of vancomycin. Significant increases in spa expression, protein A production and biofilm formation were observed during the post-exponential phase when the MDR S. aureus isolates were challenged with vancomycin. Conclusion. The polymorphism agrII, which is associated with nosocomial isolates, was the most prevalent polymorphism in MDR S. aureus. Additionally, under our study conditions, vancomycin modified hld and spa expression in these clinical isolates. Therefore, vancomycin may regulate alternative systems that jointly participate in the regulation of these virulence factors.

  7. Comparative proteomics of Staphylococcus aureus and the response of methicillin-resistant and methicillin-sensitive strains to Triton X-100

    DEFF Research Database (Denmark)

    Cordwell, Stuart J; Larsen, Martin Røssel; Cole, Rebecca T;

    2002-01-01

    Proteomics is a powerful tool for analysing differences in gene expression between bacterial strains with alternate phenotypes. Staphylococcus aureus strains are grouped on the basis of their sensitivity to methicillin. Two-dimensional gel electrophoresis was combined with MS to compare the protein...... profiles of S. aureus strains COL (methicillin-resistant) and 8325 (methicillin-sensitive). Reference mapping via this approach identified 377 proteins that corresponded to 266 distinct ORFs. Amongst these identified proteins were 14 potential virulence factors. The production of 41 'hypothetical' proteins...... was confirmed, and eight of these appeared to be unique to S. aureus. Strain COL displayed 12 protein spots, which included alkaline-shock protein 23 (Asp23) and cold-shock proteins CspABC, which either were not present in strain 8325 or were present at a significantly lower intensity in this strain...

  8. Comparative proteomics of Staphylococcus aureus and the response of methicillin-resistant and methicillin-sensitive strains to Triton X-100

    DEFF Research Database (Denmark)

    Cordwell, Stuart J; Larsen, Martin Røssel; Cole, Rebecca T

    2002-01-01

    profiles of S. aureus strains COL (methicillin-resistant) and 8325 (methicillin-sensitive). Reference mapping via this approach identified 377 proteins that corresponded to 266 distinct ORFs. Amongst these identified proteins were 14 potential virulence factors. The production of 41 'hypothetical' proteins...... was confirmed, and eight of these appeared to be unique to S. aureus. Strain COL displayed 12 protein spots, which included alkaline-shock protein 23 (Asp23) and cold-shock proteins CspABC, which either were not present in strain 8325 or were present at a significantly lower intensity in this strain....... Comparative maps were used to characterize the S. aureus response to treatment with Triton X-100 (TX-100), a detergent that has been shown to reduce methicillin resistance independently of an interaction with the mecA-encoded penicillin-binding protein 2a. In response to growth of the bacteria in the presence...

  9. Isobaric tags for relative and absolute quantitation proteomics analysis of gene regulation by SprC in Staphylococcus aureus.

    Science.gov (United States)

    Zhao, Huanqiang; Hu, Fupin; Yang, Han; Ding, Baixing; Xu, Xiaogang; He, Chunyan; Cui, Zelin; Shu, Wen; Liu, Qingzhong

    2017-09-06

    To explore the complete gene networks regulated by small RNA SprC and its targets in Staphylococcus aureus. The isobaric tags for relative and absolute quantitation and bioinformatic methods were utilized to identify and analyze the target proteins affected by SprC in S. aureus N315. Proteomic analysis showed that the expression of 44 proteins was modulated by SprC. Further, bioinformatic analysis displayed that these affected proteins mainly associated with metabolic and cellular process, biological regulation and catalytic activity. Our data provide a rich resource of SprC targets in S. aureus, although the mechanism of regulation by SprC is yet to be elucidated.

  10. Staphylococcus aureus Alpha-Toxin Induces the Formation of Dynamic Tubules Labeled with LC3 within Host Cells in a Rab7 and Rab1b-Dependent Manner

    Directory of Open Access Journals (Sweden)

    María M. López de Armentia

    2017-10-01

    Full Text Available Staphylococcus aureus is a pathogen that causes severe infectious diseases that eventually lead to septic and toxic shock. S. aureus infection is characterized by the production of virulence factors, including enzymes and toxins. After internalization S. aureus resides in a phagosome labeled with Rab7 protein. Here, we show that S. aureus generates tubular structures marked with the small GTPases Rab1b and Rab7 and by the autophagic protein LC3 at early times post-infection. As shown by live cell imaging these tubular structures are highly dynamic, extend, branch and grow in length. We have named them S. aureus induced filaments (Saf. Furthermore, we demonstrate that the formation of these filaments depends on the integrity of microtubules and the activity of the motor protein Kinesin-1 (Kif5B and the Rab-interacting lysosomal protein (RILP. Our group has previously reported that α-hemolysin, a secreted toxin of S. aureus, is responsible of the activation of the autophagic pathway induced by the bacteria. In the present report, we demonstrate that the autophagic protein LC3 is recruited to the membrane of S. aureus induced filaments and that α-hemolysin is the toxin that induces Saf formation. Interestingly, increasing the levels of intracellular cAMP significantly inhibited Saf biogenesis. Remarkably in this report we show the formation of tubular structures that emerge from the S. aureus-containing phagosome and that these tubules generation seems to be required for efficient bacteria replication.

  11. Intra-cellular Staphylococcus aureus alone causes infection in vivo

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    T Hamza

    2013-07-01

    Full Text Available Chronic and recurrent bone infections occur frequently but have not been explained. Staphylococcus aureus (S. aureus is often found among chronic and recurrent infections and may be responsible for such infections. One possible reason is that S. aureus can internalize and survive within host cells and by doing so, S. aureus can evade both host defense mechanisms and most conventional antibiotic treatments. In this study, we hypothesized that intra-cellular S. aureus could induce infections in vivo. Osteoblasts were infected with S. aureus and, after eliminating extra-cellular S. aureus, inoculated into an open fracture rat model. Bacterial cultures and radiographic observations at post-operative day 21 confirmed local bone infections in animals inoculated with intra-cellular S. aureus within osteoblasts alone. We present direct in vivo evidence that intra-cellular S. aureus could be sufficient to induce bone infection in animals; we found that intra-cellular S. aureus inoculation of as low as 102 colony forming units could induce severe bone infections. Our data may suggest that intra-cellular S. aureus can “hide” in host cells during symptom-free periods and, under certain conditions, they may escape and lead to infection recurrence. Intra-cellular S. aureus therefore could play an important role in the pathogenesis of S. aureus infections, especially those chronic and recurrent infections in which disease episodes may be separated by weeks, months, or even years.

  12. Genotypes and oxacillin resistance of Staphylococcus aureus from chicken and chicken meat in Poland.

    Science.gov (United States)

    Krupa, P; Bystroń, J; Bania, J; Podkowik, M; Empel, J; Mroczkowska, A

    2014-12-01

    The genotypes and oxacillin resistance of 263 Staphylococcus aureus isolates cultured from chicken cloacae (n = 138) and chicken meat (n = 125) was analyzed. Fifteen spa types were determined in the studied S. aureus population. Among 5 staphylococcal protein A gene (spa) types detected in S. aureus from chicken, t002, t3478, and t13620 were the most frequent. Staphylococcus aureus isolates from meat were assigned to 14 spa types. Among them, the genotypes t002, t056, t091, t3478, and t13620 were dominant. Except for 4 chicken S. aureus isolates belonging to CC398, the remaining 134 isolates were clustered into multilocus sequence clonal complex (CC) 5. Most of meat-derived isolates were assigned to CC5, CC7, and CC15, and to the newly described spa-CC12954 complex belonging to CC1. Except for t011 (CC398), all other spa types found among chicken isolates were also present in isolates from meat. Four S. aureus isolated from chicken and one from meat were identified as methicillin-resistant S. aureus (MRSA) with oxacillin minimum inhibitory concentrations from 16 to 64 μg/mL. All MRSA were assigned to spa types belonging to ST398, and included 4 animal spa t011 SCCmecV isolates and 1 meat-derived spa t899, SCCmecIV isolate. Borderline oxacillin-resistant S. aureus (BORSA) isolates, shown to grow on plates containing 2 to 3 μg/mL of oxacillin, were found within S. aureus isolates from chicken (3 isolates) and from meat (19 isolates). The spa t091 and t084 dominated among BORSA from chicken meat, whereas t548 and t002 were found within animal BORSA. We report for the first time the presence of MRSA in chicken in Poland. We demonstrate that MRSA CC398 could be found in chicken meat indicating potential of introduction of animal-associated genotypes into the food chain. We also report for the first time the possibility of transmission of BORSA isolates from chicken to meat. ©2014 Poultry Science Association Inc.

  13. The Staphylococcus aureus FASII bypass escape route from FASII inhibitors.

    Science.gov (United States)

    Morvan, Claire; Halpern, David; Kénanian, Gérald; Pathania, Amit; Anba-Mondoloni, Jamila; Lamberet, Gilles; Gruss, Alexandra; Gloux, Karine

    2017-07-17

    Antimicrobials targeting the fatty acid synthesis (FASII) pathway are being developed as alternative treatments for bacterial infections. Emergence of resistance to FASII inhibitors was mainly considered as a consequence of mutations in the FASII target genes. However, an alternative and efficient anti-FASII resistance strategy, called here FASII bypass, was uncovered. Bacteria that bypass FASII incorporate exogenous fatty acids in membrane lipids, and thus dispense with the need for FASII. This strategy is used by numerous Gram-positive low GC % bacteria, including streptococci, enterococci, and staphylococci. Some bacteria repress FASII genes once fatty acids are available, and "constitutively" shift to FASII bypass. Others, such as the major pathogen Staphylococcus aureus, can undergo high frequency mutations that favor FASII bypass. This capacity is particularly relevant during infection, as the host supplies the fatty acids needed for bacteria to bypass FASII and thus become resistant to FASII inhibitors. Screenings for anti-FASII resistance in the presence of exogenous fatty acids confirmed that FASII bypass confers anti-FASII resistance among clinical and veterinary isolates. Polymorphisms in S. aureus FASII initiation enzymes favor FASII bypass, possibly by increasing availability of acyl-carrier protein, a required intermediate. Here we review FASII bypass and consequences in light of proposed uses of anti-FASII to treat infections, with a focus on FASII bypass in S. aureus. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  14. Proteome changes of Caenorhabditis elegans upon a Staphylococcus aureus infection

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    Schoofs Liliane

    2010-02-01

    Full Text Available Abstract Background The success of invertebrates throughout evolution is an excellent illustration of the efficiency of their defence strategies. Caenorhabditis elegans has proven to be an appropriate model for transcriptome studies of host-pathogen interactions. The aim of this paper is to complement this knowledge by investigating the worm's response to a Staphylococcus aureus infection through a 2-dimensional differential proteomics approach. Results Different types of growth media in combination with either E. coli OP50 or Staphylococcus aureus were tested for an effect on the worm's lifespan. LB agar was chosen and C. elegans samples were collected 1 h, 4 h, 8 h and 24 h post S. aureus infection or E. coli incubation. Proteomics analyses resulted in the identification of 130 spots corresponding to a total of 108 differentially expressed proteins. Conclusions Exploring four time-points discloses a dynamic insight of the reaction against a gram-positive infection at the level of the whole organism. The remarkable upregulation after 8 h and 24 h of many enzymes involved in the citric acid cycle might illustrate the cost of fighting off an infection. Intriguing is the downregulation of chaperone molecules, which are presumed to serve a protective role. A comparison with a similar experiment in which C. elegans was infected with the gram-negative Aeromonas hydrophila reveals that merely 9% of the identified spots, some of which even exhibiting an opposite regulation, are present in both studies. Hence, our findings emphasise the complexity and pathogen-specificity of the worm's immune response and form a firm basis for future functional research. Reviewers This article was reviewed by Itai Yanai, Dieter Wolf and Torben Luebke (nominated by Walter Lutz.

  15. Contribution of SecDF to Staphylococcus aureus resistance and expression of virulence factors

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    Berger-Bächi Brigitte

    2011-04-01

    Full Text Available Abstract Background SecDF is an accessory factor of the conserved Sec protein translocation machinery and belongs to the resistance-nodulation-cell division (RND family of multidrug exporters. SecDF has been shown in Escherichia coli and Bacillus subtilis to be involved in the export of proteins. RND proteins can mediate resistance against various substances and might be of relevance in antimicrobial therapy. The role of RND proteins in Staphylococcus aureus has not yet been determined. Results Markerless deletion mutants were constructed to analyze the impact of the so far uncharacterized RND proteins in S. aureus. While the lack of Sa2056 and Sa2339 caused no phenotype regarding growth and resistance, the secDF mutant resulted in a pleiotropic phenotype. The secDF mutant was cold sensitive, but grew normally in rich medium at 37°C. Resistance to beta-lactams, glycopeptides and the RND substrates acriflavine, ethidium bromide and sodium dodecyl sulfate was reduced. The secDF mutant showed an aberrant cell separation and increased spontaneous and Triton X-100 induced autolysis, although the amounts of penicillin-binding proteins in the membrane were unchanged. The impact of secDF deletion on transcription and expression of specific virulence determinants varied: While coagulase transcription and activity were reduced, the opposite was observed for the autolysin Atl. A reduction of the transcription of the cell wall anchored protein A (spa was also found. The accumulation of SpA in the membrane and lowered amounts in the cell wall pointed to an impaired translocation. Conclusions The combination of different effects of secDF deletion on transcription, regulation and translocation lead to impaired cell division, reduced resistance and altered expression of virulence determinants suggesting SecDF to be of major relevance in S. aureus. Thus SecDF could be a potential target for the control and eradication of S. aureus in the future.

  16. The Inflammasome and the Epidermal Growth Factor Receptor (EGFR Are Involved in the Staphylococcus aureus-Mediated Induction of IL-1alpha and IL-1beta in Human Keratinocytes.

    Directory of Open Access Journals (Sweden)

    Maren Simanski

    Full Text Available Staphylococcus (S. aureus is an important pathogen causing various infections including those of the skin. Keratinocytes are able to sense invading S. aureus and to initiate a fast defense reaction by the rapid release of innate defense mediators such as antimicrobial peptides and cytokines. There is increasing evidence that the cytokines IL-1alpha and IL-1beta, which both signal through the IL-1 receptor, play an important role in cutaneous defense against S. aureus. The aim of this study was to gain more insight into the underlying mechanisms leading to the S. aureus-induced IL-1alpha and IL-1beta expression in keratinocytes. Infection of human primary keratinocytes with S. aureus led to the induction of gene expression and protein secretion of IL-1alpha and IL-1beta. Full S. aureus-induced IL-1 protein release required the inflammasome components caspase-1 and ASC (apoptosis-associated speck-like protein containing a CARD whereas gene induction of IL-1alpha and IL-beta by S. aureus was not dependent on caspase-1 and ASC. Since patients receiving anti-cancer therapy by inhibition of the epidermal growth factor receptor (EGFR often suffer from skin infections caused by S. aureus we additionally evaluated whether the EGFR pathway may be involved in the IL-1alpha and IL-1beta induction by S. aureus. Inactivation of the EGFR with a blocking antibody decreased the S. aureus-mediated IL-1alpha and IL-1beta induction in primary keratinocytes. Moreover, the use of siRNA experiments revealed that ADAM17 (A Disintegrin and A Metalloprotease 17, a metalloproteinase known to mediate the shedding and release of EGFR ligands, was required for full induction of IL-1alpha and IL-1beta in keratinocytes infected with S. aureus. A failure of keratinocytes to adequately upregulate IL-1alpha and IL-1beta may promote S. aureus skin infections.

  17. The Inflammasome and the Epidermal Growth Factor Receptor (EGFR) Are Involved in the Staphylococcus aureus-Mediated Induction of IL-1alpha and IL-1beta in Human Keratinocytes.

    Science.gov (United States)

    Simanski, Maren; Rademacher, Franziska; Schröder, Lena; Gläser, Regine; Harder, Jürgen

    2016-01-01

    Staphylococcus (S.) aureus is an important pathogen causing various infections including those of the skin. Keratinocytes are able to sense invading S. aureus and to initiate a fast defense reaction by the rapid release of innate defense mediators such as antimicrobial peptides and cytokines. There is increasing evidence that the cytokines IL-1alpha and IL-1beta, which both signal through the IL-1 receptor, play an important role in cutaneous defense against S. aureus. The aim of this study was to gain more insight into the underlying mechanisms leading to the S. aureus-induced IL-1alpha and IL-1beta expression in keratinocytes. Infection of human primary keratinocytes with S. aureus led to the induction of gene expression and protein secretion of IL-1alpha and IL-1beta. Full S. aureus-induced IL-1 protein release required the inflammasome components caspase-1 and ASC (apoptosis-associated speck-like protein containing a CARD) whereas gene induction of IL-1alpha and IL-beta by S. aureus was not dependent on caspase-1 and ASC. Since patients receiving anti-cancer therapy by inhibition of the epidermal growth factor receptor (EGFR) often suffer from skin infections caused by S. aureus we additionally evaluated whether the EGFR pathway may be involved in the IL-1alpha and IL-1beta induction by S. aureus. Inactivation of the EGFR with a blocking antibody decreased the S. aureus-mediated IL-1alpha and IL-1beta induction in primary keratinocytes. Moreover, the use of siRNA experiments revealed that ADAM17 (A Disintegrin and A Metalloprotease 17), a metalloproteinase known to mediate the shedding and release of EGFR ligands, was required for full induction of IL-1alpha and IL-1beta in keratinocytes infected with S. aureus. A failure of keratinocytes to adequately upregulate IL-1alpha and IL-1beta may promote S. aureus skin infections.

  18. Adhesive polypeptides of Staphylococcus aureus identified using a novel secretion library technique in Escherichia coli

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    Holm Liisa

    2011-05-01

    Full Text Available Abstract Background Bacterial adhesive proteins, called adhesins, are frequently the decisive factor in initiation of a bacterial infection. Characterization of such molecules is crucial for the understanding of bacterial pathogenesis, design of vaccines and development of antibacterial drugs. Because adhesins are frequently difficult to express, their characterization has often been hampered. Alternative expression methods developed for the analysis of adhesins, e.g. surface display techniques, suffer from various drawbacks and reports on high-level extracellular secretion of heterologous proteins in Gram-negative bacteria are scarce. These expression techniques are currently a field of active research. The purpose of the current study was to construct a convenient, new technique for identification of unknown bacterial adhesive polypeptides directly from the growth medium of the Escherichia coli host and to identify novel proteinaceous adhesins of the model organism Staphylococcus aureus. Results Randomly fragmented chromosomal DNA of S. aureus was cloned into a unique restriction site of our expression vector, which facilitates secretion of foreign FLAG-tagged polypeptides into the growth medium of E. coli ΔfliCΔfliD, to generate a library of 1663 clones expressing FLAG-tagged polypeptides. Sequence and bioinformatics analyses showed that in our example, the library covered approximately 32% of the S. aureus proteome. Polypeptides from the growth medium of the library clones were screened for binding to a selection of S. aureus target molecules and adhesive fragments of known staphylococcal adhesins (e.g coagulase and fibronectin-binding protein A as well as polypeptides of novel function (e.g. a universal stress protein and phosphoribosylamino-imidazole carboxylase ATPase subunit were detected. The results were further validated using purified His-tagged recombinant proteins of the corresponding fragments in enzyme-linked immunoassay and

  19. Genomics of Natural Populations of Staphylococcus aureus.

    Science.gov (United States)

    Fitzgerald, J Ross; Holden, Matthew T G

    2016-09-01

    Staphylococcus aureus is a major human pathogen and an important cause of livestock infections. The first S. aureus genomes to be published, 15 years ago, provided the first view of genome structure and gene content. Since then, thousands of genomes from a wide array of strains from different sources have been sequenced. Comparison of these sequences has resulted in broad insights into population structure, bacterial evolution, clone emergence and expansion, and the molecular basis of niche adaptation. Furthermore, this information is now being applied clinically in outbreak investigations to inform infection control measures and to determine appropriate treatment regimens. In this review, we summarize some of the broad insights into S. aureus biology gained from the analysis of genomes and discuss future directions and opportunities in this dynamic field of research.

  20. Evolution of methicillin-resistant Staphylococcus aureus towards increasing resistance

    DEFF Research Database (Denmark)

    Strommenger, Birgit; Bartels, Mette Damkjær; Kurt, Kevin

    2014-01-01

    To elucidate the evolutionary history of Staphylococcus aureus clonal complex (CC) 8, which encompasses several globally distributed epidemic lineages, including hospital-associated methicillin-resistant S. aureus (MRSA) and the highly prevalent community-associated MRSA clone USA300....

  1. Toxicity test and bacteriophage typing of Staphylococcus aureus ...

    African Journals Online (AJOL)

    African Journal of Biotechnology ... Strains of Staphylococcus aureus isolated from foods prepared in five families in Zaria Local ... Keywords: Staphylococcus aureus, enterotoxin production, phage typing, haemolysis and food poisoning ...

  2. Propionibacterium acnes biofilm - A sanctuary for Staphylococcus aureus?

    Science.gov (United States)

    Tyner, Harmony; Patel, Robin

    2016-08-01

    The purpose of this study was to measure the effect of combined culture of Propionibacterium acnes and Staphylococcus aureus on biofilm formation under different oxygen concentrations. We measured planktonic growth and biofilm formation of P. acnes and S. aureus alone and together under aerobic and anaerobic conditions. Both P. acnes and S. aureus grew under anaerobic conditions. When grown under anaerobic conditions, P. acnes with or without S. aureus formed a denser biomass biofilm than did S. aureus alone. Viable S. aureus was recovered from a16-day old combined P. acnes and S. aureus biofilm, but not a monomicrobial S. aureus biofilm.

  3. Inhibitory Effect of Lactobacillus plantarum Extracts on HT-29 Colon Cancer Cell Apoptosis Induced by Staphylococcus aureus and Its Alpha-Toxin.

    Science.gov (United States)

    Kim, Hangeun; Kim, Hye Sun; Park, Woo Jung; Chung, Dae Kyun

    2015-11-01

    Staphylococcus aureus plays an important role in sepsis, septic shock, pneumonia, and wound infections. Here, we demonstrate that Lactobacillus plantarum extracts inhibited S. aureusinduced cell death of a human epithelial cell line, HT-29. In particular, we have shown that S. aureus-induced cell death was abolished by neutralization of α-toxin, indicating that α-toxin is the major mediator of S. aureus-induced cell death. DNA fragmentation experiment and caspase assay revealed that the S. aureus-induced cell death was apoptosis. L. plantarum extracts inhibited the generation of effector caspase-3 and the initiator caspase-9 in S. aureusor α-toxin-induced cell death. Moreover, expression of Bcl-2, an anti-apoptotic protein, was activated in L. plantarum extract-treated cells as compared with the S. aureus- or α-toxintreated only cells. Furthermore, S. aureus-induced apoptosis was efficiently inhibited by lipoteichoic acid and peptidoglycan of L. plantarum. Together, our results suggest that L. plantarum extracts can inhibit the S. aureus-mediated apoptosis, which is associated with S. aureus spreading, in intestinal epithelial cells, and may provide a new therapeutic reagent to treat bacterial infections.

  4. Mastite com lesões sistêmicas por Staphylococus aureus subesp. aureus em coelhos Mastitis with systemic lesions due to Staphylococus aureus subesp. aureus in rabbits

    Directory of Open Access Journals (Sweden)

    Sandra Davi Traverso

    2003-04-01

    Full Text Available Em uma criação composta por 1800 coelhos, 33% das matrizes apresentaram mastite e lesões cutâneas crostosas e purulentas. Estes animais apresentavam-se entre 10 a- 12 meses de idade e em segunda parição. Quinze coelhos afetados foram sacrificados e necropsiados. Na necropsia, além das lesões cutâneas haviam microabscessos em diversos órgãos. Das amostras coletadas isolou-se Staphylococcus aureus subesp. aureus. S. aureus subesp. aureus também foi isolado de "swab" nasal coletado do tratador encarregado de fazer o diagnóstico de gestação nas coelhas. Histologicamente, havia formação de múltiplos abscessos, presença de bactérias gram positivas em vasos sangüíneos e linfáticos, além de êmbolos bacterianos nos tecidos. Nas mamas, observou-se tecido glandular normal associado a abscessos multifocais delimitados.At a commercial rabbitry which was composed of 1800 New Zealand White rabbits, 30% of the does had presented mastitis and purulent cutaneal lesions. The age of the animals ranged from 10 to 12 months and were at the second parturition. At necropsy, microabscesses were observed in several organs. Bacteriological samples collected from affected animals resulted Staphylococcus aureus subesp. aureus.. Additionally, the same agent has been isolated from a nasal swab collected from the person responsible for the pregnancy diagnosis. Histologically, there were multiple abscesses, gram positive bacteria within blood and lymphatic vessels, and bacterial emboli scattered in the tissues. In the mammas, normal glandular tissue associated with multifocal abscesses were observed.

  5. Improved accuracy of cell surface shaving proteomics in Staphylococcus aureus using a false-positive control

    DEFF Research Database (Denmark)

    Solis, Nestor; Larsen, Martin Røssel; Cordwell, Stuart J

    2010-01-01

    Proteolytic treatment of intact bacterial cells is an ideal means for identifying surface-exposed peptide epitopes and has potential for the discovery of novel vaccine targets. Cell stability during such treatment, however, may become compromised and result in the release of intracellular proteins...... that complicate the final analysis. Staphylococcus aureus is a major human pathogen, causing community and hospital-acquired infections, and is a serious healthcare concern due to the increasing prevalence of multiple antibiotic resistances amongst clinical isolates. We employed a cell surface "shaving" technique...... lysis and were removed from the trypsin-shaved data set. We identified 42 predicted S. aureus COL surface proteins from 260 surface-exposed peptides. Trypsin and proteinase-K digests were highly complementary with ten proteins identified by both, 16 specific to proteinase-K treatment, 13 specific...

  6. X-ray crystal structure of Staphylococcus aureus FemA.

    Science.gov (United States)

    Benson, Timothy E; Prince, D Bryan; Mutchler, Veronica T; Curry, Kimberly A; Ho, Andrea M; Sarver, Ronald W; Hagadorn, Jeanne C; Choi, Gil H; Garlick, Robert L

    2002-08-01

    The latter stages of peptidoglycan biosynthesis in Staphylococci involve the synthesis of a pentaglycine bridge on the epsilon amino group of the pentapeptide lysine side chain. Genetic and biochemical evidence suggest that sequential addition of these glycines is catalyzed by three homologous enzymes, FemX (FmhB), FemA, and FemB. The first protein structure from this family, Staphylococcus aureus FemA, has been solved at 2.1 A resolution by X-ray crystallography. The FemA structure reveals a unique organization of several known protein folds involved in peptide and tRNA binding. The surface of the protein also reveals an L-shaped channel suitable for a peptidoglycan substrate. Analysis of the structural features of this enzyme provides clues to the mechanism of action of S. aureus FemA.

  7. Crystal Structure of an Invasivity-Associated Domain of SdrE in S. aureus

    Science.gov (United States)

    Zhang, Hongpeng; Yang, Wei; Zhu, Zhongliang; Chen, Ke; Bai, Lei; Wei, Jie; Huang, Ailong; Wang, Deqiang

    2017-01-01

    The surface protein SdrE, a microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family protein expressed on the surface of Staphylococcus aureus (S. aureus), can recognize human complement regulator Factor H and C4BP, thus making it a potentially promising vaccine candidate. In this study, SdrE278-591 was found to directly affect S. aureus host cell invasion. Additionally, the crystal structure of SdrE278-591 at a resolution of 1.25 Å was established, with the three-dimensional structure revealing N2-N3 domains which fold in a manner similar to an IgG fold. Furthermore, a putative ligand binding site located at a conserved charged groove formed by the interface between N2 and N3 domains was identified, with β2 suspected to occupy the ligand recognizing site and undergo a structural rearrangement to allow ligand binding. Overall, these findings have further contributed to the understanding of SdrE as a key factor for S. aureus invasivity and will enable a better understanding of bacterial infection processes. PMID:28125581

  8. Staphylococcus aureus aggregation and coagulation mechanisms, and their function in host-pathogen interactions

    Science.gov (United States)

    Crosby, Heidi A.; Kwiecinski, Jakub; Horswill, Alexander R.

    2017-01-01

    The human commensal bacterium Staphylococcus aureus can cause a wide range of infections ranging from skin and soft tissue infections to invasive diseases like septicemia, endocarditis, and pneumonia. Muticellular organization almost certainly contributes to S. aureus pathogenesis mechanisms. While there has been considerable focus on biofilm formation and its role in colonizing prosthetic joints and indwelling devices, less attention has been paid to non-surface attached group behavior like aggregation and clumping. S. aureus is unique in its ability to coagulate blood, and it also produces multiple fibrinogen-binding proteins that facilitate clumping. Formation of clumps, which are large, tightly-packed groups of cells held together by fibrin(ogen), has been demonstrated to be important for S. aureus virulence and immune evasion. Clumps of cells are able to avoid detection by the host’s immune system due to a fibrin(ogen) coat that acts as a shield, and the size of the clumps facilitates evasion of phagocytosis. In addition, clumping could be an important early step in establishing infections that involve tight clusters of cells embedded in host matrix proteins, such as soft tissue abscesses and endocarditis. In this review we discuss clumping mechanisms and regulation, as well as what is known about how clumping contributes to immune evasion. PMID:27565579

  9. Characterization, prevalence and antibiogram study of Staphylococcus aureus in poultry

    OpenAIRE

    Yeasmeen Ali; Md. Ashraful Islam; Nazmul Hasan Muzahid; Mohd. Omar Faruk Sikder; Md. Amzad Hossain; Lolo Wal Marzan

    2017-01-01

    Objective: To reveal the presence of methicillin resistant Staphylococcus aureus (S. aureus) (MRSA) in poultry samples and to determine the antibiogram pattern against five antibiotics. Methods: Samples from different poultry farm of Chittagong city, Bangladesh were examined for S. aureus by different biochemical tests and confirmed as MRSA by identifying the presence of mecA gene using PCR. Antibiotic resistance pattern in S. aureus was determined by antibiotic disk diffusion method. R...

  10. Epidemiology of Staphylococcus aureus during space flight

    Science.gov (United States)

    Pierson, D. L.; Chidambaram, M.; Heath, J. D.; Mallary, L.; Mishra, S. K.; Sharma, B.; Weinstock, G. M.

    1996-01-01

    Staphylococcus aureus was isolated over 2 years from Space Shuttle mission crewmembers to determine dissemination and retention of bacteria. Samples before and after each mission were from nasal, throat, urine, and feces and from air and surface sampling of the Space Shuttle. DNA fingerprinting of samples by digestion of DNA with SmaI restriction endonuclease followed by pulsed-field gel electrophoresis showed S. aureus from each crewmember had a unique fingerprint and usually only one strain was carried by an individual. There was only one instance of transfer between crewmembers. Strains from interior surfaces after flight matched those of crewmembers, suggesting microbial fingerprinting may have forensic application.

  11. METHODS COMPARISON FOR ENTEROTOXIC STAPHYLOCOCCUS AUREUS CHARACTERIZATION

    Directory of Open Access Journals (Sweden)

    L. Decastelli

    2011-01-01

    Full Text Available The aim of this study was to compare two different methods for enterotoxic Staphylococcus aureus characterization. 110 S.aureus strains was isolated from foods and tested with ELISA method able to detect toxins type A to E in culture medium and PCR protocols able to detect the presence of genes (sea to see; seg to sej; sep; ser encoding for staphylococcal enterotoxins. 27 strains came out positive with ELISA; 68 resulted to have at least one encoding gene. sea and ser genes were detected respectively in 29,1% and 27,3% of strains.

  12. Hydrogen Sulfide and Reactive Sulfur Species Impact Proteome S-Sulfhydration and Global Virulence Regulation in Staphylococcus aureus.

    Science.gov (United States)

    Peng, Hui; Zhang, Yixiang; Palmer, Lauren D; Kehl-Fie, Thomas E; Skaar, Eric P; Trinidad, Jonathan C; Giedroc, David P

    2017-09-06

    Hydrogen sulfide (H2S) is thought to protect bacteria from oxidative stress, but a comprehensive understanding of its function in bacteria is largely unexplored. In this study, we show that the human pathogen Staphylococcus aureus (S. aureus) harbors significant effector molecules of H2S signaling, reactive sulfur species (RSS), as low molecular weight persulfides of bacillithiol, coenzyme A, and cysteine, and significant inorganic polysulfide species. We find that proteome S-sulfhydration, a post-translational modification (PTM) in H2S signaling, is widespread in S. aureus. RSS levels modulate the expression of secreted virulence factors and the cytotoxicity of the secretome, consistent with an S-sulfhydration-dependent inhibition of DNA binding by MgrA, a global virulence regulator. Two previously uncharacterized thioredoxin-like proteins, denoted TrxP and TrxQ, are S-sulfhydrated in sulfide-stressed cells and are capable of reducing protein hydrodisulfides, suggesting that this PTM is potentially regulatory in S. aureus. In conclusion, our results reveal that S. aureus harbors a pool of proteome- and metabolite-derived RSS capable of impacting protein activities and gene regulation and that H2S signaling can be sensed by global regulators to affect the expression of virulence factors.

  13. Characterization of the modular design of the autolysin/adhesin Aaa from Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Nina Hirschhausen

    Full Text Available BACKGROUND: Staphylococcus aureus is a frequent cause of serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, and sepsis. Its adherence to various host structures is crucial for the establishment of diseases. Adherence may be mediated by a variety of adhesins, among them the autolysin/adhesins Atl and Aaa. Aaa is composed of three N-terminal repeated sequences homologous to a lysin motif (LysM that can confer cell wall attachment and a C-terminally located cysteine, histidine-dependent amidohydrolase/peptidase (CHAP domain having bacteriolytic activity in many proteins. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show by surface plasmon resonance that the LysM domain binds to fibrinogen, fibronectin, and vitronectin respresenting a novel adhesive function for this domain. Moreover, we demonstrated that the CHAP domain not only mediates the bacteriolytic activity, but also adherence to fibrinogen, fibronectin, and vitronectin, thus demonstrating for the first time an adhesive function for this domain. Adherence of an S. aureus aaa mutant and the complemented aaa mutant is slightly decreased and increased, respectively, to vitronectin, but not to fibrinogen and fibronectin, which might at least in part result from an increased expression of atl in the aaa mutant. Furthermore, an S. aureus atl mutant that showed enhanced adherence to fibrinogen, fibronectin, and endothelial cells also demonstrated increased aaa expression and production of Aaa. Thus, the redundant functions of Aaa and Atl might at least in part be interchangeable. Lastly, RT-PCR and zymographic analysis revealed that aaa is negatively regulated by the global virulence gene regulators agr and SarA. CONCLUSIONS/SIGNIFICANCE: We identified novel functions for two widely distributed protein domains, LysM and CHAP, i.e. the adherence to the extracellular matrix proteins fibrinogen, fibronectin, and vitronectin. The adhesive properties of Aaa might

  14. Prevalence of clonal complexes and virulence genes among commensal and invasive Staphylococcus aureus isolates in Sweden.

    Directory of Open Access Journals (Sweden)

    Gunlög Rasmussen

    Full Text Available Staphylococcus aureus encodes a remarkable number of virulence factors which may contribute to its pathogenicity and ability to cause invasive disease. The main objective of this study was to evaluate the association between S. aureus invasiveness and bacterial genotype, in terms of the presence of virulence genes and affiliation to clonal complexes. Also, the significance of different virulence genes, mainly adhesins, for the development of infective endocarditis was investigated. DNA microarray technology was used to analyze 134 S. aureus isolates, all methicillin-susceptible, derived from three groups of clinically well-characterized patients: nasal carriers (n=46, bacteremia (n=55, and bacteremia with infective endocarditis (n=33. Invasive isolates were dominant in four of the major clonal complexes: 5, 8, 15, and 25. Of the 170 virulence genes examined, those encoding accessory gene regulator group II (agr II, capsule polysaccharide serotype 5 (cap5, and adhesins such as S. aureus surface protein G (sasG and fibronectin-binding protein B (fnbB were found to be associated with invasive disease. The same was shown for the leukocidin genes lukD/lukE, as well as the genes encoding serine protease A and B (splA/splB, staphylococcal complement inhibitor (scn and the staphylococcal exotoxin-like protein (setC or selX. In addition, there was a trend of higher prevalence of certain genes or gene clusters (sasG, agr II, cap5 among isolates causing infective endocarditis compared to other invasive isolates. In most cases, the presence of virulence genes was linked to clonal complex affiliation. In conclusion, certain S. aureus clonal lineages harboring specific sets of virulence genes seem to be more successful in causing invasive disease.

  15. 9 CFR 113.115 - Staphylococcus Aureus Bacterin-Toxoid.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Staphylococcus Aureus Bacterin-Toxoid... REQUIREMENTS Inactivated Bacterial Products § 113.115 Staphylococcus Aureus Bacterin-Toxoid. Staphylococcus... Staphylococcus aureus which has been inactivated and is nontoxic. Each serial of biological product...

  16. A pig model of acute Staphylococcus aureus induced pyemia

    DEFF Research Database (Denmark)

    Nielsen, O. L.; Iburg, T.; Aalbæk, B.;

    2009-01-01

    Background: Sepsis caused by Staphylococcus aureus constitutes an important cause of morbidity and mortality in humans, and the incidence of this disease-entity is increasing. In this paper we describe the initial microbial dynamics and lesions in pigs experimentally infected with S. aureus....... aureus isolated from man and an extension of the timeframe aiming at inducing sepsis, severe sepsis and septic shock....

  17. Detection of some virulence factors in Staphylococcus aureus ...

    African Journals Online (AJOL)

    USER

    2010-06-21

    Jun 21, 2010 ... Among a 360 raw milk samples, 86 samples contained 1250 bp ... Key words: Bovine mastitis, Staphylococcus aureus, virulence ... Some strains of S. aureus produce one or both of two ... about the occurrence of these toxins among S. aureus ..... ear infection and chicken infections were studied and it.

  18. The molecular evolution of methicillin-resistant Staphylococcus aureus

    NARCIS (Netherlands)

    Deurenberg, R H; Vink, C; Kalenic, S; Friedrich, A W; Bruggeman, C A; Stobberingh, E E

    2007-01-01

    Staphylococcus aureus is a potentially pathogenic bacterium that causes a broad spectrum of diseases. S. aureus can adapt rapidly to the selective pressure of antibiotics, and this has resulted in the emergence and spread of methicillin-resistant S. aureus (MRSA). Resistance to methicillin and other

  19. Streptokinase Treatment Reverses Biofilm-Associated Antibiotic Resistance in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Nis Pedersen Jørgensen

    2016-09-01

    Full Text Available Biofilms formed by Staphylococcus aureus is a serious complication to the use of medical implants. A central part of the pathogenesis relies on S. aureus’ ability to adhere to host extracellular matrix proteins, which adsorb to medical implants and stimulate biofilm formation. Being coagulase positive, S. aureus furthermore induces formation of fibrin fibers from fibrinogen in the blood. Consequently, we hypothesized that fibrin is a key component of the extracellular matrix of S. aureus biofilms under in vivo conditions, and that the recalcitrance of biofilm infections can be overcome by combining antibiotic treatment with a fibrinolytic drug. We quantified S. aureus USA300 biofilms grown on peg-lids in brain heart infusion (BHI broth with 0%–50% human plasma. Young (2 h and mature (24 h biofilms were then treated with streptokinase to determine if this lead to dispersal. Then, the minimal biofilm eradication concentration (MBEC of 24 h old biofilms was measured for vancomycin and daptomycin alone or in combination with 10 µg/mL rifampicin in the presence or absence of streptokinase in the antibiotic treatment step. Finally, biofilms were visualized by confocal laser scanning microscopy. Addition of human plasma stimulated biofilm formation in BHI in a dose-dependent manner, and biofilms could be partially dispersed by streptokinase. The biofilms could be eradicated with physiologically relevant concentrations of streptokinase in combination with rifampicin and vancomycin or daptomycin, which are commonly used antibiotics for treatment of S. aureus infections. Fibronolytic drugs have been used to treat thromboembolic events for decades, and our findings suggest that their use against biofilm infections has the potential to improve the efficacy of antibiotics in treatment of S. aureus biofilm infections.

  20. A coverslip-based technique for evaluating Staphylococcus aureus biofilm formation on human plasma

    Directory of Open Access Journals (Sweden)

    Jennifer N Walker

    2012-03-01

    Full Text Available The ability of the opportunistic pathogen, Staphylococcus aureus, to form biofilms is increasingly being viewed as an important contributor to chronic infections. In vitro methods for analyzing S. aureus biofilm formation have focused on bacterial attachment and accumulation on abiotic surfaces, such as in microtiter plate and flow cell assays. Microtiter plates provide a rapid measure of relative biomass levels, while flow cells have limited experimental throughput but are superior for confocal microscopy biofilm visualization. Although these assays have proven effective at identifying mechanisms involved in cell attachment and biofilm accumulation, the significance of these assays in vivo remains unclear. Studies have shown that when medical devices are implanted they are coated with host factors, such as matrix proteins, that facilitate S. aureus attachment and biofilm formation. To address the challenge of integrating existing biofilm assay features with a biotic surface, we have established an in vitro biofilm technique utilizing UV-sterilized coverslips coated with human plasma. The substratum more closely resembles the in vivo state and provides a platform for S. aureus to establish a robust biofilm. Importantly, these coverslips are amenable to confocal microscopy imaging to provide a visual reference of the biofilm growth stage, effectively merging the benefits of the microtiter and flow cell assays. We confirmed the approach using clinical S. aureus isolates and mutants with known biofilm phenotypes. Altogether, this new biofilm assay can be used to assess the function of S. aureus virulence factors associated with biofilm formation and for monitoring the efficacy of biofilm treatment modalities.

  1. Streptokinase Treatment Reverses Biofilm-Associated Antibiotic Resistance in Staphylococcus aureus

    Science.gov (United States)

    Jørgensen, Nis Pedersen; Zobek, Natalia; Dreier, Cindy; Haaber, Jakob; Ingmer, Hanne; Larsen, Ole Halfdan; Meyer, Rikke L.

    2016-01-01

    Biofilms formed by Staphylococcus aureus is a serious complication to the use of medical implants. A central part of the pathogenesis relies on S. aureus’ ability to adhere to host extracellular matrix proteins, which adsorb to medical implants and stimulate biofilm formation. Being coagulase positive, S. aureus furthermore induces formation of fibrin fibers from fibrinogen in the blood. Consequently, we hypothesized that fibrin is a key component of the extracellular matrix of S. aureus biofilms under in vivo conditions, and that the recalcitrance of biofilm infections can be overcome by combining antibiotic treatment with a fibrinolytic drug. We quantified S. aureus USA300 biofilms grown on peg-lids in brain heart infusion (BHI) broth with 0%–50% human plasma. Young (2 h) and mature (24 h) biofilms were then treated with streptokinase to determine if this lead to dispersal. Then, the minimal biofilm eradication concentration (MBEC) of 24 h old biofilms was measured for vancomycin and daptomycin alone or in combination with 10 µg/mL rifampicin in the presence or absence of streptokinase in the antibiotic treatment step. Finally, biofilms were visualized by confocal laser scanning microscopy. Addition of human plasma stimulated biofilm formation in BHI in a dose-dependent manner, and biofilms could be partially dispersed by streptokinase. The biofilms could be eradicated with physiologically relevant concentrations of streptokinase in combination with rifampicin and vancomycin or daptomycin, which are commonly used antibiotics for treatment of S. aureus infections. Fibronolytic drugs have been used to treat thromboembolic events for decades, and our findings suggest that their use against biofilm infections has the potential to improve the efficacy of antibiotics in treatment of S. aureus biofilm infections. PMID:27681928

  2. Effect of Substance P in Staphylococcus aureus and Staphylococcus epidermidis virulence: Implication for skin homeostasis

    Directory of Open Access Journals (Sweden)

    Awa eNdiaye

    2016-04-01

    Full Text Available Staphylococcus aureus and Staphylococcus epidermidis are two major skin associated bacteria, and Substance P (SP is a major skin neuropeptide. Since bacteria are known to sense and response to many human hormones, we investigated the effects of SP on Staphylococci virulence in reconstructed human epidermis model and HaCaT keratinocytes. We show that SP is stimulating the virulence of Staphylococcus aureus and Staphylococcus epidermidis in a reconstructed human epidermis model. qRT-PCR array analysis of 64 genes expressed by keratinocytes in the response to bacterial infection revealed a potential link between the action of SP on Staphylococci and skin physiopathology. qRT-PCR and direct assay of cathelicidin and human β-defensin 2 secretion also provided that demonstration that the action of SP on bacteria is independent of antimicrobial peptide expression by keratinocytes. Considering an effect of SP on S. aureus and S. epidermidis, we observed that SP increases the adhesion potential of both bacteria on keratinocytes. However, SP modulates the virulence of S. aureus and S. epidermidis through different mechanisms. The response of S. aureus is associated with an increase in Staphylococcal Enterotoxin C2 (SEC2 production and a reduction of exolipase processing whereas in S. epidermidis the effect of SP appears mediated by a rise in biofilm formation activity. The Thermo unstable ribosomal Elongation factor Ef-Tu was identified as the SP-interacting protein in S. aureus and S. epidermidis. SP appears as an inter-kingdom communication factor involved in the regulation of bacterial virulence and essential for skin microflora homeostasis.

  3. Multidrug Efflux Pumps in Staphylococcus aureus: an Update.

    Science.gov (United States)

    Costa, Sofia Santos; Viveiros, Miguel; Amaral, Leonard; Couto, Isabel

    2013-01-01

    The emergence of infections caused by multi- or pan-resistant bacteria in the hospital or in the community settings is an increasing health concern. Albeit there is no single resistance mechanism behind multiresistance, multidrug efflux pumps, proteins that cells use to detoxify from noxious compounds, seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria. During the last decades, experimental data has established their contribution to low level resistance to antimicrobials in bacteria and their potential role in the appearance of MDR phenotypes, by the extrusion of multiple, unrelated compounds. Recent studies suggest that efflux pumps may be used by the cell as a first-line defense mechanism, avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration occurs, that allows survival in the presence of that agent. In this paper we review the current knowledge on MDR efflux pumps and their intricate regulatory network in Staphylococcus aureus, a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that S. aureus MDR efflux pumps, either chromosomal or plasmid-encoded, have on resistance towards different antimicrobial agents and on the selection of drug - resistant strains. We will also discuss the many questions that still remain on the role of each specific efflux pump and the need to establish appropriate methodological approaches to address all these questions.

  4. Autophagy mediates tolerance to Staphylococcus aureus alpha-toxin.

    Science.gov (United States)

    Maurer, Katie; Reyes-Robles, Tamara; Alonzo, Francis; Durbin, Joan; Torres, Victor J; Cadwell, Ken

    2015-04-01

    Resistance and tolerance are two defense strategies employed by the host against microbial threats. Autophagy-mediated degradation of bacteria has been extensively described as a major resistance mechanism. Here we find that the dominant function of autophagy proteins during infections with the epidemic community-associated methicillin-resistant Staphylococcus aureus USA300 is to mediate tolerance rather than resistance. Atg16L1 hypomorphic mice (Atg16L1(HM)), which have reduced autophagy, were highly susceptible to lethality in both sepsis and pneumonia models of USA300 infection. Autophagy confers protection by limiting the damage caused by α-toxin, particularly to endothelial cells. Remarkably, Atg16L1(HM) mice display enhanced survival rather than susceptibility upon infection with α-toxin-deficient S. aureus. These results identify an essential role for autophagy in tolerance to Staphylococcal disease and highlight how a single virulence factor encoded by a pathogen can determine whether a given host factor promotes tolerance or resistance.

  5. SpA, ClfA, and FnbA Genetic Variations Lead to Staphaurex Test-Negative Phenotypes in Bovine Mastitis Staphylococcus aureus Isolates▿

    Science.gov (United States)

    Stutz, Katrin; Stephan, Roger; Tasara, Taurai

    2011-01-01

    Staphylococcus aureus encodes many proteins that act as virulence factors, leading to a variety of diseases, including mastitis in cows. Among these virulence factors, SpA, ClfA, ClfB, FnbA, and FnbB are important for the ability of S. aureus to adhere to and invade host cells as well as to evade host immune responses. The interaction between these S. aureus surface proteins and human immunoglobulin G and fibrinogen that are coupled to latex particles is utilized to induce latex agglutination reactions, which are used widely in diagnostic kits for confirmation of presumptive S. aureus isolates. In this study, the Staphaurex latex agglutination test was performed on a collection of confirmed bovine mastitis S. aureus isolates. Notably, 54% (43/79 isolates) of these isolates exhibited latex agglutination-negative phenotypes (Staphaurex-negative result). To gain insights into the reasons for the high frequency of Staphaurex-negative bovine mastitis S. aureus isolates, the spa, clfA, clfB, fnbA, and fnbB genes were examined. Specific genetic changes in spa, clfA, and fnbA, as well as a loss of fnbB, which may impair SpA, ClfA, FnbA, and FnbB functions in latex agglutination reactions, were detected in Staphaurex-negative S. aureus isolates. The genetic changes included a premature stop codon in the spa gene, leading to a truncated SpA protein that is unable to participate in S. aureus cell-mediated agglutination of latex particles. In addition, clfA and fnbA genetic polymorphisms were detected that were linked to ClfA and FnbA amino acid changes that may significantly reduce fibrinogen-binding activity. The genetic variations in these S. aureus isolates might also have implications for their bovine mastitis virulence capacity. PMID:21147952

  6. Application of a Novel "Pan-Genome"-Based Strategy for Assigning RNAseq Transcript Reads to Staphylococcus aureus Strains.

    Science.gov (United States)

    Chaves-Moreno, Diego; Wos-Oxley, Melissa L; Jáuregui, Ruy; Medina, Eva; Oxley, Andrew P A; Pieper, Dietmar H

    2015-01-01

    Understanding the behaviour of opportunistic pathogens such as Staphylococcus aureus in their natural human niche holds great medical interest. With the development of sensitive molecular methods and deep-sequencing technology, it is now possible to robustly assess the global transcriptome of bacterial species in their human habitat. However, as the genomes of the colonizing strains are often not available compiling the pan-genome for the species of interest may provide an effective method to reliably and rapidly compile the transcriptome of a bacterial species. The pan-genome of S. aureus and its associated core and accessory components were compiled based on 25 genomes and comprises a total of 65,557 proteins clustering into 4,198 Orthologous Groups (OGs). The generated gene catalogue was used to assign RNAseq-derived sequence reads to S. aureus in a variety of in vitro and in vivo samples. In all cases, the number of reads that could be assigned to S. aureus was greater using the OG database than using a reference genome. Growth of two S. aureus strains in synthetic nasal medium confirmed that both strains experienced strong iron starvation. Traits such as purine metabolism appeared to be more affected in a typical nasal colonizer than in a strain representative of the S. aureus USA300 lineage. Mapping sequencing reads from a metatranscriptome generated from the human anterior nares allowed the identification of genes highly expressed by S. aureus in vivo. The OG database generated in this study represents a useful tool to obtain a snapshot of the functional attributes of S. aureus under different in vitro and in vivo conditions. The approach proved to be advantageous to assign sequencing reads to bacterial strains when RNAseq data is derived from samples where strain information and/or the corresponding genome/s are unavailable.

  7. Application of a Novel "Pan-Genome"-Based Strategy for Assigning RNAseq Transcript Reads to Staphylococcus aureus Strains.

    Directory of Open Access Journals (Sweden)

    Diego Chaves-Moreno

    Full Text Available Understanding the behaviour of opportunistic pathogens such as Staphylococcus aureus in their natural human niche holds great medical interest. With the development of sensitive molecular methods and deep-sequencing technology, it is now possible to robustly assess the global transcriptome of bacterial species in their human habitat. However, as the genomes of the colonizing strains are often not available compiling the pan-genome for the species of interest may provide an effective method to reliably and rapidly compile the transcriptome of a bacterial species. The pan-genome of S. aureus and its associated core and accessory components were compiled based on 25 genomes and comprises a total of 65,557 proteins clustering into 4,198 Orthologous Groups (OGs. The generated gene catalogue was used to assign RNAseq-derived sequence reads to S. aureus in a variety of in vitro and in vivo samples. In all cases, the number of reads that could be assigned to S. aureus was greater using the OG database than using a reference genome. Growth of two S. aureus strains in synthetic nasal medium confirmed that both strains experienced strong iron starvation. Traits such as purine metabolism appeared to be more affected in a typical nasal colonizer than in a strain representative of the S. aureus USA300 lineage. Mapping sequencing reads from a metatranscriptome generated from the human anterior nares allowed the identification of genes highly expressed by S. aureus in vivo. The OG database generated in this study represents a useful tool to obtain a snapshot of the functional attributes of S. aureus under different in vitro and in vivo conditions. The approach proved to be advantageous to assign sequencing reads to bacterial strains when RNAseq data is derived from samples where strain information and/or the corresponding genome/s are unavailable.

  8. Application of a Novel “Pan-Genome”-Based Strategy for Assigning RNAseq Transcript Reads to Staphylococcus aureus Strains

    Science.gov (United States)

    Chaves-Moreno, Diego; Wos-Oxley, Melissa L.; Jáuregui, Ruy; Medina, Eva

    2015-01-01

    Understanding the behaviour of opportunistic pathogens such as Staphylococcus aureus in their natural human niche holds great medical interest. With the development of sensitive molecular methods and deep-sequencing technology, it is now possible to robustly assess the global transcriptome of bacterial species in their human habitat. However, as the genomes of the colonizing strains are often not available compiling the pan-genome for the species of interest may provide an effective method to reliably and rapidly compile the transcriptome of a bacterial species. The pan-genome of S. aureus and its associated core and accessory components were compiled based on 25 genomes and comprises a total of 65,557 proteins clustering into 4,198 Orthologous Groups (OGs). The generated gene catalogue was used to assign RNAseq-derived sequence reads to S. aureus in a variety of in vitro and in vivo samples. In all cases, the number of reads that could be assigned to S. aureus was greater using the OG database than using a reference genome. Growth of two S. aureus strains in synthetic nasal medium confirmed that both strains experienced strong iron starvation. Traits such as purine metabolism appeared to be more affected in a typical nasal colonizer than in a strain representative of the S. aureus USA300 lineage. Mapping sequencing reads from a metatranscriptome generated from the human anterior nares allowed the identification of genes highly expressed by S. aureus in vivo. The OG database generated in this study represents a useful tool to obtain a snapshot of the functional attributes of S. aureus under different in vitro and in vivo conditions. The approach proved to be advantageous to assign sequencing reads to bacterial strains when RNAseq data is derived from samples where strain information and/or the corresponding genome/s are unavailable. PMID:26717500

  9. Vancomycin-induced biofilm formation by methicillin-resistant Staphylococcus aureus is associated with the secretion of membrane vesicles.

    Science.gov (United States)

    He, Xinlong; Yuan, Fenglai; Lu, Feng; Yin, Yinyan; Cao, Jun

    2017-09-01

    Chronic burn wound infections caused by Stapyhylococcus aureus (S. aureus) are largely associated with biofilm formation. However, the mechanism by which S. aureus form biofilm in clinical environments is far less understood. In the present study we addressed the association between biofilm formation and membrane vesicle (MV) secretion of S. aureus during vancomycin treatment. A representative methicillin-resistant S. aureus (MRSA) strain BWMR22 obtained from a chronic burn wound was used in this study. Transmission electron microscope was used to observe the MV secretion. Fourier transform infrared spectroscopy was used to analyze the chemical component of MV. Biofilm formation was assayed under conditions of sub-inhibitory concentrations of vancomycin. Functional potencies of MV in surface adhesion and auto-aggregation were assayed in the presence of additional purified MVs. Biofilm formation by S. aureus BWMR22 was enhanced in the presence of sub-inhibitory concentration of vancomycin. Vancomycin treatment caused an increase in the chemical composition of protein relative to carbohydrates of secreted MVs, a property which was highly associated with bacterial hydrophobicity, surface adhesion, and intercellular aggregation. These findings suggest that MV secretion is correlated with biofilm formation by MRSA especially under clinical conditions with improper vancomycin chemotherapy. This study first demonstrates a potential role of MVs in the biofilm formation by S. aureus, stresses on the importance of avoiding low dose of antibiotic therapy in controlling of S. aureus infections, and provides further information to reveal the mechanisms behind MRSA infections. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Persistent Staphylococcus aureus Isolates from Two Independent Cases of Bacteremia Display Increased Bacterial Fitness and Novel Immune Evasion Phenotypes

    Science.gov (United States)

    Richards, R. L.; Haigh, R. D.; Pascoe, B.; Sheppard, S. K.; Price, F.; Jenkins, D.; Rajakumar, K.

    2015-01-01

    Staphylococcus aureus bacteremia cases are complicated by bacterial persistence and treatment failure despite the confirmed in vitro susceptibility of the infecting strain to administered antibiotics. A high incidence of methicillin-resistant S. aureus (MRSA) bacteremia cases are classified as persistent and are associated with poorer patient outcomes. It is still unclear how S. aureus evades the host immune system and resists antibiotic treatment for the prolonged duration of a persistent infection. In this study, the genetic changes and associated phenotypic traits specific to S. aureus persistent bacteremia were identified by comparing temporally dispersed isolates from persistent infections (persistent isolates) originating from two independent persistent S. aureus bacteremia cases with the initial infection isolates and with three resolved S. aureus bacteremia isolates from the same genetic background. Several novel traits were associated specifically with both independent sets of persistent S. aureus isolates compared to both the initial isolates and the isolates from resolved infections (resolved isolates). These traits included (i) increased growth under nutrient-poor conditions; (ii) increased tolerance of iron toxicity; (iii) higher expression of cell surface proteins involved in immune evasion and stress responses; and (iv) attenuated virulence in a Galleria mellonella larva infection model that was not associated with small-colony variation or metabolic dormancy such as had been seen previously. Whole-genome sequence analysis identified different single nucleotide mutations within the mprF genes of all the isolates with the adaptive persistence traits from both independent cases. Overall, our data indicate a novel role for MprF function during development of S. aureus persistence by increasing bacterial fitness and immune evasion. PMID:26056388

  11. Molecular Epidemiology of Staphylococcus aureus among Patients with Skin and Soft Tissue Infections in Two Chinese Hospitals

    Institute of Scientific and Technical Information of China (English)

    Fei-Fei Gu; Ye Chen; De-Ping Dong; Zhen Song; Xiao-Kui Guo; Yu-Xing Ni; Li-Zhong Han

    2016-01-01

    Background:Staphylococcus aureus is one of the predominant causes of skin and soft tissue infections (SSTIs),but limited data were available regarding the characterization of S.aureus from SSTIs patients in Jiangsu Province in China.We aimed to investigate the molecular epidemiology ofS.aureus among SSTIs patients in two hospitals of Jiangsu Province.Methods:Sixty-two patients with SSTIs from two Chinese hospitals in Jiangsu Province were enrolled in this study,and 62 S.aureus isolates were collected from February 2014 to January 2015.S.aureus isolates were characterized by antimicrobial susceptibility testing,toxin gene detection,and molecular typing with sequence type,Staphylococcus protein A gene type,accessorygeneregulator(agr)group,and Staphylococcal cassette chromosome mec type.Results:Sixteen (25.8%) methicillin-resistant S.aureus (MRSA) isolates were detected,and there was no isolate found resistant to vancomycin,teicoplanin,sulfamethoxazole-trimethoprim,and linezolid.The sei was the toxin gene most frequently found,and no lukS/F-PV-positive isolates were detected among the SSTIs' patients.Molecular analysis revealed that ST398 (10/62,16.1%;2 MRSA and 8 methicillin-susceptible S.aureus) to be the dominant clone,followed by ST5 (8/62,12.9%) and ST7 (8/62,12.9%).Conclusions:The livestock ST398 was the most common clone among patients with S.aureus SSTIs in Jiangsu Province,China.Surveillance and further studies on the important livestock ST398 clone in human infections are necessarily requested.

  12. Persistent Staphylococcus aureus isolates from two independent cases of bacteremia display increased bacterial fitness and novel immune evasion phenotypes.

    Science.gov (United States)

    Richards, R L; Haigh, R D; Pascoe, B; Sheppard, S K; Price, F; Jenkins, D; Rajakumar, K; Morrissey, J A

    2015-08-01

    Staphylococcus aureus bacteremia cases are complicated by bacterial persistence and treatment failure despite the confirmed in vitro susceptibility of the infecting strain to administered antibiotics. A high incidence of methicillin-resistant S. aureus (MRSA) bacteremia cases are classified as persistent and are associated with poorer patient outcomes. It is still unclear how S. aureus evades the host immune system and resists antibiotic treatment for the prolonged duration of a persistent infection. In this study, the genetic changes and associated phenotypic traits specific to S. aureus persistent bacteremia were identified by comparing temporally dispersed isolates from persistent infections (persistent isolates) originating from two independent persistent S. aureus bacteremia cases with the initial infection isolates and with three resolved S. aureus bacteremia isolates from the same genetic background. Several novel traits were associated specifically with both independent sets of persistent S. aureus isolates compared to both the initial isolates and the isolates from resolved infections (resolved isolates). These traits included (i) increased growth under nutrient-poor conditions; (ii) increased tolerance of iron toxicity; (iii) higher expression of cell surface proteins involved in immune evasion and stress responses; and (iv) attenuated virulence in a Galleria mellonella larva infection model that was not associated with small-colony variation or metabolic dormancy such as had been seen previously. Whole-genome sequence analysis identified different single nucleotide mutations within the mprF genes of all the isolates with the adaptive persistence traits from both independent cases. Overall, our data indicate a novel role for MprF function during development of S. aureus persistence by increasing bacterial fitness and immune evasion.

  13. Molecular Epidemiology of Staphylococcus aureus among Patients with Skin and Soft Tissue Infections in Two Chinese Hospitals

    Science.gov (United States)

    Gu, Fei-Fei; Chen, Ye; Dong, De-Ping; Song, Zhen; Guo, Xiao-Kui; Ni, Yu-Xing; Han, Li-Zhong

    2016-01-01

    Background: Staphylococcus aureus is one of the predominant causes of skin and soft tissue infections (SSTIs), but limited data were available regarding the characterization of S. aureus from SSTIs patients in Jiangsu Province in China. We aimed to investigate the molecular epidemiology of S. aureus among SSTIs patients in two hospitals of Jiangsu Province. Methods: Sixty-two patients with SSTIs from two Chinese hospitals in Jiangsu Province were enrolled in this study, and 62 S. aureus isolates were collected from February 2014 to January 2015. S. aureus isolates were characterized by antimicrobial susceptibility testing, toxin gene detection, and molecular typing with sequence type, Staphylococcus protein A gene type, accessory gene regulator (agr) group, and Staphylococcal cassette chromosome mec type. Results: Sixteen (25.8%) methicillin-resistant S. aureus (MRSA) isolates were detected, and there was no isolate found resistant to vancomycin, teicoplanin, sulfamethoxazole-trimethoprim, and linezolid. The sei was the toxin gene most frequently found, and no lukS/F-PV-positive isolates were detected among the SSTIs’ patients. Molecular analysis revealed that ST398 (10/62, 16.1%; 2 MRSA and 8 methicillin-susceptible S. aureus) to be the dominant clone, followed by ST5 (8/62, 12.9%) and ST7 (8/62, 12.9%). Conclusions: The livestock ST398 was the most common clone among patients with S. aureus SSTIs in Jiangsu Province, China. Surveillance and further studies on the important livestock ST398 clone in human infections are necessarily requested. PMID:27647191

  14. Staphylococcus aureus entrance into the dairy chain: Tracking S. aureus from dairy cow to cheese

    Directory of Open Access Journals (Sweden)

    Judith Kümmel

    2016-10-01

    Full Text Available Staphylococcus aureus is one of the most important contagious mastitis pathogens in dairy cattle. Due to its zoonotic potential, control of S. aureus is not only of great economic importance in the dairy industry but also a significant public health concern. The aim of this study was to decipher the potential of bovine udder associated S. aureus as reservoir for S. aureus contamination in dairy production and processing. From 18 farms, delivering their milk to an alpine dairy plant for the production of smeared semi-hard and hard cheese. 1176 quarter milk (QM samples of all cows in lactation (n = 294 and representative samples form bulk tank milk (BTM of all farms were surveyed for coagulase positive (CPS and coagulase negative Staphylococci (CNS. Furthermore, samples from different steps of the cheese manufacturing process were tested for CPS and CNS. As revealed by chemometric-assisted FTIR spectroscopy and molecular subtyping (spa typing and multi locus sequence typing, dairy cattle represent indeed an important, yet underreported, entrance point of S. aureus into the dairy chain. Our data clearly show that certain S. aureus subtypes are present in primary production as well as in the cheese processing at the dairy plant. However, although a considerable diversity of S. aureus subtypes was observed in QM and BTM at the farms, only certain S. aureus subtypes were able to enter and persist in the cheese manufacturing at the dairy plant and could be isolated from cheese until day fourteen of ripening. Farm strains belonging to the FTIR cluster B1 and B3, which show genetic characteristics (t2953, ST8, enterotoxin profile: sea/sed/sej of the recently described S. aureus genotype B, most successfully contaminated the cheese production at the dairy plant. Thus our study fosters the hypothesis that genotype B S. aureus represent a specific challenge in control of S. aureus in the dairy chain that requires effective clearance strategies and hygienic

  15. Staphylococcus aureus Entrance into the Dairy Chain: Tracking S. aureus from Dairy Cow to Cheese

    Science.gov (United States)

    Kümmel, Judith; Stessl, Beatrix; Gonano, Monika; Walcher, Georg; Bereuter, Othmar; Fricker, Martina; Grunert, Tom; Wagner, Martin; Ehling-Schulz, Monika

    2016-01-01

    Staphylococcus aureus is one of the most important contagious mastitis pathogens in dairy cattle. Due to its zoonotic potential, control of S. aureus is not only of great economic importance in the dairy industry but also a significant public health concern. The aim of this study was to decipher the potential of bovine udder associated S. aureus as reservoir for S. aureus contamination in dairy production and processing. From 18 farms, delivering their milk to an alpine dairy plant for the production of smeared semi-hard and hard cheese. one thousand hundred seventy six one thousand hundred seventy six quarter milk (QM) samples of all cows in lactation (n = 294) and representative samples form bulk tank milk (BTM) of all farms were surveyed for coagulase positive (CPS) and coagulase negative Staphylococci (CNS). Furthermore, samples from different steps of the cheese manufacturing process were tested for CPS and CNS. As revealed by chemometric-assisted FTIR spectroscopy and molecular subtyping (spa typing and multi locus sequence typing), dairy cattle represent indeed an important, yet underreported, entrance point of S. aureus into the dairy chain. Our data clearly show that certain S. aureus subtypes are present in primary production as well as in the cheese processing at the dairy plant. However, although a considerable diversity of S. aureus subtypes was observed in QM and BTM at the farms, only certain S. aureus subtypes were able to enter and persist in the cheese manufacturing at the dairy plant and could be isolated from cheese until day 14 of ripening. Farm strains belonging to the FTIR cluster B1 and B3, which show genetic characteristics (t2953, ST8, enterotoxin profile: sea/sed/sej) of the recently described S. aureus genotype B, most successfully contaminated the cheese production at the dairy plant. Thus, our study fosters the hypothesis that genotype B S. aureus represent a specific challenge in control of S. aureus in the dairy chain that requires

  16. Staphylococcus aureus Entrance into the Dairy Chain: Tracking S. aureus from Dairy Cow to Cheese.

    Science.gov (United States)

    Kümmel, Judith; Stessl, Beatrix; Gonano, Monika; Walcher, Georg; Bereuter, Othmar; Fricker, Martina; Grunert, Tom; Wagner, Martin; Ehling-Schulz, Monika

    2016-01-01

    Staphylococcus aureus is one of the most important contagious mastitis pathogens in dairy cattle. Due to its zoonotic potential, control of S. aureus is not only of great economic importance in the dairy industry but also a significant public health concern. The aim of this study was to decipher the potential of bovine udder associated S. aureus as reservoir for S. aureus contamination in dairy production and processing. From 18 farms, delivering their milk to an alpine dairy plant for the production of smeared semi-hard and hard cheese. one thousand hundred seventy six one thousand hundred seventy six quarter milk (QM) samples of all cows in lactation (n = 294) and representative samples form bulk tank milk (BTM) of all farms were surveyed for coagulase positive (CPS) and coagulase negative Staphylococci (CNS). Furthermore, samples from different steps of the cheese manufacturing process were tested for CPS and CNS. As revealed by chemometric-assisted FTIR spectroscopy and molecular subtyping (spa typing and multi locus sequence typing), dairy cattle represent indeed an important, yet underreported, entrance point of S. aureus into the dairy chain. Our data clearly show that certain S. aureus subtypes are present in primary production as well as in the cheese processing at the dairy plant. However, although a considerable diversity of S. aureus subtypes was observed in QM and BTM at the farms, only certain S. aureus subtypes were able to enter and persist in the cheese manufacturing at the dairy plant and could be isolated from cheese until day 14 of ripening. Farm strains belonging to the FTIR cluster B1 and B3, which show genetic characteristics (t2953, ST8, enterotoxin profile: sea/sed/sej) of the recently described S. aureus genotype B, most successfully contaminated the cheese production at the dairy plant. Thus, our study fosters the hypothesis that genotype B S. aureus represent a specific challenge in control of S. aureus in the dairy chain that requires

  17. Staphylococcus aureus spa type t437

    DEFF Research Database (Denmark)

    Glasner, C; Pluister, G; Westh, H;

    2015-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) belonging to the multilocus sequence type clonal complex 59 (MLST CC59) is the predominant community-associated MRSA clone in Asia. This clone, which is primarily linked with the spa type t437, has so far only been reported in low numbers among...

  18. Meticillin-resistant Staphylococcus aureus (MRSA)

    DEFF Research Database (Denmark)

    Stefani, Stefania; Chung, Doo Ryeon; Lindsay, Jodi A;

    2012-01-01

    This article reviews recent findings on the global epidemiology of healthcare-acquired/associated (HA), community-acquired/associated (CA) and livestock-associated (LA) meticillin-resistant Staphylococcus aureus (MRSA) and aims to reach a consensus regarding the harmonisation of typing methods...

  19. Meningitis causada por staphylococcus aureus catalasa negativa

    OpenAIRE

    Álvarez Moreno, Carlos Arturo; Arroyo A., Claudia Patricia; Rodríguez, Elizabeth; Martínez R., Luz Marina; Quevedo S., Ruth

    2011-01-01

    En un paciente con cáncer se aisló del liquido cefaloraquideo y ascitico un coco gram positivo coagulasa positivo. El germen aislado mostró las características típicas de un Staphylococcus aureus, a excepción de la actividad de la catalasa, la cual no pudo ser encontrada.

  20. Staphylococcus aureus infections; Lead by the nose

    NARCIS (Netherlands)

    H.F.L. Wertheim (Heiman)

    2005-01-01

    textabstractAn overview and the latest insights regarding S. aureus nasal carriage, associated risks of developing infections and possible preventive measures, will be given in Chapter 2. Since mupirocin efficacy studies in preventing nosocomial infections have only been performed in surgical and

  1. Staphylococcus aureus resistente a la meticilina (SARM)

    Centers for Disease Control (CDC) Podcasts

    2007-10-22

    Datos importantes sobre las infecciones por SARM en Estados Unidos, en las escuelas y los entornos médicos. (Title: Methicillin-resistant Staphylococcus aureus (MRSA)Created: 10/2007).  Created: 10/22/2007 by National Center for Preparedness, Detection, and Control of Infectious Diseases.   Date Released: 11/9/2007.

  2. Methicillin-resistant Staphylococcus aureus transmission

    DEFF Research Database (Denmark)

    Andersen, Leif Percival; Nielsen, Xiaohui

    2015-01-01

    INTRODUCTION: Even though methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of nosocomial infections, it may often be difficult to evaluate the exact route of transmission. METHODS: In this study, we describe four cases of nosocomial transmission of MRSA in a hospital with a low...

  3. Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease.

    Science.gov (United States)

    Kiedrowski, Megan R; Crosby, Heidi A; Hernandez, Frank J; Malone, Cheryl L; McNamara, James O; Horswill, Alexander R

    2014-01-01

    Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc) enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the development of an infection imaging approach based on Nuc-activatable probes. Interestingly, S. aureus strains encode a second nuclease (Nuc2) that has received limited attention. With the growing interest in bacterial nucleases, we sought to characterize Nuc2 in more detail through localization, expression, and biochemical studies. Fluorescence microscopy and alkaline phosphatase localization approaches using Nuc2-GFP and Nuc2-PhoA fusions, respectively, demonstrated that Nuc2 is membrane bound with the C-terminus facing the extracellular environment, indicating it is a signal-anchored Type II membrane protein. Nuc2 enzyme activity was detectable on the S. aureus cell surface using a fluorescence resonance energy transfer (FRET) assay, and in time courses, both nuc2 transcription and enzyme activity peaked in early logarithmic growth and declined in stationary phase. Using a mouse model of S. aureus pyomyositis, Nuc2 activity was detected with activatable probes in vivo in nuc mutant strains, demonstrating that Nuc2 is produced during infections. To assess Nuc2 biochemical properties, the protein was purified and found to cleave both single- and double-stranded DNA, and it exhibited thermostability and calcium dependence, paralleling the properties of Nuc. Purified Nuc2 prevented biofilm formation in vitro and modestly decreased biomass in dispersal experiments. Altogether, our findings confirm that S. aureus encodes a second, surface-attached and functional DNase that is expressed during infections and displays similar biochemical properties to the secreted Nuc enzyme.

  4. Effect of a glucose impulse on the CcpA regulon in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Engelmann Susanne

    2009-05-01

    Full Text Available Abstract Background The catabolite control protein A (CcpA is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram-positive bacteria. It functions as a catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in Staphylococcus aureus, focussing on short-time effects of glucose under stable pH conditions. Results The addition of glucose to exponentially growing S. aureus increased the expression of genes and enzymes of the glycolytic pathway, while genes and proteins of the tricarboxylic acid (TCA cycle, required for the complete oxidation of glucose, were repressed via CcpA. Phosphotransacetylase and acetate kinase, converting acetyl-CoA to acetate with a concomitant substrate-level phosphorylation, were neither regulated by glucose nor by CcpA. CcpA directly repressed genes involved in utilization of amino acids as secondary carbon sources. Interestingly, the expression of a larger number of genes was found to be affected by ccpA inactivation in the absence of glucose than after glucose addition, suggesting that glucose-independent effects due to CcpA may have a particular impact in S. aureus. In the presence of glucose, CcpA was found to regulate the expression of genes involved in metabolism, but also that of genes coding for virulence determinants. Conclusion This study describes the CcpA regulon of exponentially growing S. aureus cells. As in other bacteria, CcpA of S. aureus seems to control a large regulon that comprises metabolic genes as well as virulence determinants that are affected in their expression by CcpA in a glucose-dependent as well as -independent manner.

  5. Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease.

    Directory of Open Access Journals (Sweden)

    Megan R Kiedrowski

    Full Text Available Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the development of an infection imaging approach based on Nuc-activatable probes. Interestingly, S. aureus strains encode a second nuclease (Nuc2 that has received limited attention. With the growing interest in bacterial nucleases, we sought to characterize Nuc2 in more detail through localization, expression, and biochemical studies. Fluorescence microscopy and alkaline phosphatase localization approaches using Nuc2-GFP and Nuc2-PhoA fusions, respectively, demonstrated that Nuc2 is membrane bound with the C-terminus facing the extracellular environment, indicating it is a signal-anchored Type II membrane protein. Nuc2 enzyme activity was detectable on the S. aureus cell surface using a fluorescence resonance energy transfer (FRET assay, and in time courses, both nuc2 transcription and enzyme activity peaked in early logarithmic growth and declined in stationary phase. Using a mouse model of S. aureus pyomyositis, Nuc2 activity was detected with activatable probes in vivo in nuc mutant strains, demonstrating that Nuc2 is produced during infections. To assess Nuc2 biochemical properties, the protein was purified and found to cleave both single- and double-stranded DNA, and it exhibited thermostability and calcium dependence, paralleling the properties of Nuc. Purified Nuc2 prevented biofilm formation in vitro and modestly decreased biomass in dispersal experiments. Altogether, our findings confirm that S. aureus encodes a second, surface-attached and functional DNase that is expressed during infections and displays similar biochemical properties to the secreted Nuc enzyme.

  6. Relationship between Vancomycin-Resistant Staphylococcus aureus, Vancomycin-Intermediate S. aureus, High Vancomycin MIC, and Outcome in Serious S. aureus Infections

    OpenAIRE

    Holmes, Natasha E.; Johnson, Paul D. R.; Howden, Benjamin P.

    2012-01-01

    Vancomycin has been used successfully for over 50 years for the treatment of Staphylococcus aureus infections, particularly those involving methicillin-resistant S. aureus. It has proven remarkably reliable, but its efficacy is now being questioned with the emergence of strains of S. aureus that display heteroresistance, intermediate resistance, and, occasionally, complete vancomycin resistance. More recently, an association has been established between poor outcome and infections with strain...

  7. Intravital two-photon microscopy of host-pathogen interactions in a mouse model of Staphylococcus aureus skin abscess formation.

    Science.gov (United States)

    Liese, Jan; Rooijakkers, Suzan H M; van Strijp, Jos A G; Novick, Richard P; Dustin, Michael L

    2013-06-01

    Staphylococcus (S.) aureus is a frequent cause of severe skin infections. The ability to control the infection is largely dependent on the rapid recruitment of neutrophils (PMN). To gain more insight into the dynamics of PMN migration and host-pathogen interactions in vivo, we used intravital two-photon (2-P) microscopy to visualize S. aureus skin infections in the mouse. Reporter S. aureus strains expressing fluorescent proteins were developed, which allowed for detection of the bacteria in vivo. By employing LysM-EGFP mice to visualize PMN, we observed the rapid appearance of PMN in the extravascular space of the dermis and their directed movement towards the focus of infection, which led to the delineation of an abscess within 1 day. Moreover, tracking of transferred labelled bone-marrow neutrophils showed that PMN localization to the site of infection is dependent on the presence of G-protein-coupled receptors on the PMN, whereas Interleukin-1 receptor was required on host cells other than PMN. Furthermore, the S. aureus complement inhibitor Ecb could block PMN accumulation at thesite of infection. Our results establish that 2-P microscopy is a powerful tool to investigate the orchestration of the immune cells, S. aureus location and gene expression in vivo on a single cell level.

  8. Mechanism of Gene Regulation by a Staphylococcus aureus Toxin

    Directory of Open Access Journals (Sweden)

    Hwang-Soo Joo

    2016-10-01

    Full Text Available The virulence of many bacterial pathogens, including the important human pathogen Staphylococcus aureus, depends on the secretion of frequently large amounts of toxins. Toxin production involves the need for the bacteria to make physiological adjustments for energy conservation. While toxins are primarily targets of gene regulation, such changes may be accomplished by regulatory functions of the toxins themselves. However, mechanisms by which toxins regulate gene expression have remained poorly understood. We show here that the staphylococcal phenol-soluble modulin (PSM toxins have gene regulatory functions that, in particular, include inducing expression of their own transport system by direct interference with a GntR-type repressor protein. This capacity was most pronounced in PSMs with low cytolytic capacity, demonstrating functional specification among closely related members of that toxin family during evolution. Our study presents a molecular mechanism of gene regulation by a bacterial toxin that adapts bacterial physiology to enhanced toxin production.

  9. Contribution of the mannan backbone of cryptococcal glucuronoxylomannan and a glycolytic enzyme of Staphylococcus aureus to contact-mediated killing of Cryptococcus neoformans.

    Science.gov (United States)

    Ikeda, Reiko; Saito, Fumito; Matsuo, Miki; Kurokawa, Kenji; Sekimizu, Kazuhisa; Yamaguchi, Masashi; Kawamoto, Susumu

    2007-07-01

    The fungal pathogen Cryptococcus neoformans is killed by the bacterium Staphylococcus aureus, and the killing is inhibited by soluble capsular polysaccharides. To investigate the mechanism of killing, cells in coculture were examined by scanning and transmission electron microscopy. S. aureus attached to the capsule of C. neoformans, and the ultrastructure of the attached C. neoformans cells was characteristic of dead cells. To identify the molecules that contributed to the fungal-bacterial interaction, we treated each with NaIO(4) or protease. Treatment of C. neoformans with NaIO(4) promoted adherence. It was inferred that cleavage of xylose and glucuronic acid side chains of glucuronoxylomannan (GXM) allowed S. aureus to recognize mannose residues in the backbone, which resisted periodate oxidation. On the other hand, treatment of S. aureus with protease decreased adherence, suggesting that protein contributed to attachment in S. aureus. In confirmation, side chain-cleaved polysaccharide or defined alpha-(1-->3)-mannan inhibited the killing at lower concentrations than native GXM did. Also, these polysaccharides reduced the adherence of the two species and induced clumping of pure S. aureus cells. alpha-(1-->3)-Mannooligosaccharides with a degree of polymerization (DP) of >/=3 induced cluster formation of S. aureus in a dose-dependent manner. Surface plasmon resonance analyses showed interaction of GXM and surface protein from S. aureus; the interaction was inhibited by oligosaccharides with a DP of > or =3. Conformations of alpha-(1-->3) oligosaccharides were predicted. The three-dimensional structures of mannooligosaccharides larger than triose appeared curved and could be imagined to be recognized by a hypothetical staphylococcal lectin. Native polyacrylamide gel electrophoresis of staphylococcal protein followed by electroblotting, enzyme-linked immunolectin assay, protein staining, and N-terminal amino acid sequencing suggested that the candidate protein was

  10. Staphylococcus aureus Shifts toward Commensalism in Response to Corynebacterium Species.

    Science.gov (United States)

    Ramsey, Matthew M; Freire, Marcelo O; Gabrilska, Rebecca A; Rumbaugh, Kendra P; Lemon, Katherine P

    2016-01-01

    Staphylococcus aureus-human interactions result in a continuum of outcomes from commensalism to pathogenesis. S. aureus is a clinically important pathogen that asymptomatically colonizes ~25% of humans as a member of the nostril and skin microbiota, where it resides with other bacteria including commensal Corynebacterium species. Commensal Corynebacterium spp. are also positively correlated with S. aureus in chronic polymicrobial diabetic foot infections, distinct from acute monomicrobial S. aureus infections. Recent work by our lab and others indicates that microbe-microbe interactions between S. aureus and human skin/nasal commensals, including Corynebacterium species, affect S. aureus behavior and fitness. Thus, we hypothesized that S. aureus interactions with Corynebacterium spp. diminish S. aureus virulence. We tested this by assaying for changes in S. aureus gene expression during in vitro mono- versus coculture with Corynebacterium striatum, a common skin and nasal commensal. We observed a broad shift in S. aureus gene transcription during in vitro growth with C. striatum, including increased transcription of genes known to exhibit increased expression during human nasal colonization and decreased transcription of virulence genes. S. aureus uses several regulatory pathways to transition between commensal and pathogenic states. One of these, the quorum signal accessory gene regulator (agr) system, was strongly inhibited in response to Corynebacterium spp. Phenotypically, S. aureus exposed to C. striatum exhibited increased adhesion to epithelial cells, reflecting a commensal state, and decreased hemolysin activity, reflecting an attenuation of virulence. Consistent with this, S. aureus displayed diminished fitness in experimental in vivo coinfection with C. striatum when compared to monoinfection. These data support a model in which S. aureus shifts from virulence toward a commensal state when exposed to commensal Corynebacterium species.

  11. Rapid first-line discrimination of methicillin resistant Staphylococcus aureus strains using MALDI-TOF MS

    DEFF Research Database (Denmark)

    Østergaard, Claus; Grønvall Kjær Hansen, Sanne; Møller, Jens K

    2015-01-01

    Fast and reliable discrimination of methicillin-resistant Staphylococcus aureus (MRSA) isolates is essential in identifying an outbreak. Molecular typing methods, such as S. aureus protein A (spa) typing, multi locus sequence typing (MLST) and pulse field gel electrophoresis (PFGE) are generally...... used for this purpose. These methods are all relatively time-consuming and not performed routinely in all laboratories. The aim of this study is to examine whether MALDI-TOF MS can be used as a fast, simple and easily implemented method for first-line discrimination of MRSA isolates. Mass spectra from...... 600 clinical MRSA isolates were included in the study, representing 89 spa types, associated with 16 different known clonal complexes. All spectra were obtained directly from colony material obtained from overnight cultures without prior protein extraction. We identified 43 useful discriminatory m/z...

  12. Quercitrin, an Inhibitor of Sortase A, Interferes with the Adhesion of Staphylococcal aureus

    Directory of Open Access Journals (Sweden)

    Bingrun Liu

    2015-04-01

    Full Text Available Sortase A (SrtA is a cysteine transpeptidase of most Gram-positive bacteria that is responsible for the anchorage of many surface protein virulence factors to the cell wall layer. SrtA mutants are unable to display surface proteins and are defective in the establishment of infections without affecting microbial viability. In this study, we report that quercitrin (QEN, a natural compound that does not affect Staphylococcus aureus growth, can inhibit the catalytic activity of SrtA in fibrinogen (Fg cell-clumping and immobilized fibronectin (Fn adhesion assays. Molecular dynamics simulations and mutagenesis assays suggest that QEN binds to the binding sites of the SrtA G167A and V193A mutants. These findings indicate that QEN is a potential lead compound for the development of new anti-virulence agents against S. aureus infections.

  13. Alterations in the transcriptome and antibiotic susceptibility of Staphylococcus aureus grown in the presence of diclofenac

    Directory of Open Access Journals (Sweden)

    Nagarajan Vijayaraj

    2011-07-01

    Full Text Available Abstract Background Diclofenac is a non-steroidal anti-inflammatory drug (NSAID which has been shown to increase the susceptibility of various bacteria to antimicrobials and demonstrated to have broad antimicrobial activity. This study describes transcriptome alterations in S. aureus strain COL grown with diclofenac and characterizes the effects of this NSAID on antibiotic susceptibility in laboratory, clinical and diclofenac reduced-susceptibility (DcRS S. aureus strains. Methods Transcriptional alterations in response to growth with diclofenac were measured using S. aureus gene expression microarrays and quantitative real-time PCR. Antimicrobial susceptibility was determined by agar diffusion MICs and gradient plate analysis. Ciprofloxacin accumulation was measured by fluorescence spectrophotometry. Results Growth of S. aureus strain COL with 80 μg/ml (0.2 × MIC of diclofenac resulted in the significant alteration by ≥2-fold of 458 genes. These represented genes encoding proteins for transport and binding, protein and DNA synthesis, and the cell envelope. Notable alterations included the strong down-regulation of antimicrobial efflux pumps including mepRAB and a putative emrAB/qacA-family pump. Diclofenac up-regulated sigB (σB, encoding an alternative sigma factor which has been shown to be important for antimicrobial resistance. Staphylococcus aureus microarray metadatabase (SAMMD analysis further revealed that 46% of genes differentially-expressed with diclofenac are also σB-regulated. Diclofenac altered S. aureus susceptibility to multiple antibiotics in a strain-dependent manner. Susceptibility increased for ciprofloxacin, ofloxacin and norfloxacin, decreased for oxacillin and vancomycin, and did not change for tetracycline or chloramphenicol. Mutation to DcRS did not affect susceptibility to the above antibiotics. Reduced ciprofloxacin MICs with diclofenac in strain BB255, were not associated with increased drug accumulation

  14. Cyclic peptide inhibitors of the β-sliding clamp in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Kjelstrup, Susanne; Hansen, Paula Melo Paulon; Thomsen, Line Elnif;

    2013-01-01

    Interaction between pairs of Staphylococcus aureus replication proteins was detected in an Escherichia coli based two-hybrid analysis. A reverse two-hybrid system was constructed for selection of compounds that hindered interaction between interacting protein pairs. A number of cyclic peptides......, from a library generated by the split intein-mediated circular ligation of peptides and proteins technology, were found to interfere with dimerization of the β-sliding clamp of the replisome. Two 8-mer peptides were analyzed in more detail. Both inhibited DNA replication, led to SOS induction, altered...

  15. Preventing Staphylococcus aureus sepsis through the inhibition of its agglutination in blood.

    Science.gov (United States)

    McAdow, Molly; Kim, Hwan Keun; Dedent, Andrea C; Hendrickx, Antoni P A; Schneewind, Olaf; Missiakas, Dominique M

    2011-10-01

    Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. When suspended in human or animal plasma, staphylococci are known to agglutinate, however the bacterial factors responsible for agglutination and their possible contribution to disease pathogenesis have not yet been revealed. Using a mouse model for S. aureus sepsis, we report here that staphylococcal agglutination in blood was associated with a lethal outcome of this disease. Three secreted products of staphylococci--coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA)--were required for agglutination. Coa and vWbp activate prothrombin to cleave fibrinogen, whereas ClfA allowed staphylococci to associate with the resulting fibrin cables. All three virulence genes promoted the formation of thromboembolic lesions in heart tissues. S. aureus agglutination could be disrupted and the lethal outcome of sepsis could be prevented by combining dabigatran-etexilate treatment, which blocked Coa and vWbp activity, with antibodies specific for ClfA. Together these results suggest that the combined administration of direct thrombin inhibitors and ClfA-antibodies that block S. aureus agglutination with fibrin may be useful for the prevention of staphylococcal sepsis in humans.

  16. Diversity and evolution of blaZ from Staphylococcus aureus and coagulase-negative staphylococci

    DEFF Research Database (Denmark)

    Olsen, John E.; Christensen, Henrik; Aarestrup, Frank Møller

    2006-01-01

    NS) and Staphylococcus aureus of bovine origin. Methods: blaZ was detected in 143 strains of penicillin-resistant S. aureus and CoNS from five Danish cattle herds (n = 25/23), random CoNS isolates from Denmark (n = 37), a collection of S. aureus from six different countries (n = 52), humans in Denmark (n = 3) and beta...... and sequences retrieved from public databases were compared. A phylogenetic tree showed that blaZ exists in three evolutionary lines: one group was of plasmid origin, one group was of chromosomal origin and one intermediate group. Sixty-nine sequence types were demonstrated. They translated into 11 BlaZ protein...... types. The major types all contained strains of both human and bovine origin, and more than one Staphylococcus species, demonstrating a shared gene pool. In a comparison of S. aureus and CoNS obtained from five Danish cattle herds, the same type of blaZ was only detected in one case. Conclusions...

  17. Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin.

    Science.gov (United States)

    Fiaschi, Luigi; Di Palo, Benedetta; Scarselli, Maria; Pozzi, Clarissa; Tomaszewski, Kelly; Galletti, Bruno; Nardi-Dei, Vincenzo; Arcidiacono, Letizia; Mishra, Ravi P N; Mori, Elena; Pallaoro, Michele; Falugi, Fabiana; Torre, Antonina; Fontana, Maria Rita; Soriani, Marco; Bubeck Wardenburg, Juliane; Grandi, Guido; Rappuoli, Rino; Ferlenghi, Ilaria; Bagnoli, Fabio

    2016-06-01

    Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus.

  18. Involvement of superoxide and myeloperoxidase in oxygen-dependent killing of Staphylococcus aureus by neutrophils.

    Science.gov (United States)

    Hampton, M B; Kettle, A J; Winterbourn, C C

    1996-09-01

    We have used a quantitative assay that measures independent rate constants for phagocytosis and killing of Staphylococcus aureus to investigate the involvement of superoxide and myeloperoxidase in bacterial killing by human neutrophils. To inhibit superoxide-dependent processes, superoxide dismutase was cross-linked to immunoglobulin G and the conjugate was attached to the surface of S. aureus via protein A in its cell wall. Myeloperoxidase was inhibited with azide, and myeloperoxidase-deficient neutrophils were used. Adding the NADPH oxidase inhibitor diphenyleneiodonium, to prevent superoxide production, decreased the killing rate to 25%, indicating that oxidative killing mechanisms predominate in this system. The rate constant for killing of S. aureus with superoxide dismutase attached was 70% of that for control bacteria linked to inactivated enzyme. Superoxide dismutase had no effect in the presence of diphenyleneiodonium. The rate of killing was decreased to 33% in the presence of azide and to 40% with myeloperoxidase-deficient neutrophils. Superoxide dismutase had no effect in the presence of azide. On the assumption that the oxidative and nonoxidative components of killing can be considered separately, the oxidative rate was decreased by almost half by superoxide dismutase and was about six times lower when myeloperoxidase was inactive. We conclude that myeloperoxidase-dependent processes are strongly favored by human neutrophils as their prime mechanism of oxidative killing of S. aureus and that superoxide makes a direct contribution to killing. Our results also suggest that superoxide acts in conjunction with a myeloperoxidase-dependent pathway.

  19. Molecular Typing of Staphylococcus aureus Isolated from Patients with Autosomal Dominant Hyper IgE Syndrome

    Directory of Open Access Journals (Sweden)

    Inka Sastalla

    2017-06-01

    Full Text Available Autosomal dominant hyper IgE syndrome (AD-HIES is a primary immunodeficiency caused by a loss-of-function mutation in the Signal Transducer and Activator of Transcription 3 (STAT3. This immune disorder is clinically characterized by increased susceptibility to cutaneous and sinopulmonary infections, in particular with Candida and Staphylococcus aureus. It has recently been recognized that the skin microbiome of patients with AD-HIES is altered with an overrepresentation of certain Gram-negative bacteria and Gram-positive staphylococci. However, these alterations have not been characterized at the species- and strain-level. Since S. aureus infections are influenced by strain-specific expression of virulence factors, information on colonizing strain characteristics may provide insights into host-pathogen interactions and help guide management strategies for treatment and prophylaxis. The aim of this study was to determine whether the immunodeficiency of AD-HIES selects for unique strains of colonizing S. aureus. Using multi-locus sequence typing (MLST, protein A (spa typing, and PCR-based detection of toxin genes, we performed a detailed analysis of the S. aureus isolates (n = 13 found on the skin of twenty-one patients with AD-HIES. We found a low diversity of sequence types, and an abundance of strains that expressed methicillin resistance, Panton-Valentine leukocidin (PVL, and staphylococcal enterotoxins K and Q (SEK, SEQ. Our results indicate that patients with AD-HIES may often carry antibiotic-resistant strains that harbor key virulence factors.

  20. Effect of negative pressure on growth, secretion and biofilm formation of Staphylococcus aureus.

    Science.gov (United States)

    Li, Tongtong; Wang, Guoqi; Yin, Peng; Li, Zhirui; Zhang, Licheng; Liu, Jianheng; Li, Ming; Zhang, Lihai; Han, Li; Tang, Peifu

    2015-10-01

    Negative pressure wound therapy (NPWT) has gained popularity in the management of contaminated wounds as an effective physical therapy, although its influence on the bacteria in the wounds remains unclear. In this study, we attempted to explore the effect of negative pressure conditions on Staphylococcus aureus, the most frequently isolated pathogen during wound infection. S. aureus was cultured in Luria-Bertani medium at subatmospheric pressure of -125 mmHg for 24 h, with the bacteria grown at ambient pressure as the control. The application of negative pressure was found to slow down the growth rate and inhibit biofilm development of S. aureus, which was confirmed by static biofilm assays. Furthermore, decreases in the total amount of virulence factors and biofilm components were observed, including α-hemolysin, extracellular adherence protein, polysaccharide intercellular adhesin and extracellular DNA. With quantitative RT-PCR analysis, we also revealed a significant inhibition in the transcription of virulence and regulatory genes related to wound infections and bacterial biofilms. Together, these findings indicated that negative pressure could inhibit the growth, virulence and biofilm formation of S. aureus. A topical subatmospheric pressure condition, such as NPWT, may be a potential antivirulence and antibiofilm strategy in the field of wound care.

  1. Complete genome analysis of two new bacteriophages isolated from impetigo strains of Staphylococcus aureus.

    Science.gov (United States)

    Botka, Tibor; Růžičková, Vladislava; Konečná, Hana; Pantůček, Roman; Rychlík, Ivan; Zdráhal, Zbyněk; Petráš, Petr; Doškař, Jiří

    2015-08-01

    Exfoliative toxin A (ETA)-coding temperate bacteriophages are leading contributors to the toxic phenotype of impetigo strains of Staphylococcus aureus. Two distinct eta gene-positive bacteriophages isolated from S. aureus strains which recently caused massive outbreaks of pemphigus neonatorum in Czech maternity hospitals were characterized. The phages, designated ϕB166 and ϕB236, were able to transfer the eta gene into a prophageless S. aureus strain which afterwards converted into an ETA producer. Complete phage genome sequences were determined, and a comparative analysis of five designed genomic regions revealed major variances between them. They differed in the genome size, number of open reading frames, genome architecture, and virion protein patterns. Their high mutual sequence similarity was detected only in the terminal regions of the genome. When compared with the so far described eta phage genomes, noticeable differences were found. Thus, both phages represent two new lineages of as yet not characterized bacteriophages of the Siphoviridae family having impact on pathogenicity of impetigo strains of S. aureus.

  2. CcpA Affects Infectivity of Staphylococcus aureus in a Hyperglycemic Environment

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    Markus Bischoff

    2017-05-01

    Full Text Available Many bacteria regulate the expression of virulence factors via carbon catabolite responsive elements. In Gram-positive bacteria, the predominant mediator of carbon catabolite repression is the catabolite control protein A (CcpA. Hyperglycemia is a widespread disorder that predisposes individuals to an array of symptoms and an increased risk of infections. In hyperglycemic individuals, the bacterium Staphylococcus aureus causes serious, life-threatening infections. The importance of CcpA in regulating carbon catabolite repression in S. aureus suggests it may be important for infections in hyperglycemic individuals. To test this suggestion, hyperglycemic non-obese diabetic (NOD; blood glucose level ≥20 mM mice were challenged with the mouse pathogenic S. aureus strain Newman and the isogenic ccpA deletion mutant (MST14, and the effects on infectivity were determined. Diabetic NOD mice challenged with the ccpA deletion mutant enhanced the symptoms of infection in an acute murine pneumonia model relative to the parental strain. Interestingly, when diabetic NOD mice were used in footpad or catheter infection models, infectivity of the ccpA mutant decreased relative to the parental strain. These differences greatly diminished when normoglycemic NOD mice (blood glucose level ≤ 10 mM were used. These data suggest that CcpA is important for infectivity of S. aureus in hyperglycemic individuals.

  3. Presence of toxic shock toxin in toxic shock and other clinical strains of Staphylococcus aureus.

    OpenAIRE

    Reeves, M W; Pine, L; Feeley, J. C.; Wells, D.E.

    1984-01-01

    Toxic shock toxin (TST), also known as pyrogenic exotoxin C (Schlievert et al., J. Infect. Dis. 143:509-516, 1981) and staphylococcal enterotoxin F (Bergdoll et al., Lancet i:1017-1021, 1981), was purified from toxic shock strains of Staphylococcus aureus by preparative isoelectric focusing and by chromatofocusing. Neither method produced an absolutely pure protein as determined by silver staining of sodium dodecyl sulfate-acrylamide gels, although chromatofocusing was the better method of th...

  4. Rapid detection of methicillin resistance in Staphylococcus aureus isolates by the MRSA-screen latex agglutination test

    NARCIS (Netherlands)

    W.B. van Leeuwen (Willem); C. van Pelt (Cindy); A. Luijendijk (Ad); H.A. Verbrugh (Henri); W.H.F. Goessens (Wil)

    1999-01-01

    textabstractThe slide agglutination test MRSA-Screen (Denka Seiken Co., Niigata, Japan) was compared with the mecA PCR ("gold standard") for the detection of methicillin resistance in Staphylococcus aureus. The MRSA-Screen test detected the penicillin-binding protein 2a

  5. Detection of alpha-toxin and other virulence factors in biofilms of staphylococcus aureus on polystyrene and a human epidermalmodel

    NARCIS (Netherlands)

    P.M. Den Reijer (P. Martijn); J.A. Haisma (Janneke); N. Lemmens-den Toom; J. Willemse (José); R.A. Koning; J.A.A. Demmers (Jeroen); D.H. Dekkers (Dick); E.J. Rijkers; A. El Ghalbzouri (Abdoelwaheb); P.H. Nibbering (Peter); W.J.B. van Wamel (Willem)

    2016-01-01

    textabstractBackground & Aim: The ability of Staphylococcus aureus to successfully colonize (a)biotic surfaces may be explained by biofilm formation and the actions of virulence factors. The aim of the present study was to establish the presence of 52 proteins, including virulence factors such as al

  6. Roles of X-linked inhibitor of apoptosis protein in the apoptosis of human monocytic cells induced by t-toxin from Staphylococcus aureus%X连锁凋亡抑制蛋白通路在金黄色葡萄球菌α-毒素诱导人外周血单核细胞凋亡过程中的作用

    Institute of Scientific and Technical Information of China (English)

    喻博; 佡剑非; 张萌; 阚亮; 王佳贺

    2013-01-01

    目的 研究金黄色葡萄球菌的主要毒力因子α-毒素感染人外周血单核细胞后细胞的凋亡率及X连锁凋亡抑制蛋白(XIAP)、凋亡抑制蛋白1/2 (cIAP1/2)、Survivin、Bcl-2、Bax和半胱氨酸的天冬氨酸蛋白酶(caspase-3)等的表达.方法 以Annexin V异硫氰酸荧光素(FITC)/碘化丙啶(PI)双染流式细胞仪检测人外周血单核细胞的凋亡率,Western blot法检测XIAP、cIAP1/2、Survivin、Bc1-2、Bax和caspase-3等的表达,采用荧光比色法测定细胞内caspase-3蛋白酶活性.结果 α-毒素能够以时间依赖的方式诱导人外周血单核细胞凋亡,α-毒素感染30、60和90 min凋亡率与感染0 min比较,其差异有统计学意义(P<0.05或P<0.001).随着作用时间的延长,XIAP、cIAP1/2、Survivin和Bcl-2表达逐渐下降,而Bax和caspase-3表达逐渐增加.结论 α-毒素可诱导人外周血单核细胞凋亡,XIAP信号通路在金黄色葡萄球菌的致病过程中起重要作用.%Objective To investigate the roles of X-linked inhibitor of apoptosis protein (XIAP),cellular inhibitor of apoptosis proteinl/2 (cIAP1/2),Survivin,Bcl-2,Bax,and cysteine aspartic acid specific protease-3 (caspase-3) in the apoptosis of human monocytic cells induced by α-toxin from Staphylococcus aureus.Methods Apoptosis rates of human monocytic cells were detected by Annexin V fluorescein isothiocyanate/propidium iodide assay.Western blot was performed to detect the expressions of XIAP,cIAP1/2,Survivin,Bcl-2,Bax,and caspase-3.Results The results showed that α-toxin induced apoptosis in human monocytic cells in a time-dependent manner.The apoptosis rates in the groups of 30,60 and 90 min increased significantly than those in the group of 0 min(P < 0.05 or P < 0.001).With the time extension,there was downregulation of XIAP,cIAP1/2,Survivin and Bcl-2,and upregulation of Bax and caspase-3.Conclusion α-toxin from Staphylococcus aureus can induce the apoptosis of human monocytic cells.It is

  7. Cyclic peptide inhibitors of the β-sliding clamp in Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Susanne Kjelstrup

    Full Text Available Interaction between pairs of Staphylococcus aureus replication proteins was detected in an Escherichia coli based two-hybrid analysis. A reverse two-hybrid system was constructed for selection of compounds that hindered interaction between interacting protein pairs. A number of cyclic peptides, from a library generated by the split intein-mediated circular ligation of peptides and proteins technology, were found to interfere with dimerization of the β-sliding clamp of the replisome. Two 8-mer peptides were analyzed in more detail. Both inhibited DNA replication, led to SOS induction, altered cell morphology and cell death. The peptides were active when added to bacterial cultures indicating that they could traverse the bacterial membrane to find their intracellular target. Peptide specificity was confirmed by overproduction of the putative target (DnaN which resulted in resistance. The minimum inhibitory concentration was ∼50 μg/ml for S. aureus cells. These compounds may serve as lead candidates for future development into novel classes of antibiotics as well as provide information on the function of the S. aureus replication process.

  8. Modulation of connexin expression and gap junction communication in astrocytes by the gram-positive bacterium S. aureus.

    Science.gov (United States)

    Esen, Nilufer; Shuffield, Debbie; Syed, Mohsin M D; Kielian, Tammy

    2007-01-01

    Gap junctions establish direct intercellular conduits between adjacent cells and are formed by the hexameric organization of protein subunits called connexins (Cx). It is unknown whether the proinflammatory milieu that ensues during CNS infection with S. aureus, one of the main etiologic agents of brain abscess in humans, is capable of eliciting regional changes in astrocyte homocellular gap junction communication (GJC) and, by extension, influencing neuron homeostasis at sites distant from the primary focus of infection. Here we investigated the effects of S. aureus and its cell wall product peptidoglycan (PGN) on Cx43, Cx30, and Cx26 expression, the main Cx isoforms found in astrocytes. Both bacterial stimuli led to a time-dependent decrease in Cx43 and Cx30 expression; however, Cx26 levels were elevated following bacterial exposure. Functional examination of dye coupling, as revealed by single-cell microinjections of Lucifer yellow, demonstrated that both S. aureus and PGN inhibited astrocyte GJC. Inhibition of protein synthesis with cyclohexamide (CHX) revealed that S. aureus directly modulates, in part, Cx43 and Cx30 expression, whereas Cx26 levels appear to be regulated by a factor(s) that requires de novo protein production; however, CHX did not alter the inhibitory effects of S. aureus on astrocyte GJC. The p38 MAPK inhibitor SB202190 was capable of partially restoring the S. aureus-mediated decrease in astrocyte GJC to that of unstimulated cells, suggesting the involvement of p38 MAPK-dependent pathway(s). These findings could have important implications for limiting the long-term detrimental effects of abscess formation in the brain which may include seizures and cognitive deficits. Copyright 2006 Wiley-Liss, Inc.

  9. Staphylococcus aureus shifts towards commensalism in response to Corynebacterium species

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    Matthew M Ramsey

    2016-08-01

    Full Text Available Staphylococcus aureus–human interactions result in a continuum of outcomes from commensalism to pathogenesis. S. aureus is a clinically important pathogen that asymptomatically colonizes ~25% of humans as a member of the nostril and skin microbiota, where it resides with other bacteria including commensal Corynebacterium species. Commensal Corynebacterium spp. are also positively correlated with S. aureus in chronic polymicrobial diabetic foot infections, distinct from acute monomicrobial S. aureus infections. Recent work by our lab and others indicates that microbe-microbe interactions between S. aureus and human skin/nasal commensals, including Corynebacterium species, affect S. aureus behavior and fitness. Thus, we hypothesized that S. aureus interactions with Corynebacterium spp. diminish S. aureus virulence. We tested this by assaying for changes in S. aureus gene expression during in vitro mono- versus coculture with Corynebacterium striatum, a common skin and nasal commensal. We observed a broad shift in S. aureus gene transcription during in vitro growth with C. striatum, including increased transcription of genes known to exhibit increased expression during human nasal colonization and decreased transcription of virulence genes. S. aureus uses several regulatory pathways to transition between commensal and pathogenic states. One of these, the quorum signal accessory gene regulator (agr system, was strongly inhibited in response to Corynebacterium spp. Phenotypically, S. aureus exposed to C. striatum exhibited increased adhesion to epithelial cells, reflecting a commensal state, and decreased hemolysin activity, reflecting an attenuation of virulence. Consistent with this, S. aureus displayed diminished fitness in experimental in vivo coinfection with C. striatum when compared to monoinfection. These data support a model in which S. aureus shifts from virulence towards a commensal state when exposed to commensal Corynebacterium species.

  10. Prevalence, Virulence Genes, Antimicrobial Susceptibility, and Genetic Diversity of Staphylococcus aureus from Retail Aquatic Products in China.

    Science.gov (United States)

    Rong, Dongli; Wu, Qingping; Xu, Mingfang; Zhang, Jumei; Yu, Shubo

    2017-01-01

    Staphylococcus aureus is an important food-borne opportunistic pathogen that frequently causes severe blood and tissue infections or even fatal illnesses. Although S. aureus has been extensively studied in livestock and poultry foods in China, limited information has been reported in aquatic products. Accordingly, in this study, we aimed to characterize S. aureus in aquatic products purchased from retail markets in China. In total, 320 aquatic food samples were collected from 32 provincial capitals in China. The results showed that 119 samples (37.2%, 119/320) were positive for S. aureus by both qualitative and quantitative analyses. The contamination levels of 78.2% of samples ranged from 0.3 to 10 MPN/g, and six samples exceeded 110 MPN/g. A total of 119 S. aureus isolates from positive samples were selected to evaluate virulence factors, antibiotic resistance, and molecular characteristics. All S. aureus isolates were evaluated for the presence of 11 virulence genes by multiplex polymerase chain reaction, and α-hemolysin (hlα, 84.9%), fibronectin-binding protein A (fnbA, 79.0%), S. aureus enterotoxin E (see, 53.8%), and Panton-Valentine leucocidin (pvl, 50.4%) were identified as the major genes. These genes formed 56 different profiles, with the major profile identified as pvl-hlα-fnbA (28.6%). The antimicrobial susceptibility of all isolates was analyzed through the disk diffusion method, and the results showed high resistance to β-lactams, macrolides and tetracyclines, but susceptibility to linezolid and vancomycin. In addition, 26 sequence types (STs) were obtained via multilocus sequence typing, including seven novel STs, among which ST1 (20.2%), ST15 (18.5%), and ST188 (13.4%) were the most common STs. All the isolates were mecC negative, but nine isolates carrying mecA were evaluated by staphylococcal cassette chromosome mec (SCCmec) typing, all of which were SCCmecIII or SCCmecIV types. Isolates of SCCmecIII showed a high prevalence and were multidrug

  11. Characterization of Staphylococcus aureus EssB, an integral membrane component of the Type VII secretion system: atomic resolution crystal structure of the cytoplasmic segment

    OpenAIRE

    Zoltner, Martin; Fyfe, Paul K.; Palmer, Tracy; Hunter, William N.

    2012-01-01

    The Type VII protein translocation/secretion system, unique to Gram-positive bacteria, is a key virulence determinant in Staphylococcus aureus. We aim to characterize the architecture of this secretion machinery and now describe the present study of S. aureus EssB, a 52 kDa bitopic membrane protein essential for secretion of the ESAT-6 (early secretory antigenic target of 6 kDa) family of proteins, the prototypic substrate of Type VII secretion. Full-length EssB was heterologously expressed i...

  12. BIOMOLECULAR IDENTIFICATION OF METHICILLIN-RESISTANT STRAINS OF STAPHYLOCOCCUS AUREUS (MRSA ISOLATED FROM MEAT AND MEAT PROCESSING ENVIRONMENTS

    Directory of Open Access Journals (Sweden)

    V. D’Orio

    2008-09-01

    Full Text Available 371 samples from meat and meat-environments were collected and examined for the detection of methicillin-resistant Staphylococcus aureus (MRSA. The structural gene for penicillin-binding protein 2a (mecA gene, was amplified by PCR and detected by agarose gel electrophoresis. 96 samples (25.8%, contained S. aureus and 2 of them (2.08% were mecA positive. Further assays are necessary to evaluate the spread of MRSA in food and food-environments.

  13. Impact of sub-inhibitory antibiotics on fibronectin-mediated host cell adhesion and invasion by Staphylococcus aureus

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    Rasigade Jean

    2011-12-01

    Full Text Available Abstract Background Staphylococcus aureus is a well-armed pathogen prevalent in severe infections such as endocarditis and osteomyelitis. Fibronectin-binding proteins A and B, encoded by fnbA/B, are major pathogenesis determinants in these infections through their involvement in S. aureus adhesion to and invasion of host cells. Sub-minimum inhibitory concentrations (sub-MICs of antibiotics, frequently occurring in vivo because of impaired drug diffusion at the infection site, can alter S. aureus phenotype. We therefore investigated their impact on S. aureus fibronectin-mediated adhesiveness and invasiveness. Methods After in vitro challenge of S. aureus 8325-4 and clinical isolates with sub-MICs of major anti-staphylococcal agents, we explored fnbA/B transcription levels, bacterial adhesiveness to immobilised human fibronectin and human osteoblasts in culture, and bacterial invasion of human osteoblasts. Results Oxacillin, moxifloxacin and linezolid led to the development of a hyper-adhesive phenotype in the fibronectin adhesion assay that was consistent with an increase in fnbA/B transcription. Conversely, rifampin treatment decreased fibronectin binding in all strains tested without affecting fnbA/B transcription. Gentamicin and vancomycin had no impact on fibronectin binding or fnbA/B transcription levels. Only oxacillin-treated S. aureus displayed a significantly increased adhesion to cultured osteoblasts, but its invasiveness did not differ from that of untreated controls. Conclusion Our findings demonstrate that several antibiotics at sub-MICs modulate fibronectin binding in S. aureus in a drug-specific fashion. However, hyper- and hypo- adhesive phenotypes observed in controlled in vitro conditions were not fully confirmed in whole cell infection assays. The relevance of adhesion modulation during in vivo infections is thus still uncertain and requires further investigations.

  14. Altered Competitive Fitness, Antimicrobial Susceptibility, and Cellular Morphology in a Triclosan-Induced Small-Colony Variant of Staphylococcus aureus.

    Science.gov (United States)

    Forbes, Sarah; Latimer, Joe; Bazaid, Abdulrahman; McBain, Andrew J

    2015-08-01

    Staphylococcus aureus can produce small-colony variants (SCVs) that express various phenotypes. While their significance is unclear, SCV propagation may be influenced by relative fitness, antimicrobial susceptibility, and the underlying mechanism. We have investigated triclosan-induced generation of SCVs in six S. aureus strains, including methicillin-resistant S. aureus (MRSA). Parent strains (P0) were repeatedly passaged on concentration gradients of triclosan using a solid-state exposure system to generate P10. P10 was subsequently passaged without triclosan to generate X10. Susceptibility to triclosan and 7 antibiotics was assessed at all stages. For S. aureus ATCC 6538, SCVs were further characterized by determining microbicide susceptibility and competitive fitness. Cellular morphology was examined using electron microscopy, and protein expression was evaluated through proteomics. Triclosan susceptibility in all SCVs (which could be generated from 4/6 strains) was markedly decreased, while antibiotic susceptibility was significantly increased in the majority of cases. An SCV of S. aureus ATCC 6538 exhibited significantly increased susceptibility to all tested microbicides. Cross-wall formation was impaired in this bacterium, while expression of FabI, a target of triclosan, and IsaA, a lytic transglycosylase involved in cell division, was increased. The P10 SCV was 49% less fit than P0. In summary, triclosan exposure of S. aureus produced SCVs in 4/6 test bacteria, with decreased triclosan susceptibility but with generally increased antibiotic susceptibility. An SCV derived from S. aureus ATCC 6538 showed reduced competitive fitness, potentially due to impaired cell division. In this SCV, increased FabI expression could account for reduced triclosan susceptibility, while IsaA may be upregulated in response to cell division defects.

  15. Impact of cell wall peptidoglycan O-acetylation on the pathogenesis of Staphylococcus aureus in septic arthritis.

    Science.gov (United States)

    Baranwal, Gaurav; Mohammad, Majd; Jarneborn, Anders; Reddy, Bommana Raghunath; Golla, Archana; Chakravarty, Sumana; Biswas, Lalitha; Götz, Friedrich; Shankarappa, Sahadev; Jin, Tao; Biswas, Raja

    2017-10-01

    Staphylococcus aureus (S. aureus) is one of the most common pathogen causing septic arthritis. To colonize the joints and establish septic arthritis this bacterium needs to resist the host innate immune responses. Lysozyme secreted by neutrophils and macrophages is an important defense protein present in the joint synovial fluids. S. aureus is known to be resistant to lysozyme due to its peptidoglycan modification by O-acetylation of N-acetyl muramic acid. In this study we have investigated the role of O-acetylated peptidoglycan in septic arthritis. Using mouse models for both local and hematogenous S. aureus arthritis we compared the onset and progress of the disease induced by O-acetyl transferase mutant and the parenteral wild type SA113 strain. The disease progression was assessed by observing the clinical parameters including body weight, arthritis, and functionality of the affected limbs. Further X-ray and histopathological examinations were performed to monitor the synovitis and bone damage. In local S. aureus arthritis model, mice inoculated with the ΔoatA strain developed milder disease (in terms of knee swelling, motor and movement functionality) compared to mice inoculated with the wild type SA113 strain. X-ray and histopathological data revealed that ΔoatA infected mice knee joints had significantly lesser joint destruction, which was accompanied by reduced bacterial load in knee joints. Similarly, in hematogenous S. aureus arthritis model, ΔoatA mutant strain induced significantly less severe clinical septic arthritis compared to its parental strain, which is in accordance with radiological findings. Our data indicate that peptidoglycan O-acetylation plays an important role in S. aureus mediated septic arthritis. Copyright © 2017 Elsevier GmbH. All rights reserved.