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Sample records for aureus surface protein

  1. Presence of Pseudomonas aeruginosa influences biofilm formation and surface protein expression of Staphylococcus aureus.

    Science.gov (United States)

    Kumar, Amit; Ting, Yen Peng

    2015-11-01

    Although Staphylococcus aureus and Pseudomonas aeruginosa can individually colonize and infect their hosts, the commensalistic effect of the two is more tenacious and lethal. In this study, it was shown that in co-culture with P. aeruginosa, a sub-population of S. aureus exhibited improved resistance to kanamycin by selection of small colony variant (SCV) phenotype. Additionally, biofilm formation by the two bacteria was denser in the co-culture, compared with biofilm formed in individual pure cultures. Using Atomic Force Microscope (AFM) force spectroscopy for single cells, it was demonstrated that S. aureus cultured in the presence of P. aeruginosa bound more tenaciously to substrates. Surface-shaved peptides were isolated and identified using ultra-performance liquid chromatography-quadrupole-time of flight and a homology search program spider. Results indicated that serine-rich adhesin, extracellular matrix binding protein and other putative adhesion proteins could be responsible for the enhanced attachment of S. aureus in the co-culture. Besides, several other proteins were differentially expressed, indicating the occurrence of a range of other interactions. Of particular interest was a multidrug resistant protein named ABC transporter permease which is known to expel xenobiotics out of the cells. Positive regulation of this protein could be involved in the SCV selection of S. aureus in the co-culture. PMID:25925222

  2. 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.

  3. 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.

  4. The Surface Protein Pls of Methicillin-Resistant Staphylococcus aureus Is a Virulence Factor in Septic Arthritis

    OpenAIRE

    Josefsson, Elisabet; Juuti, Katri; Bokarewa, Maria; Kuusela, Pentti

    2005-01-01

    Pls, a surface protein of certain methicillin-resistant Staphylococcus aureus strains, is associated with poor bacterial adherence to solid-phase fibronectin and immunoglobulin G, as well as with reduced invasion of cultured epithelial cells. Here the importance of Pls for the development of septic arthritis and sepsis was investigated by using a mouse model. Mice inoculated with a pls knockout mutant developed a much milder arthritis and showed less grave weight reduction than mice infected ...

  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. 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.

  7. [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.

  8. Fibrinogen is a ligand for the Staphylococcus aureus Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMM) Bone sialoprotein-binding protein (Bbp)

    OpenAIRE

    Foster, Timothy

    2011-01-01

    PUBLISHED MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) are bacterial surface proteins mediating adherence of the microbes to components of the extracellular matrix of the host. On Staphylococci the MSCRAMMs often have multiple ligands. Consequently we hypothesized that the S. aureus MSCRAMM Bbp (bone sialoprotein-binding protein) might recognize host molecules other than the identified bone protein. A ligand screen revealed that Bbp binds human fibrinogen (...

  9. Fibrinogen Is a Ligand for the Staphylococcus aureus Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMM) Bone Sialoprotein-binding Protein (Bbp)

    OpenAIRE

    Vazquez, Vanessa; Liang, Xiaowen; Horndahl, Jenny K.; Ganesh, Vannakambadi K.; Smeds, Emanuel; Foster, Timothy J.; Hook, Magnus

    2011-01-01

    Microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) are bacterial surface proteins mediating adherence of the microbes to components of the extracellular matrix of the host. On Staphylococci, the MSCRAMMs often have multiple ligands. Consequently, we hypothesized that the Staphylococcus aureus MSCRAMM bone sialoprotein-binding protein (Bbp) might recognize host molecules other than the identified bone protein. A ligand screen revealed that Bbp binds human fibrinogen ...

  10. 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.

  11. The cell surface proteome of Staphylococcus aureus

    NARCIS (Netherlands)

    Dreisbach, Annette; van Dijl, Jan Maarten; Buist, Girbe

    2011-01-01

    The Gram-positive bacterium Staphylococcus aureus is a wide spread opportunistic pathogen that can cause a range of life-threatening diseases. To obtain a better understanding of the global mechanisms for pathogenesis and to identify novel targets for therapeutic interventions, the S. aureus proteom

  12. Occurrence of methicillin-resistant Staphylococcus aureus in surface waters near industrial hog operation spray fields.

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    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.

  13. 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. PMID:27261430

  14. Staphylococcus aureus and Pseudomonas aeruginosa express and secrete human surfactant proteins.

    Science.gov (United States)

    Bräuer, Lars; Schicht, Martin; Worlitzsch, Dieter; Bensel, Tobias; Sawers, R Gary; Paulsen, Friedrich

    2013-01-01

    Surfactant proteins (SP), originally known from human lung surfactant, are essential to proper respiratory function in that they lower the surface tension of the alveoli. They are also important components of the innate immune system. The functional significance of these proteins is currently reflected by a very large and growing number of publications. The objective goal of this study was to elucidate whether Staphylococcus aureus and Pseudomonas aeruginosa is able to express surfactant proteins. 10 different strains of S. aureus and P. aeruginosa were analyzed by means of RT-PCR, Western blot analysis, ELISA, immunofluorescence microscopy and immunoelectron microscopy. The unexpected and surprising finding revealed in this study is that different strains of S. aureus and P. aeruginosa express and secrete proteins that react with currently commercially available antibodies to known human surfactant proteins. Our results strongly suggest that the bacteria are either able to express 'human-like' surfactant proteins on their own or that commercially available primers and antibodies to human surfactant proteins detect identical bacterial proteins and genes. The results may reflect the existence of a new group of bacterial surfactant proteins and DNA currently lacking in the relevant sequence and structure databases. At any rate, our knowledge of human surfactant proteins obtained from immunological and molecular biological studies may have been falsified by the presence of bacterial proteins and DNA and therefore requires critical reassessment. PMID:23349731

  15. Staphylococcus aureus and Pseudomonas aeruginosa express and secrete human surfactant proteins.

    Directory of Open Access Journals (Sweden)

    Lars Bräuer

    Full Text Available Surfactant proteins (SP, originally known from human lung surfactant, are essential to proper respiratory function in that they lower the surface tension of the alveoli. They are also important components of the innate immune system. The functional significance of these proteins is currently reflected by a very large and growing number of publications. The objective goal of this study was to elucidate whether Staphylococcus aureus and Pseudomonas aeruginosa is able to express surfactant proteins. 10 different strains of S. aureus and P. aeruginosa were analyzed by means of RT-PCR, Western blot analysis, ELISA, immunofluorescence microscopy and immunoelectron microscopy. The unexpected and surprising finding revealed in this study is that different strains of S. aureus and P. aeruginosa express and secrete proteins that react with currently commercially available antibodies to known human surfactant proteins. Our results strongly suggest that the bacteria are either able to express 'human-like' surfactant proteins on their own or that commercially available primers and antibodies to human surfactant proteins detect identical bacterial proteins and genes. The results may reflect the existence of a new group of bacterial surfactant proteins and DNA currently lacking in the relevant sequence and structure databases. At any rate, our knowledge of human surfactant proteins obtained from immunological and molecular biological studies may have been falsified by the presence of bacterial proteins and DNA and therefore requires critical reassessment.

  16. Staphylococcus aureus and Pseudomonas aeruginosa Express and Secrete Human Surfactant Proteins

    OpenAIRE

    Bräuer, Lars; Schicht, Martin; Worlitzsch, Dieter; Bensel, Tobias; Sawers, R. Gary; Paulsen, Friedrich

    2013-01-01

    Surfactant proteins (SP), originally known from human lung surfactant, are essential to proper respiratory function in that they lower the surface tension of the alveoli. They are also important components of the innate immune system. The functional significance of these proteins is currently reflected by a very large and growing number of publications. The objective goal of this study was to elucidate whether Staphylococcus aureus and Pseudomonas aeruginosa is able to express surfactant prot...

  17. Nanoadhesion of Staphylococcus aureus onto Titanium Implant Surfaces

    OpenAIRE

    Aguayo, S.; Donos, N.; Spratt, D.; Bozec, L.

    2015-01-01

    Adhesion of bacteria to dental implant surfaces is the critical initial step in the process of biofilm colonization; however, the specific nanoadhesive interactions occurring during the first contact between bacterial cells and biomaterial substrates remain poorly understood. In this report, we utilize single-cell force spectroscopy to characterize the dynamics of the initial interaction between living Staphylococcus aureus cells and machined titanium surfaces at the nanoscale. Values for max...

  18. 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

    with either trypsin or proteinase-K combined with LC-MS/MS. Trypsin-derived data were controlled using a "false-positive" strategy where cells were incubated without protease, removed by centrifugation and the resulting supernatants digested. Peptides identified in this fraction most likely result from cell...... 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...

  19. 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.

  20. 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.

  1. Nanoadhesion of Staphylococcus aureus onto Titanium Implant Surfaces.

    Science.gov (United States)

    Aguayo, S; Donos, N; Spratt, D; Bozec, L

    2015-08-01

    Adhesion of bacteria to dental implant surfaces is the critical initial step in the process of biofilm colonization; however, the specific nanoadhesive interactions occurring during the first contact between bacterial cells and biomaterial substrates remain poorly understood. In this report, we utilize single-cell force spectroscopy to characterize the dynamics of the initial interaction between living Staphylococcus aureus cells and machined titanium surfaces at the nanoscale. Values for maximum adhesion force were found to increase from 0-s (-0.27 ± 0.30 nN) to 60-s (-9.15 ± 0.78 nN) surface delays, with similar results observed for total adhesion work (7.39 ± 2.38 and 988.06 ± 117.08 aJ, respectively). Single unbinding events observed at higher surface delays were modeled according to the wormlike chain model, obtaining molecular contour-length predictions of 314.06 ± 9.27 nm. Average single-bond rupture forces of -0.95 ± 0.04 nN were observed at increased contact times. Short- and long-range force components of bacterial adhesion were obtained by Poisson analysis of single unbinding event peaks, yielding values of -0.75 ± 0.04 and -0.58 ± 0.15 nN, respectively. Addition of 2-mg/mL chlorhexidine to the buffer solution resulted in the inhibition of specific adhesive events but an increased overall adhesion force and work. These results suggest that initial attachment of S. aureus to smooth titanium is mostly mediated by short-range attractive forces observed at higher surface delays. PMID:26130256

  2. 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.

  3. PSC: protein surface classification.

    Science.gov (United States)

    Tseng, Yan Yuan; Li, Wen-Hsiung

    2012-07-01

    We recently proposed to classify proteins by their functional surfaces. Using the structural attributes of functional surfaces, we inferred the pairwise relationships of proteins and constructed an expandable database of protein surface classification (PSC). As the functional surface(s) of a protein is the local region where the protein performs its function, our classification may reflect the functional relationships among proteins. Currently, PSC contains a library of 1974 surface types that include 25,857 functional surfaces identified from 24,170 bound structures. The search tool in PSC empowers users to explore related surfaces that share similar local structures and core functions. Each functional surface is characterized by structural attributes, which are geometric, physicochemical or evolutionary features. The attributes have been normalized as descriptors and integrated to produce a profile for each functional surface in PSC. In addition, binding ligands are recorded for comparisons among homologs. PSC allows users to exploit related binding surfaces to reveal the changes in functionally important residues on homologs that have led to functional divergence during evolution. The substitutions at the key residues of a spatial pattern may determine the functional evolution of a protein. In PSC (http://pocket.uchicago.edu/psc/), a pool of changes in residues on similar functional surfaces is provided.

  4. 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

  5. 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.

  6. 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.

    2015-01-01

    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 (MSE). 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. PMID:25987705

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

    Directory of Open Access Journals (Sweden)

    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.

  8. High Level Expression and Purification of Atl, the Major Autolytic Protein of Staphylococcus aureus

    OpenAIRE

    Singh, Vineet K.

    2014-01-01

    Staphylococcus aureus is a major human and animal pathogen. Autolysins regulate the growth, turnover, cell lysis, biofilm formation, and the pathogenicity of S. aureus. Atl is the major autolysin in S. aureus. The biochemical and structural studies of staphylococcal Atl have been limited due to difficulty in cloning, high level overexpression, and purification of this protein. This study describes successful cloning, high level over-expression, and purification of two forms of fully functiona...

  9. Expression and inducibility in Staphylococcus aureus of the mecA gene, which encodes a methicillin-resistant S. aureus-specific penicillin-binding protein.

    OpenAIRE

    Ubukata, K; Nonoguchi, R; Matsuhashi, M; Konno, M

    1989-01-01

    A beta-lactam-sensitive strain of Staphylococcus aureus could be converted to methicillin resistance by the introduction of a plasmid carrying the 4.3-kilobase HindIII chromosomal DNA fragment which encoded the mecA gene from a methicillin-resistant S. aureus. Transformant cells produced methicillin-resistant S. aureus-specific penicillin-binding protein constitutively, and additional insertion of an inducible penicillinase plasmid caused production of the pencillin-binding protein to become ...

  10. Tolerance of Salmonella Enteritidis and Staphylococcus aureus to surface cleaning and household bleach

    NARCIS (Netherlands)

    Kusumaningrum, H.D.; Paltinaite, R.; Koomen, A.J.; Hazeleger, W.C.; Rombouts, F.M.; Beumer, R.R.

    2003-01-01

    Effective cleaning and sanitizing of food preparation sites is important because pathogens are readily spread to food contact surfaces after preparation of contaminated raw products. Tolerance of Salmonella Enteritidis and Staphylococcus aureus to surface cleaning by wiping with regular, microfiber,

  11. 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, G; 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. Microbiol

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  13. 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

  14. 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.

  15. 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. PMID:27354296

  16. 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

  17. Contamination of environmental surfaces by Staphylococcus aureus in a dermatological ward and its preventive measures.

    Science.gov (United States)

    Oie, Shigeharu; Yanagi, Chikashige; Matsui, Hiroto; Nishida, Tomoko; Tomita, Masaaki; Kamiya, Akira

    2005-01-01

    We investigated contamination of environmental surfaces by Staphylococcus aureus from April 1 to the end of June in 2002 in the dermatological ward (37 beds) of a university hospital. For surfaces contaminated by high levels of S. aureus, disinfection methods were evaluated. 100-10(5) colony forming units (cfu) of methicillin-resistant S. aureus (MRSA) or methicillin-sensitive S. aureus (MSSA) were detected on items such as an immersion bathtub (examined area, about 900 cm2), foot washbowl, stretcher for an immersion bath, and chair for the shower. After disinfection, no S. aureus was detected on smooth surfaces such as the immersion bathtub and foot washbowl; however, S. aureus was detected even after disinfection on porous surfaces made of sponge-like materials (polyethylene foam) such as the stretcher for the immersion bath and the shower chair. Scanning electron microscopy of the porous surfaces showed formation of a large amount of coccus and bacillus biofilms on the walls of pores in the multi-pore structure. Material that is porous should not be used in patient care settings because it is not possible to disinfect it properly. PMID:15635175

  18. 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...

  19. Structural Analysis of Serine/Threonine Protein Kinase B and Multiple Peptide Resistant Factor of Staphylococcus aureus

    OpenAIRE

    Rakette, Sonja

    2012-01-01

    Staphylococcus aureus (S. aureus) infections are becoming increasingly problematic. The bacteria gain resistance to new antibiotics in relatively short time periods. Besides the hospital associated methicillin-resistant S. aureus (HA-MRSA), infections with community-associated MRSA (CA-MRSA) are increasing. The only serine/threonine kinase PknB of S. aureus is composed of an intracellular kinase domain, a transmembrane helix and three extracellular penicillin-binding protein and serine/th...

  20. Are gym surfaces reservoirs for Staphylococcus aureus? A point prevalence survey.

    Science.gov (United States)

    Markley, John Daniel; Edmond, Michael B; Major, Yvette; Bearman, Gonzalo; Stevens, Michael P

    2012-12-01

    We sought to identify staphylococcal contamination of gymnasium surfaces. Various environmental surfaces were cultured at a university fitness center. Ten out of 99 samples yielded Staphylococcus aureus, all of which were methicillin-susceptible. Gym surfaces may be colonized with staphylococci and could play a role in community transmission of staphylococcal species.

  1. 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...... and revealed various mutations of spa, all of which included a deletion of immunoglobulin G binding domain C, in which the upper primer for spa typing is located, while two strains were truly spa negative. This is the first report demonstrating that nontypeability of S. aureus by spa sequencing is due either...

  2. Transfer and Decontamination of S. aureus in Transmission Routes Regarding Hands and Contact Surfaces

    Science.gov (United States)

    Arinder, Pernilla; Johannesson, Pär; Karlsson, Ingela; Borch, Elisabeth

    2016-01-01

    Hand hygiene, cleaning and disinfection are pre-requirements for hygiene management in hospital settings and the food industry. In order to facilitate risk management, different contamination scenarios and interventions need to be evaluated. In the present study data on transfer rates and reductions of Staphylococcus aureus were provided in an experimental set-up using artificial skin. Using this methodology, test persons were not exposed with pathogenic bacteria. An exposure assessment model was developed and applied to evaluate different contamination routes and hygiene interventions. The transfer rates of S. aureus from inoculated VITRO-SKIN® to fomites were calculated from blotting series. The VITRO-SKIN® was more prone to spread bacteria than fomites. When different surfaces were cleaned, the reduction of S. aureus varied between <1 and 7 log CFU. It could not be concluded that a certain coupon material, cleaning agent, cleaning wipe, soiling or humidity consistently resulted in a high or low reduction of S. aureus. The reduction of S. aureus and E. coli during hand washing was evaluated on artificial skin, VITRO-SKIN®. The reduction of E. coli on VITRO-SKIN® was similar to the log reduction obtained when washing human hands. The S. aureus count on a human hand was both calculated in different scenarios describing different contamination routes starting from a contaminated hand using the exposure assessment model, and measured on an experimental setup using VITRO-SKIN® for validation. A linear relationship was obtained between the analysed level of S. aureus and the calculated level. However, the calculated levels of S. aureus on the VITRO-SKIN® in the scenarios were 1–1.5 log lower than the analysed level. One of the scenarios was used to study the effect of interventions like hand washing and cleaning of surfaces. PMID:27280772

  3. The staphylococcal surface-glycopolymer wall teichoic acid (WTA) is crucial for complement activation and immunological defense against Staphylococcus aureus infection.

    Science.gov (United States)

    Kurokawa, Kenji; Takahashi, Kazue; Lee, Bok Luel

    2016-10-01

    Staphylococcus aureus is a Gram-positive bacterial pathogen that is decorated by glycopolymers, including wall teichoic acid (WTA), peptidoglycan, lipoteichoic acid, and capsular polysaccharides. These bacterial surface glycopolymers are recognized by serum antibodies and a variety of pattern recognition molecules, including mannose-binding lectin (MBL). Recently, we demonstrated that human serum MBL senses staphylococcal WTA. Whereas MBL in infants who have not yet fully developed adaptive immunity binds to S. aureus WTA and activates complement serum, MBL in adults who have fully developed adaptive immunity cannot bind to WTA because of an inhibitory effect of serum anti-WTA IgG. Furthermore, we showed that human anti-WTA IgGs purified from pooled adult serum IgGs triggered activation of classical complement-dependent opsonophagocytosis against S. aureus. Because the epitopes of WTA that are recognized by anti-WTA IgG and MBL have not been determined, we constructed several S. aureus mutants with altered WTA glycosylation. Our intensive biochemical studies provide evidence that the β-GlcNAc residues of WTA are required for the induction of anti-WTA IgG-mediated opsonophagocytosis and that both β- and α-GlcNAc residues are required for MBL-mediated complement activation. The molecular interactions of other S. aureus cell wall components and host recognition proteins are also discussed. In summary, in this review, we discuss the biological importance of S. aureus cell surface glycopolymers in complement activation and host defense responses. PMID:27424796

  4. 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

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

    International Nuclear Information System (INIS)

    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. (letter)

  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. Cleavage of Complement C3b to iC3b on the Surface of Staphylococcus aureus Is Mediated by Serum Complement Factor I

    OpenAIRE

    Cunnion, K. M.; Hair, P. S.; Buescher, E. S.

    2004-01-01

    Complement-mediated opsonization of Staphylococcus aureus bearing the dominant capsule serotypes, serotypes 5 and 8, remains incompletely understood. We have previously shown that complement plays a vital role in the efficient phagocytosis of a serotype 5 S. aureus strain and that the opsonic fragments of the central complement protein C3, C3b and iC3b, were present on the bacterial surface after incubation in human serum. In the present studies, C3b and iC3b were found on several serotype 5 ...

  8. Ultrastructural alteration of the cell surface of Staphylococcus aureus cultured in a different salt condition

    Directory of Open Access Journals (Sweden)

    Kanemasa,Yasuhiro

    1974-10-01

    Full Text Available Staphylococcus aureus growing in a normal NaGI medium has a specific NaGI tolerance property to grow in the medium contain. ing NaGl in as high a concentration as over 10%. In our comparative study of the cells proliferating in the normal NaGI medium and 10% NaGl medium, we have observed the following differences aside from the changes of lipid composition in the cytoplasmic membrane previously reported. 1. S. aureus grown in high NaGl medium undergoes changes as to increase its size and reduce its surface area. 2. The thickness and weight of cell wall are increased to about 1. 7 times and 1. 32 times, respectively. 3. The protoplast prepared from S. aureus growing in the high NaGI medium shows a weaker resistance to hypotonic condition than that from normal cell.

  9. G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA.

    Science.gov (United States)

    Stoltenburg, Regina; Krafčiková, Petra; Víglaský, Viktor; Strehlitz, Beate

    2016-01-01

    Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting Protein A on the cell surface. The full-length aptamer and one of its truncated variants could be demonstrated to specifically bind to Protein A-expressing intact cells of S. aureus, and thus have the potential to expand the portfolio of aptamers that can act as an analytical agent for the specific recognition and rapid detection of the bacterial pathogen. The functionality of the aptamer was found to be based on a very complex, but also highly variable structure. Two structural key elements were identified. The aptamer sequence contains several G-clusters allowing folding into a G-quadruplex structure with the potential of dimeric and multimeric assembly. An inverted repeat able to form an imperfect stem-loop at the 5'-end also contributes essentially to the aptameric function. PMID:27650576

  10. 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

  11. 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...

  12. 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...

  13. 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

  14. Staphylococcus aureus manganese transport protein C (MntC is an extracellular matrix- and plasminogen-binding protein.

    Directory of Open Access Journals (Sweden)

    Natália Salazar

    Full Text Available 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.

  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. Hydrophobic patches on protein surfaces

    NARCIS (Netherlands)

    Lijnzaad, P.

    2007-01-01

    Hydrophobicity is a prime determinant of the structure and function of proteins. It is the driving force behind the folding of soluble proteins, and when exposed on the surface, it is frequently involved in recognition and binding of ligands and other proteins. The energetic cost of exposing hydroph

  17. 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.

  18. Bap: a family of surface proteins involved in biofilm formation.

    Science.gov (United States)

    Lasa, Iñigo; Penadés, José R

    2006-03-01

    A group of surface proteins sharing several structural and functional features is emerging as an important element in the biofilm formation process of diverse bacterial species. The first member of this group of proteins was identified in a Staphylococcus aureus mastitis isolate and was named Bap (biofilm-associated protein). As common structural features, Bap-related proteins: (i) are present on the bacterial surface; (ii) show a high molecular weight; (iii) contain a core domain of tandem repeats; (iv) confer upon bacteria the capacity to form a biofilm; (v) play a relevant role in bacterial infectious processes; and (vi) can occasionally be contained in mobile elements. This review summarizes recent studies that have identified and assigned roles to Bap-related proteins in biofilm biology and virulence.

  19. 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

  20. 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.

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

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    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

  2. 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.

  3. 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.

  4. Reassessing the Role of Staphylococcus aureus Clumping Factor and Fibronectin-Binding Protein by Expression in Lactococcus lactis

    OpenAIRE

    Que, Yok-Ai; François, Patrice; Haefliger, Jacques-Antoine; Entenza, José-Manuel; Vaudaux, Pierre; Moreillon, Philippe

    2001-01-01

    Since Staphylococcus aureus expresses multiple pathogenic factors, studying their individual roles in single-gene-knockout mutants is difficult. To circumvent this problem, S. aureus clumping factor A (clfA) and fibronectin-binding protein A (fnbA) genes were constitutively expressed in poorly pathogenic Lactococcus lactis using the recently described pOri23 vector. The recombinant organisms were tested in vitro for their adherence to immobilized fibrinogen and fibronectin and in vivo for the...

  5. Antibody Responses in Patients with Staphylococcal Septicemia against Two Staphylococcus aureus Fibrinogen Binding Proteins: Clumping Factor and an Extracellular Fibrinogen Binding Protein

    OpenAIRE

    Colque-Navarro, Patricia; Palma, Marco; Söderquist, Bo; Flock, Jan-Ingmar; Möllby, Roland

    2000-01-01

    We analyzed the serum antibody responses against two Staphylococcus aureus fibrinogen binding proteins, the cell-bound clumping factor (Clf) and an extracellular fibrinogen binding protein (Efb). The material consisted of 105 consecutive serum samples from 41 patients suffering from S. aureus septicemia and 72 serum samples from healthy individuals. An enzyme-linked immunosorbent assay (ELISA) was developed. Healthy individuals showed variable levels of antibodies against the studied antigens...

  6. Differential Roles of Poly-N-Acetylglucosamine Surface Polysaccharide and Extracellular DNA in Staphylococcus aureus and Staphylococcus epidermidis Biofilms▿

    OpenAIRE

    Era A Izano; Amarante, Matthew A.; Kher, William B.; Kaplan, Jeffrey B.

    2007-01-01

    Staphylococcus aureus and Staphylococcus epidermidis are major human pathogens of increasing importance due to the dissemination of antibiotic-resistant strains. Evidence suggests that the ability to form matrix-encased biofilms contributes to the pathogenesis of S. aureus and S. epidermidis. In this study, we investigated the functions of two staphylococcal biofilm matrix polymers: poly-N-acetylglucosamine surface polysaccharide (PNAG) and extracellular DNA (ecDNA). We measured the ability o...

  7. 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.

  8. Nanorough titanium surfaces reduce adhesion of Escherichia coli and Staphylococcus aureus via nano adhesion points.

    Science.gov (United States)

    Lüdecke, Claudia; Roth, Martin; Yu, Wenqi; Horn, Uwe; Bossert, Jörg; Jandt, Klaus D

    2016-09-01

    Microbial adhesion to natural and synthetic materials surfaces is a key issue e.g. in food industry, sewage treatment and most importantly in the biomedical field. The current development and progress in nanoscale structuring of materials surfaces to control microbial adhesion requires an advanced understanding of the microbe-material-interaction. This study aimed to investigate the nanostructure of the microbe-material-interface and link it to microbial adhesion kinetics as function of titanium surface nanoroughness to gain new insight into controlling microbial adhesion via materials' surface nanoroughness. Adhesion of Escherichia coli and Staphylococcus aureus was statistically significantly reduced (p≤0.05) by 55.6 % and 40.5 %, respectively, on physical vapor deposited titanium thin films with a nanoroughness of 6nm and the lowest surface peak density compared to 2nm with the highest surface peak density. Cross-sectioning of the microbial cells with a focused ion beam (FIB) and SEM imaging provided for the first time direct insight into the titanium-microbe-interface. High resolution SEM micrographs gave evidence that the surface peaks are the loci of initial contact between the microbial cells and the material's surface. In a qualitative model we propose that the initial microbial adhesion on nanorough surfaces is controlled by the titanium surface peak density via nano adhesion points. This new understanding will help towards the design of materials surfaces for controlling microbial adhesion. PMID:27288816

  9. SURFACE: a database of protein surface regions for functional annotation

    OpenAIRE

    Ferrè, Fabrizio; Ausiello, Gabriele; Zanzoni, Andreas; Helmer-Citterich, Manuela

    2004-01-01

    The SURFACE (SUrface Residues and Functions Annotated, Compared and Evaluated, URL http://cbm.bio.uniroma2.it/surface/) database is a repository of annotated and compared protein surface regions. SURFACE contains the results of a large-scale protein annotation and local structural comparison project. A non-redundant set of protein chains is used to build a database of protein surface patches, defined as putative surface functional sites. Each patch is annotated with sequence and structure-der...

  10. The Sbi Protein Contributes to Staphylococcus aureus Inflammatory Response during Systemic Infection.

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    Cintia Daniela Gonzalez

    Full Text Available Staphylococcus aureus is an important human pathogen that causes infections that may present high morbidity and mortality. Among its many virulence factors protein A (SpA and Staphylococcal binding immunoglobulin protein (Sbi bind the Fc portion of IgG interfering with opsonophagocytosis. We have previously demonstrated that SpA interacts with the TNF-α receptor (TNFR 1 through each of the five IgG binding domains and induces the production of pro-inflammatory cytokines and chemokines. The IgG binding domains of Sbi are homologous to those of SpA, which allow us to hypothesize that Sbi might also have a role in the inflammatory response induced by S. aureus. We demonstrate that Sbi is a novel factor that participates in the induction of the inflammatory response during staphylococcal infections via TNFR1 and EGFR mediated signaling as well as downstream MAPKs. The expression of Sbi significantly contributed to IL-6 production and modulated CXCL-1 expression as well as neutrophil recruitment to the site of infection, thus demonstrating for the first time its relevance as a pro-inflammatory staphylococcal antigen in an in vivo model.

  11. Spread of Staphylococcus aureus between medical staff and high-frequency contact surfaces in a large metropolitan hospital

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    Li-sha Shi

    2015-12-01

    Conclusion: Cross-contamination of S. aureus or MRSA on medical workers' hands and contact surfaces was demonstrated within and between departments of a large metropolitan hospital. Improvements are needed in medical staff hygiene habits and in the cleaning of high-frequency contact surfaces to help prevent and control nosocomial infections.

  12. 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.

  13. 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. PMID:27085951

  14. 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

  15. Localized surface plasmon resonance-based hybrid Au-Ag nanoparticles for detection of Staphylococcus aureus enterotoxin B

    Science.gov (United States)

    Zhu, Shaoli; Du, ChunLei; Fu, Yongqi

    2009-09-01

    A triangular hybrid Au-Ag nanoparticles array was proposed for the purpose of biosensing in this paper. Constructing the hybrid nanoparticles, an Au thin film is capped on the Ag nanoparticles which are attached on glass substrate. The hybrid nanoparticles array was designed by means of finite-difference and time-domain (FDTD) algorithm-based computational numerical calculation and optimization. Sensitivity of refractive index of the hybrid nanoparticles array was obtained by the computational calculation and experimental detection. Moreover, the hybrid nanoparticles array can prevent oxidation of the pure Ag nanoparticles from atmosphere environment because the Au protective layer was deposited on top of the Ag nanoparticles so as to isolate the Ag particles from the atmosphere. We presented a novel surface covalent link method between the localized surface plasmon resonance (LSPR) effect-based biosensors with hybrid nanoparticles array and the detected target molecules. The generated surface plasmon wave from the array carries the biological interaction message into the corresponding spectra. Staphylococcus aureus enterotoxin B (SEB), a small protein toxin was directly detected at nanogramme per milliliter level using the triangular hybrid Au-Ag nanoparticles. Hence one more option for the SEB detection is provided by this way.

  16. ADHESION AND SURFACE GROWTH OF STAPHYLOCOCCUS AUREUS AND LACTOBACILLUS PLANTARUM ON VARIOUS METALS

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    Tsveteslava V. Ignatova-Ivanova

    2015-06-01

    Full Text Available Background: One of the major drawbacks in the use of biomedical materials is the occurrence of biomaterial-centered infections. Adhesion of microorganisms to an implant is mediated by their physico-chemical surface properties and the properties of the biomaterial surface itself. Subsequent surface growth of the microorganisms will lead to a mature biofilm and infection, which is difficult to eradicate by antibiotics. Objective: The purpose of this research is to examine the adhesion in the combined cultivation of Staphylococcus aureus and the Lactobacillus plantarum probiotic bacterium on the surface of different metals (copper, aluminium, low-carbon steel, and zinc. Methods: The precise weighing (with an allowance of 0,0001 g of the metal plates before and after the treatment found a minimum negative change in their weight, which may be caused by reduction resulting from corrosion processes, on one hand, or growth because of the forming of a biofilm, on the other. The structure of the layer over the metal plates was analysed by SEM (scanning electron microscopy JSM 5510. Results: The thinnest biofilm for both bacteria was registered on the surface of the copper plate. When a combined culture is used on the surface of the aluminium and the steel plates, the pathogenic bacterium is adhered predominantly. On the zinc plate it is only the probiotic bacterium that adheres. Conclusion: This is an initial research on this problem of significance for the doctors and it is about to be further examined

  17. 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.

  18. 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

    2015-01-01

    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 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. PMID:26411347

  19. 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.

  20. 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

  1. 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

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

    Directory of Open Access Journals (Sweden)

    Jaione Valle

    Full Text Available 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.

  3. Surface plasmon resonance detection of E. coli and methicillin-resistant S. aureus using bacteriophages.

    Science.gov (United States)

    Tawil, Nancy; Sacher, Edward; Mandeville, Rosemonde; Meunier, Michel

    2012-01-01

    Early diagnosis and appropriate treatment of Escherichia coli (E. coli) O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA) are key elements in preventing resultant life-threatening illnesses, such as hemorrhagic colitis, hemolytic uremic syndrome, and septicemia. In this report, we describe the use of surface plasmon resonance (SPR) for the biodetection of pathogenic bacteria, using bacteriophages as the recognition elements. T4 bacteriophages were used to detect E. coli, while a novel, highly specific phage was used to detect MRSA. We found that the system permits label-free, real-time, specific, rapid and cost-effective detection of pathogens, for concentrations of 10(3) colony forming units/milliliter, in less than 20 min. This system promises to become a diagnostic tool for bacteria that cause major public concern for food safety, bioterrorism, and nosocomial infections.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Cutting edge: members of the Staphylococcus aureus extracellular fibrinogen-binding protein family inhibit the interaction of C3d with complement receptor 2.

    Science.gov (United States)

    Ricklin, Daniel; Ricklin-Lichtsteiner, Salome K; Markiewski, Maciej M; Geisbrecht, Brian V; Lambris, John D

    2008-12-01

    Staphylococcus aureus expresses a highly diversified arsenal of immune evasion proteins, many of which target the complement system. The extracellular fibrinogen-binding protein (Efb) and the Efb homologous protein (Ehp) have previously been demonstrated to bind to C3 and inhibit complement activation and amplification. In this study we present the first evidence that Efb and Ehp are also capable of inhibiting the interaction of C3d with complement receptor 2 (CR2), which plays an important role in B cell activation and maturation. The C-terminal domain of Efb efficiently blocked this interaction both in surface plasmon resonance-based competition studies and cellular assays and prevented the CR2-mediated stimulation of B cells. Furthermore, analyses of the available structural data were consistent with a molecular mechanism that reflects both steric and electrostatic effects on the C3d-CR2 interaction. Our study therefore suggests that S. aureus may disrupt both the innate and adaptive immune responses with a single protein module. PMID:19017934

  6. 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

  7. Evaluation of different methods to recover methicillin-resistant Staphylococcus aureus from hospital environmental surfaces.

    LENUS (Irish Health Repository)

    Dolan, A

    2011-11-01

    The environment is implicated as a source of healthcare-associated infections (HAIs) and there is a need for evidence-based approaches to environmental sampling to assess cleanliness and improve infection prevention and control. We assessed, in vitro, different approaches to sampling the environment for meticillin-resistant Staphylococcus aureus (MRSA). In a laboratory-based investigation, the recovery of MRSA from two common hospital environments using six different sampling methods was evaluated, with a wild-type strain of MRSA. A 100 cm(2) section of mattress and a laboratory bench surface were contaminated with known inocula of MRSA. Bacteria were recovered by sampling at 30 min after inoculation, using either saline-moistened cotton swabs, neutralising buffer swabs, eSwabs or macrofoam swabs, which were all enriched in tryptone soya broth, or by sampling with direct contact plates or chromogenic \\'sweep\\' plates. The sensitivity (i.e. the minimum number of bacteria inoculated on to a surface which subsequently produced a positive result) of each method was determined for each surface. The most sensitive methods were eSwabs and macrofoam swabs, requiring 6.1 × 10(-1) and 3.9 × 10(-1) MRSA\\/cm(2), respectively, to produce a positive result from the bench surface. The least sensitive swabbing method was saline-moistened cotton swabs, requiring 1.1 × 10(3) MRSA\\/cm(2) of mattress. The recovery of bacteria from environmental samples varies with the swabs and methodology used and negative culture results do not exclude a pathogen-free environment. Greater standardisation is required to facilitate the assessment of cleanliness of healthcare environments.

  8. Biofilm formation by Staphylococcus aureus from food contact surfaces in a meat-based broth and sensitivity to sanitizers

    Directory of Open Access Journals (Sweden)

    Evandro Leite de Souza

    2014-01-01

    Full Text Available This study assessed the capacity of adhesion, the detachment kinetic and the biofilm formation by Staphylococcus aureus isolated from food services on stainless steel and polypropylene surfaces (2 x 2 cm when cultivated in a meat-based broth at 28 and 7 ºC. It was also to study the efficacy of the sanitizers sodium hypochlorite (250 mg/L and peracetic acid (30 mg/L in inactivating the bacterial cells in the preformed biofilm. S. aureus strains adhered in high numbers regardless the assayed surface kind and incubation temperature over 72 h. Cells detachment of surfaces revealed high persistence over the incubation period. Number of cells needed for biofilm formation was noted at all experimental systems already after 3 days. Peracetic acid and sodium hypochlorite were not efficient in completely removing the cells of S. aureus adhered on polypropylene and stainless steel surfaces. From these results, the assayed strains revealed high capacity to adhere and form biofilm on polypropylene and stainless steel surfaces under different growth conditions. Moreover, the cells in biofilm matrix were resistant for total removal when submitted to the exposure to sanitizers.

  9. 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.

  10. 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.

  11. Effects of Silver Sulphadiazine on Production of Extracellular Proteins by Strains of Staphylococcus Aureus Isolated from Burns Wound.

    Science.gov (United States)

    Javid Khojasteh, Vahideh; Alfakhri, Souad; Foster, Howard Anthony

    2016-01-01

    Previous studies had shown that sub-inhibitory concentrations of silver sulphadiazine (AgSD) stimulated the production of Toxic Shock Syndrome Toxin-1 in certain strains (responder strains) of Staphylococcus aureus and that protease production was also affected. No changes were detected in other strains (non-responders). Extracellular proteins from eleven responder and non-responder strains grown with and without AgSD were separated by SDS PAGE. There were three classes of response, responder strains that showed enhancement of synthesis of certain proteins, non-responder strains that showed no change and responder strains that showed a general decrease in exoprotein production in the early stages of growth. The results showed that the effects of AgSD were complex and that S. aureus strains were heterogenous with respect to their response to sub-inhibitory concentrations of AgSD. PMID:27642339

  12. Ice nucleation protein as a bacterial surface display protein

    OpenAIRE

    Sarhan Mohammed A.A.

    2011-01-01

    Surface display technology can be defined as that phenotype (protein or peptide) which is linked to a genotype (DNA or RNA) through an appropriate anchoring motif. A bacterial surface display system is based on expressing recombinant proteins fused to sorting signals (anchoring motifs) that direct their incorporation on the cell surface.

  13. 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.

  14. 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. PMID:27040789

  15. Femtosecond laser surface texturing of titanium as a method to reduce the adhesion of Staphylococcus aureus and biofilm formation

    Science.gov (United States)

    Cunha, Alexandre; Elie, Anne-Marie; Plawinski, Laurent; Serro, Ana Paula; Botelho do Rego, Ana Maria; Almeida, Amélia; Urdaci, Maria C.; Durrieu, Marie-Christine; Vilar, Rui

    2016-01-01

    The aim of the present work was to investigate the possibility of using femtosecond laser surface texturing as a method to reduce the colonization of Grade 2 Titanium alloy surfaces by Staphylococcus aureus and the subsequent formation of biofilm. The laser treatments were carried out with a Yb:KYW chirped-pulse-regenerative amplification laser system with a central wavelength of 1030 nm and a pulse duration of 500 fs. Two types of surface textures, consisting of laser-induced periodic surface structures (LIPSS) and nanopillars, were produced. The topography, chemical composition and phase constitution of these surfaces were investigated by atomic force microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, micro-Raman spectroscopy, and X-ray diffraction. Surface wettability was assessed by the sessile drop method using water and diiodomethane as testing liquids. The response of S. aureus put into contact with the laser treated surfaces in controlled conditions was investigated by epifluorescence microscopy and scanning electron microscopy 48 h after cell seeding. The results achieved show that the laser treatment reduces significantly the bacterial adhesion to the surface as well as biofilm formation as compared to a reference polished surfaces and suggest that femtosecond laser texturing is a simple and promising method for endowing dental and orthopedic titanium implants with antibacterial properties, reducing the risk of implant-associated infections without requiring immobilized antibacterial substances, nanoparticles or coatings.

  16. Enhanced Levels of Staphylococcus aureus Stress Protein GroEL and DnaK Homologs Early in Infection of Human Epithelial Cells

    OpenAIRE

    Qoronfleh, M. Walid; Bortner, Carol A.; Schwartzberg, Paul; Wilkinson, Brian J.

    1998-01-01

    Antibodies to Staphylococcus aureus heat shock proteins (Hsps) are present in the sera of patients with S. aureus endocarditis (M. W. Qoronfleh, W. Weraarchakul, and B. J. Wilkinson, Infect. Immun. 61:1567–1570, 1993). Although these proteins are immunogenic, their role in infection has not been established. We developed a cell culture system as a model to examine the potential involvement of staphylococcal Hsps in the initial events of infection. This study supports a model in which a clinic...

  17. Resistance to Quinupristin-Dalfopristin Due to Mutation of L22 Ribosomal Protein in Staphylococcus aureus

    OpenAIRE

    Malbruny, Brigitte; Canu, Annie; Bozdogan, Bülent; Fantin, Bruno; Zarrouk, Virginie; Dutka-Malen, Sylvie; Feger, Celine; Leclercq, Roland

    2002-01-01

    The mechanism of resistance to the streptogramin antibiotics quinupristin and dalfopristin was studied in a Staphylococcus aureus clinical isolate selected under quinupristin-dalfopristin therapy, in four derivatives of S. aureus RN4220 selected in vitro, and in a mutant selected in a model of rabbit aortic endocarditis. For all strains the MICs of erythromycin, quinupristin, and quinupristin-dalfopristin were higher than those for the parental strains but the MICs of dalfopristin and lincomy...

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

    Directory of Open Access Journals (Sweden)

    Olivier Poupel

    Full Text Available 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

  19. Competitive Protein Adsorption - Multilayer Adsorption and Surface Induced Protein Aggregation

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hou, Xiaolin

    2009-01-01

    In this study, competitive adsorption of albumin and IgG (immunoglobulin G) from human serum solutions and protein mixtures onto polymer surfaces is studied by means of radioactive labeling. By using two different radiolabels (125I and 131I), albumin and IgG adsorption to polymer surfaces...... is monitored simultaneously and the influence from the presence of other human serum proteins on albumin and IgG adsorption, as well as their mutual influence during adsorption processes, is investigated. Exploring protein adsorption by combining analysis of competitive adsorption from complex solutions...... of high concentration with investigation of single protein adsorption and interdependent adsorption between two specific proteins enables us to map protein adsorption sequences during competitive protein adsorption. Our study shows that proteins can adsorb in a multilayer fashion onto the polymer surfaces...

  20. Three-Dimensional Structure and Biophysical Characterization of Staphylococcus aureus Cell Surface Antigen-Manganese Transporter MntC

    Energy Technology Data Exchange (ETDEWEB)

    Gribenko, Alexey; Mosyak, Lidia; Ghosh, Sharmistha; Parris, Kevin; Svenson, Kristine; Moran, Justin; Chu, Ling; Li, Sheng; Liu, Tong; Woods, Jr., Virgil L.; Jansen, Kathrin U.; Green, Bruce A.; Anderson, Annaliesa S.; Matsuka, Yury V. [Pfizer; (UCSD)

    2013-08-23

    MntC is a metal-binding protein component of the Mn2 +-specific mntABC transporter from the pathogen Staphylococcus aureus. The protein is expressed during the early stages of infection and was proven to be effective at reducing both S. aureus and Staphylococcus epidermidis infections in a murine animal model when used as a vaccine antigen. MntC is currently being tested in human clinical trials as a component of a multiantigen vaccine for the prevention of S. aureus infections. To better understand the biological function of MntC, we are providing structural and biophysical characterization of the protein in this work. The three-dimensional structure of the protein was solved by X-ray crystallography at 2.2 Å resolution and suggests two potential metal binding modes, which may lead to reversible as well as irreversible metal binding. Precise Mn2 +-binding affinity of the protein was determined from the isothermal titration calorimetry experiments using a competition approach. Differential scanning calorimetry experiments confirmed that divalent metals can indeed bind to MntC reversibly as well as irreversibly. Finally, Mn2 +-induced structural and dynamics changes have been characterized using spectroscopic methods and deuterium–hydrogen exchange mass spectroscopy. Results of the experiments show that these changes are minimal and are largely restricted to the structural elements involved in metal coordination. Therefore, it is unlikely that antibody binding to this antigen will be affected by the occupancy of the metal-binding site by Mn2 +.

  1. 金黄色葡萄球菌IsdB基因缺失序列比较与宿主特异性分析%GENE DELETIONS OF IRON RESPONSIVE SURFACE DETERMINANT ISDB PROTEIN OF STAPHYLOCOCCUS AUREUS ARE NOT RELATED TO HOST TROPISM

    Institute of Scientific and Technical Information of China (English)

    王登峰; 吴建勇; 李建军; 杨学云; 王治才

    2012-01-01

    对新疆地区分离的牛源金黄色葡萄球菌XJ43(CC97)、XJ108(CC398)和XJ69(CC479)的IsdB基因序列测定、比对发现,在编码非功能结构域的277-314碱基(5'-3')间存在不同序列的缺失,并导致各编码蛋白质的二级结构出现一定差异;Blast分析显示,NCBI中登录的金黄色葡萄球菌不同菌株IsdB基因序列相似性大于95%,但与新分离的三种序列型相比,除医源菌株USA300 TCH959(CC8)与XJ69(CC479)的IsdB基因序列缺失一致外,其他医源菌株与XJ43缺失的序列均不一致;牛源或羊源CC133、CC425、CC151和属人畜共患CC398 XJ108菌株IsdB基因缺失的序列一致,表明这种序列缺失与金黄色葡萄球菌的宿主特异性无绝对相关性,但含有IsdB基因序列缺失的菌株在反刍动物乳腺感染中出现频率较高。%Comparison of IsdB gene sequences of Staphylococcus aureus strains XJ43(CC97),XJ108(CC398) and XJ69(CC479) of Xinjiang origin revealed that gene deletions of different lengths existed between 277-314 base pairs,which constituted three sequence types and led to changes in protein secondary structure.Further Blast analysis of genomes of Staphylococcus aureus deposited in NCBI confirmed also identified their difference from XJ43 strain despite of their sequence consensus at more than 95%.The same gene deletion was found in bovine strains CC133 and 425,sheep strain 151 and zoonotic strain CC398 but not in human strains USA300 and TCH959(CC8).The deletion of IsdB gene sequences was not related to animal species although it occurred more frequently in ruminant mastitis strains.

  2. 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.

  3. Neurocognitive derivation of protein surface property from protein aggregate parameters

    OpenAIRE

    Mishra, Hrishikesh; Lahiri, Tapobrata

    2011-01-01

    Current work targeted to predicate parametric relationship between aggregate and individual property of a protein. In this approach, we considered individual property of a protein as its Surface Roughness Index (SRI) which was shown to have potential to classify SCOP protein families. The bulk property was however considered as Intensity Level based Multi-fractal Dimension (ILMFD) of ordinary microscopic images of heat denatured protein aggregates which was known to have potential to serve as...

  4. In Vitro Resistance of Staphylococcus aureus to Thrombin-Induced Platelet Microbicidal Protein Is Associated with Alterations in Cytoplasmic Membrane Fluidity

    OpenAIRE

    Bayer, Arnold S.; Prasad, Rajendra; Chandra, Jyotsna; Koul, Anjni; Smriti, M.; Varma, Archana; Skurray, Ronald A.; Firth, Neville; Brown, Melissa H.; Koo, Su-Pin; Yeaman, Michael R.

    2000-01-01

    Platelet microbicidal proteins (PMPs) are small, cationic peptides which possess potent microbicidal activities against common bloodstream pathogens, such as Staphylococcus aureus. We previously showed that S. aureus strains exhibiting resistance to thrombin-induced PMP (tPMP-1) in vitro have an enhanced capacity to cause human and experimental endocarditis (T. Wu, M. R. Yeaman, and A. S. Bayer, Antimicrob. Agents Chemother. 38:729–732, 1994; A. S. Bayer et al., Antimicrob. Agents Chemother. ...

  5. Interactions between whey proteins and kaolinite surfaces

    International Nuclear Information System (INIS)

    The nature of the interactions between whey proteins and kaolinite surfaces was investigated by adsorption-desorption experiments at room temperature, performed at the isoelectric point (IEP) of the proteins and at pH 7. It was found that kaolinite is a strong adsorbent for proteins, reaching the maximum adsorption capacity at the IEP of each protein. At pH 7.0, the retention capacity decreased considerably. The adsorption isotherms showed typical Langmuir characteristics. X-ray diffraction data for the protein-kaolinite complexes showed that protein molecules were not intercalated in the mineral structure, but immobilized at the external surfaces and the edges of the kaolinite. Fourier transform IR results indicate the absence of hydrogen bonding between kaolinite surfaces and the polypeptide chain. The adsorption patterns appear to be related to electrostatic interactions, although steric effects should be also considered

  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. Role of GapC in the pathogenesis of Staphylococcus aureus.

    Science.gov (United States)

    Kerro-Dego, Oudessa; Prysliak, Tracy; Perez-Casal, Jose; Potter, Andrew A

    2012-05-01

    Staphylococcus aureus is recognized worldwide as a major pathogen causing clinical or subclinical intramammary infections in lactating cows, sheep and goats. S. aureus produces a wide arsenal of cell surface and extracellular proteins involved in virulence. Among these are two conserved proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity named glyceraldehyde-3-phosphate dehydrogenase-B (GapB) and -C (GapC). In this study, we used the S. aureus wild type strain RN6390 and its isogenic gapC mutant H330 in in vitro and in vivo studies and determined that the S. aureus GapC protein plays a role on adherence to and internalization into bovine mammary epithelial (MAC-T) cells. In addition, we found that S. aureus H330 did not caused mastitis after an experimental infection of ovine mammary glands. Together, these results show that GapC is important in the pathogenesis of S. aureus mastitis. PMID:22176759

  8. Effect of pH on surface energy of glass and Teflon and theoretical prediction of Staphylococcus aureus adhesion

    Energy Technology Data Exchange (ETDEWEB)

    Hamadi, F. [Equipe de recherche: Microbiologie et biochimie appliquee a l' agroalimentaire, l' environnement et la sante. Faculte de Sciences et Techniques, B. P 523, Beni, Mellal (Morocco); Latrache, H., E-mail: latracheh@yahoo.fr [Equipe de recherche: Microbiologie et biochimie appliquee a l' agroalimentaire, l' environnement et la sante. Faculte de Sciences et Techniques, B. P 523, Beni, Mellal (Morocco); Zekraoui, M. [Equipe de recherche: Genie industriel, agroalimentaire et environnement. Faculte de Sciences et Techniques, B. P 523, Beni, Mellal (Morocco); Ellouali, M. [Equipe de recherche: Microbiologie et biochimie appliquee a l' agroalimentaire, l' environnement et la sante. Faculte de Sciences et Techniques, B. P 523, Beni, Mellal (Morocco); Bengourram, J. [Equipe de recherche: Genie industriel, agroalimentaire et environnement. Faculte de Sciences et Techniques, B. P 523, Beni, Mellal (Morocco)

    2009-05-05

    The surface energy of glass and Teflon at various pH values was examined. Contact angle was used to determine physico-chemical substratum properties. The surface energy of both substratums including, hydrophobicity, and electron donor/electron acceptor (Lewis acid-base properties) were found to depend on pH of contact solution. The maximum of hydrophobicity (higher negative value of {Delta}G{sub iwi}) was obtained at pH 11 and pH 6.5 for glass and Teflon respectively. The electron donor property was higher at pH 5 and pH 3 for glass and Teflon respectively. Moreover, prediction of Staphylococcus aureus adhesion on both substratums was estimated by calculating the total interaction free energy ({Delta}G{sup Tot}). Based on the value of {Delta}G{sup Tot}, S. aureus should adhere to glass at pH 2, pH 3 and pH 11 with the maximal adhesion obtained at pH 3 and pH 11. For Teflon, regardless of pH values, S. aureus should be able to attach on this substratum with the high adhesion level at pH 5. The relation between surface energy of substratum and the total interaction free energy was also examined. Based on this relation and the value of the components of total interaction free energy, we show that adhesion to glass could be governed by both short range forces (Lewis acid-bases forces) and by long range forces (van der Waals forces) and the adhesion to Teflon could be mediated only by the short range forces.

  9. Effect of pH on surface energy of glass and Teflon and theoretical prediction of Staphylococcus aureus adhesion

    International Nuclear Information System (INIS)

    The surface energy of glass and Teflon at various pH values was examined. Contact angle was used to determine physico-chemical substratum properties. The surface energy of both substratums including, hydrophobicity, and electron donor/electron acceptor (Lewis acid-base properties) were found to depend on pH of contact solution. The maximum of hydrophobicity (higher negative value of ΔGiwi) was obtained at pH 11 and pH 6.5 for glass and Teflon respectively. The electron donor property was higher at pH 5 and pH 3 for glass and Teflon respectively. Moreover, prediction of Staphylococcus aureus adhesion on both substratums was estimated by calculating the total interaction free energy (ΔGTot). Based on the value of ΔGTot, S. aureus should adhere to glass at pH 2, pH 3 and pH 11 with the maximal adhesion obtained at pH 3 and pH 11. For Teflon, regardless of pH values, S. aureus should be able to attach on this substratum with the high adhesion level at pH 5. The relation between surface energy of substratum and the total interaction free energy was also examined. Based on this relation and the value of the components of total interaction free energy, we show that adhesion to glass could be governed by both short range forces (Lewis acid-bases forces) and by long range forces (van der Waals forces) and the adhesion to Teflon could be mediated only by the short range forces.

  10. Neurocognitive derivation of protein surface property from protein aggregate parameters

    Science.gov (United States)

    Mishra, Hrishikesh; Lahiri, Tapobrata

    2011-01-01

    Current work targeted to predicate parametric relationship between aggregate and individual property of a protein. In this approach, we considered individual property of a protein as its Surface Roughness Index (SRI) which was shown to have potential to classify SCOP protein families. The bulk property was however considered as Intensity Level based Multi-fractal Dimension (ILMFD) of ordinary microscopic images of heat denatured protein aggregates which was known to have potential to serve as protein marker. The protocol used multiple ILMFD inputs obtained for a protein to produce a set of mapped outputs as possible SRI candidates. The outputs were further clustered and largest cluster centre after normalization was found to be a close approximation of expected SRI that was calculated from known PDB structure. The outcome showed that faster derivation of individual protein’s surface property might be possible using its bulk form, heat denatured aggregates. PMID:21572883

  11. 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

  12. Site-specific immobilization of protein layers on gold surfaces via orthogonal sortases.

    Science.gov (United States)

    Raeeszadeh-Sarmazdeh, Maryam; Parthasarathy, Ranganath; Boder, Eric T

    2015-04-01

    We report a site-specific, sortase-mediated ligation to immobilize proteins layer-by-layer on a gold surface. Recombinant fluorescent proteins with a Sortase A recognition tag at the C-terminus were immobilized on peptide-modified gold surfaces. We used two sortases with different substrate specificities (Streptococcus pyogenes Sortase A and Staphylococcus aureus Sortase A) to immobilize layers of GFP and mCherry site-specifically on the gold surface. Surfaces were characterized using fluorescence and atomic force microscopy after immobilizing each layer of protein. Fluorescent micrographs showed that both protein immobilization on the modified gold surface and protein oligomerization are sortase-dependent. AFM images showed that either homogenous protein monolayers or layers of protein oligomers can be generated using appropriately tagged substrate proteins. Using Sortase A variants with orthogonal peptide substrate specificities, site-specific immobilization of appropriately tagged GFP onto a layer of immobilized mCherry was achieved without disruption of the underlying protein layer.

  13. Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein

    Energy Technology Data Exchange (ETDEWEB)

    Dooley, J.S.G.; Trust, T.J.

    1988-02-01

    The surface protein composition of members of a serogroup of Aeromonas hydrophila was examined. Immunoblotting with antiserum raised against formalinized whole cells of A. hydrophila TF7 showed a 52K S-layer protein to be the major surface protein antigen, and impermeant Sulfo-NHS-Biotin cell surface labeling showed that the 52K S-layer protein was the only protein accessible to the Sulfo-NHS-Biotin label and effectively masked underlying outer membrane (OM) proteins. In its native surface conformation the 52K S-layer protein was only weakly reactive with a lactoperoxidase /sup 125/I surface iodination procedure. A UV-induced rough lipopolysaccharide (LPS) mutant of TF7 was found to produce an intact S layer, but a deep rough LPS mutant was unable to maintain an array on the cell surface and excreted the S-layer protein into the growth medium, indicating that a minimum LPS oligosaccharide size required for A. hydrophila S-layer anchoring. The native S layer was permeable to /sup 125/I in the lactoperoxidase radiolabeling procedure, and two major OM proteins of molecular weights 30,000 and 48,000 were iodinated. The 48K species was a peptidoglycan-associated, transmembrane protein which exhibited heat-modifiable SDS solubilization behavior characteristic of a porin protein. A 50K major peptidoglycan-associated OM protein which was not radiolabeled exhibited similar SDS heat modification characteristics and possibly represents a second porin protein.

  14. Functional dynamics of cell surface membrane proteins

    Science.gov (United States)

    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

  15. Proteins in solution: Fractal surfaces in solutions

    Directory of Open Access Journals (Sweden)

    R. Tscheliessnig

    2016-02-01

    Full Text Available The concept of the surface of a protein in solution, as well of the interface between protein and 'bulk solution', is introduced. The experimental technique of small angle X-ray and neutron scattering is introduced and described briefly. Molecular dynamics simulation, as an appropriate computational tool for studying the hydration shell of proteins, is also discussed. The concept of protein surfaces with fractal dimensions is elaborated. We finish by exposing an experimental (using small angle X-ray scattering and a computer simulation case study, which are meant as demonstrations of the possibilities we have at hand for investigating the delicate interfaces that connect (and divide protein molecules and the neighboring electrolyte solution.

  16. S-Layer-Mediated Display of the Immunoglobulin G-Binding Domain of Streptococcal Protein G on the Surface of Caulobacter crescentus: Development of an Immunoactive Reagent▿

    OpenAIRE

    Nomellini, John F.; Duncan, Gillian; Dorocicz, Irene R.; Smit, John

    2007-01-01

    The immunoglobulin G (IgG)-binding streptococcal protein G is often used for immunoprecipitation or immunoadsorption-based assays, as it exhibits binding to a broader spectrum of host species IgG and IgG subclasses than the alternative, Staphylococcus aureus protein A. Caulobacter crescentus produces a hexagonally arranged paracrystalline protein surface layer (S-layer) composed of a single secreted protein, RsaA, that is notably tolerant of heterologous peptide insertions while maintaining t...

  17. 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....

  18. Different staphylococcal species contain various numbers of penicillin-binding proteins ranging from four (Staphylococcus aureus) to only one (Staphylococcus hyicus).

    OpenAIRE

    Canepari, P; Varaldo, P E; Fontana, R; Satta, G

    1985-01-01

    The penicillin-binding proteins of a total of 25 staphylococcal strains belonging to five different species were analyzed. All strains of the same species showed an identical penicillin-binding protein pattern which clearly differed from that of strains of the other species. Staphylococcus aureus, Staphylococcus intermedius, Staphylococcus simulans, and the dolphin strains were found to contain two to four penicillin-binding proteins. Strains of Staphylococcus hyicus exhibited only one penici...

  19. Membrane Disruption by Antimicrobial Fatty Acids Releases Low-Molecular-Weight Proteins from Staphylococcus aureus

    OpenAIRE

    Parsons, Joshua B; Yao, Jiangwei; Frank, Matthew W.; Jackson, Pamela; Rock, Charles O

    2012-01-01

    The skin represents an important barrier for pathogens and is known to produce fatty acids that are toxic toward Gram-positive bacteria. A screen of fatty acids as growth inhibitors of Staphylococcus aureus revealed structure-specific antibacterial activity. Fatty acids like oleate (18:1Δ9) were nontoxic, whereas palmitoleate (16:1Δ9) was a potent growth inhibitor. Cells treated with 16:1Δ9 exhibited rapid membrane depolarization, the disruption of all major branches of macromolecular synthes...

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Atomic force microscopy study on the attachment of E. coli and S. aureus to a patterned surface of different materials

    Science.gov (United States)

    Zhang, Hailong; Komaromy, Andras; Boysen, Reinhard I.; Rius, Gemma; Borrise, Xavier; Perez-Murano, Francesc; Hearn, Milton T. W.; Nicolau, Dan V.

    2007-12-01

    Many bacterial species are able to colonize the surfaces of biomedical tools or devices and form biofilms creating a source of infection and other deleterious effects. Biofilms constitute environments in which bacteria grow and are protected from the host's immune system and antimicrobial medications. The bacterial adhesion, which is an important and first step in biofilm formation, is influenced by several physico-chemical and topographical factors at the interfaces between the bacterial cell and the surface. Therefore, the mechanism of initial adhesion needs to be investigated to better understand the events of anchorage and film formation as bacteria colonise surfaces. In this work, atomic force microscopy (AFM) in the tapping mode of imaging has been employed to investigate the attachment of bacteria onto a structured surface patterned with different hydrophilic and hydrophobic areas. The interactions of Escherichia coli and Staphylococcus aureus with these structures were also monitored by fluorescence microscopy. AFM was successfully employed for the study of the cell responses to both nanotopography and the surface chemistry via observation of various cell functions; including extracellular polymeric substance (EPS) mediated cellular adhesion.

  2. 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.

  3. 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. PMID:26299923

  4. Teicoplanin-resistant Staphylococcus aureus expresses a novel membrane protein and increases expression of penicillin-binding protein 2 complex.

    OpenAIRE

    Shlaes, D M; Shlaes, J H; VINCENT, S; Etter, L; Fey, P. D.; Goering, R. V.

    1993-01-01

    In the recent clinical trials of teicoplanin therapy of endocarditis caused by Staphylococcus aureus, at least one instance of the emergence of teicoplanin-resistant strains during therapy has been reported (G.W. Kaatz, S. M. Seo, N. J. Dorman, and S. A. Lerner, J. Infect. Dis 162:103-108, 1990). We have confirmed, using conventional electrophoresis of EcoRI-digested chromosomal DNA and pulsed-field gel electrophoresis of SmaI-digested chromosomal DNA, that the resistant strain (12873) (MIC, ...

  5. A structural basis for Staphylococcal complement subversion: X-ray structure of the complement-binding domain of Staphylococcus aureus protein Sbi in complex with ligand C3d

    OpenAIRE

    Clark, E. A.; Crennell, S.; Upadhyay, A.; Zozulya, A. V.; Mackay, J. D.; Svergun, D.I.; Bagby, S; van den Elsen, J. M.

    2011-01-01

    The structure of the complement-binding domain of Staphylococcus aureus protein Sbi (Sbi-IV) in complex with ligand C3d is presented. The 1.7 Å resolution structure reveals the molecular details of the recognition of thioester-containing fragment C3d of the central complement component C3, involving interactions between residues of Sbi-IV helix α2 and the acidic concave surface of C3d. The complex provides a structural basis for the binding preference of Sbi for native C3 over C3b and explain...

  6. 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.

  7. What is the best method? Recovery of methicillin-resistant Staphylococcus aureus and extended-spectrum β-lactamase-producing Escherichia coli from inanimate hospital surfaces.

    Science.gov (United States)

    Claro, Tânia; Galvin, Sandra; Cahill, Orla; Fitzgerald-Hughes, Deirdre; Daniels, Stephen; Humphreys, Hilary

    2014-07-01

    Environmental sampling in hospitals, when required, needs to be reliable. We evaluated different methods of sampling methicillin-resistant Staphylococcus aureus and extended-spectrum β-lactamase-producing Escherichia coli on 5 materials of the hospital setting. Petrifilms and contact plates were superior to swabs for all of the surfaces studied.

  8. Protein-mediated surface structuring in biomembranes

    Directory of Open Access Journals (Sweden)

    Maggio B.

    2005-01-01

    Full Text Available The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein, integral (Folch-Lees proteolipid protein and amphitropic (c-Fos and c-Jun proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase, in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

  9. 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

    Directory of Open Access Journals (Sweden)

    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.

  10. Organising Atoms, Clusters and Proteins on Surfaces

    Science.gov (United States)

    Palmer, Richard E.

    2008-10-01

    This talk will discuss new developments in the creation of nanoscale surface features and their applications in biomedicine. Electron-surface interactions and plasma methods play a crucial role in both the production and analysis of these ``atomic architectures.'' At the extreme limit, electron injection from the tip of a scanning tunnelling microscope (STM) enables bond-selective manipulation of individual polyatomic molecules [1]. On a more practical level, the controlled deposition of size-selected clusters [2], generated by magnetron sputtering and gas condensation followed by mass selection, represents a surprisingly efficient route to the fabrication of surface features of size 1-10 nm, the size scale of biological molecules such as proteins. STM and AFM measurements show the clusters can act as binding sites for individual protein molecules. For example, the pinning of size-selected AuN clusters (N = 1--2000) to the (hydrophobic) graphite surface presents bindings site for sulphur atoms and thus for the cysteine residues in protein molecules. Systematic studies of different proteins [3] provide ``ground rules'' for residue-specific protein immobilisation by clusters and have led to the development of a novel biochip for protein screening by a spin-off company. The 3D atomic structure of the clusters is highly relevant to such applications. We show that measurement of the scattered electron beam intensity - specifically, the high angle annular dark field (HAADF) signal - in the scanning transmission electron microscope (STEM) allows us (a) to count the number of atoms in a cluster on the surface and (b) to determine a 3D atom-density map of the cluster when an aberration-corrected STEM is used [4]. 1. P.A. Sloan and R.E. Palmer, Nature 434 367 (2005). 2. S. Pratontep, P. Preece, C. Xirouchaki, R.E. Palmer, C.F. Sanz-Navarro, S.D. Kenny and R. Smith, Phys. Rev. Lett. 90 055503 (2003). 3. R.E. Palmer, S. Pratontep and H.-G. Boyen, Nature Materials 2 443 (2003

  11. Real-time PCR detection of Staphylococcus aureus in milk and meat using new primers designed from the heat shock protein gene htrA sequence.

    Science.gov (United States)

    Chiang, Yu-Cheng; Fan, Chih-Ming; Liao, Wan-Wen; Lin, Chien-Ku; Tsen, Hau-Yang

    2007-12-01

    Staphylococcus aureus may cause foodborne disease outbreaks and staphylococcal infections and is one of the major causes of mastitis. Rapid and reliable methods for detection of this microorganism in milk and other foods are needed. In this study, we designed a primer set from the sequence of the heat shock protein gene htrA, a gene coding for high-temperature-requirement A (HtrA) protein, and used it for real-time PCR detection of S. aureus isolates: 16 reference strains and 40 strains isolated from food-poisoning cases. All strains tested generated positive results. Bacterial strains other than S. aureus, including strains of other Staphylococcus species, did not produce positive results. When this primer set was used for the real-time PCR detection of S. aureus in milk and meat samples without the preenrichment step, samples with target cell numbers greater than 10(3) CFU/ml or CFU/g could be detected, indicating the potential quantitative ability of this real-time PCR assay. With a 10-h preenrichment step, however, a detection limit of 1 CFU/ml or CFU/g could be obtained.

  12. Survival of Staphylococcus aureus exposed to UV radiation on the surface of ceramic tiles coated with TiO2.

    Science.gov (United States)

    Szczawiński, J; Tomaszewski, H; Jackowska-Tracz, A; Szczawińska, M E

    2011-01-01

    The aim of this study was to determine and compare the antimicrobial activity of UV radiation of wavelength 253.7 nm (used in typical germicidal lamps) against Staphylococcus aureus on the surfaces of conventionally produced white ceramic wall tiles (matt and shiny) and the same tiles coated with TiO2 using three different methods: RF diode sputtering, atmospheric pressure chemical vapour deposition (APCVD) and spray pyrolysis deposition (SPD). Results clearly indicate that the bactericidal action of UV radiation is much stronger on the surfaces of tiles coated with TiO2 than on the tiles uncovered. The strongest bactericidal effect of UV radiation was found for film prepared by APCVD. Results of experiments for shiny and matt tiles did not differ statistically. The use of ceramic wall tiles coated with TiO2 films in hospitals, veterinary clinics, laboratories, food processing plants and other places where UV radiation is applied for disinfection should greatly improve the efficiency of this treatment.

  13. Computational Protein Design with Explicit Consideration of Surface Hydrophobic Patches

    OpenAIRE

    Jacak, Ron; Leaver-Fay, Andrew; Kuhlman, Brian

    2011-01-01

    De novo protein design requires the identification of amino-acid sequences that favor the target folded conformation and are soluble in water. One strategy for promoting solubility is to disallow hydrophobic residues on the protein surface during design. However, naturally occurring proteins often have hydrophobic amino acids on their surface that contribute to protein stability via the partial burial of hydrophobic surface area or play a key role in the formation of protein-protein interacti...

  14. 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

    1999-01-01

    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 treatmen

  15. 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...

  16. Identification and characterization of the surface proteins of Clostridium difficile

    International Nuclear Information System (INIS)

    Several clostridial proteins were detected on the clostridial cell surface by sensitive radioiodination techniques. Two major proteins and six minor proteins comprised the radioiodinated proteins on the clostridial cell surface. Cellular fractionation of surface radiolabeled C. difficile determined that the radioiodinated proteins were found in the cell wall fraction of C. difficile and surprisingly were also present in the clostridial membrane. Furthermore, an interesting phenomenon of disulfide-crosslinking of the cell surface proteins of C. difficile was observed. Disulfide-linked protein complexes were found in both the membrane and cell wall fractions. In addition, the cell surface proteins of C. difficile were found to be released into the culture medium. In attempts to further characterize the clostridial proteins recombinant DNA techniques were employed. In addition, the role of the clostridial cell surface proteins in the interactions of C. difficile with human PMNs was also investigated

  17. Identification and characterization of the surface proteins of Clostridium difficile

    Energy Technology Data Exchange (ETDEWEB)

    Dailey, D.C.

    1988-01-01

    Several clostridial proteins were detected on the clostridial cell surface by sensitive radioiodination techniques. Two major proteins and six minor proteins comprised the radioiodinated proteins on the clostridial cell surface. Cellular fractionation of surface radiolabeled C. difficile determined that the radioiodinated proteins were found in the cell wall fraction of C. difficile and surprisingly were also present in the clostridial membrane. Furthermore, an interesting phenomenon of disulfide-crosslinking of the cell surface proteins of C. difficile was observed. Disulfide-linked protein complexes were found in both the membrane and cell wall fractions. In addition, the cell surface proteins of C. difficile were found to be released into the culture medium. In attempts to further characterize the clostridial proteins recombinant DNA techniques were employed. In addition, the role of the clostridial cell surface proteins in the interactions of C. difficile with human PMNs was also investigated.

  18. Genetic polymorphism of the C-reactive protein (CRP) gene and a deep infection focus determine maximal serum CRP level in Staphylococcus aureus bacteremia

    OpenAIRE

    Mölkänen, T.; Rostila, A.; Ruotsalainen, E.; Alanne, M.; Perola, M.; Järvinen, A.

    2010-01-01

    Abstract C-reactive protein (CRP) is widely used in early detection of sepsis or organ dysfunction. Several single nucleotide polymorphisms (SNPs) in the CRP gene are shown to be associated with variability of basal CRP. To clarify the effect of these SNPs to CRP response in systemic infections, we compared genetic and clinical data on patients with Staphylococcus aureus bacteremia (SAB). Six SNPs in the CRP gene region (rs2794521, rs30912449, rs1800947, rs1130864, rs1205 and rs309...

  19. Protein adsorption on materials surfaces with nano-topography

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Protein adsorption behavior on the surfaces of biomedical materials is highly related to the biocompatibility of the materials. In the past, numerous research reports were mainly focused on the effect of chemical components of a material's surface on protein adsorption. The effect of surface topography on protein adsorption, the topic of this review, has recently receuvedkeen interest. The influence of surface nano-topographic factors, including roughness, curvature and geometry, on protein adsorption as well as the protein adsorption behavior, such as the amount of protein adsorbed, the activity and morphology of adsorbed protein, is introduced.

  20. Fibronectin binding proteins contribute to the adherence of Staphylococcus aureus to intact endothelium in vivo

    NARCIS (Netherlands)

    Kerdudou, Sylvain; Laschke, Matthias W; Sinha, Bhanu; Preissner, Klaus T; Menger, Michael D; Herrmann, Mathias

    2006-01-01

    Staphylococcal adhesins mediate attachment to matrix proteins and endothelial cells in vitro, yet, their role in primary adherence to the physiologic vessel wall has not been studied in vivo, and complex endocarditis models yielded ambiguous results. Recently, we developed a hamster model to study i

  1. Protein surface patterning using nanoscale PEG hydrogels.

    Science.gov (United States)

    Hong, Ye; Krsko, Peter; Libera, Matthew

    2004-12-01

    We have used focused electron-beam cross-linking to create nanosized hydrogels and thus present a new method with which to bring the attractive biocompatibility associated with macroscopic hydrogels into the submicron length-scale regime. Using amine-terminated poly(ethylene glycol) thin films on silicon substrates, we generate nanohydrogels with lateral dimensions of order 200 nm which can swell by a factor of at least five, depending on the radiative dose. With the focused electron beam, high-density arrays of such nanohydrogels can be flexibly patterned onto silicon surfaces. Significantly, the amine groups remain functional after e-beam exposure, and we show that they can be used to covalently bind proteins and other molecules. We use bovine serum albumin to amplify the number of amine groups, and we further demonstrate that different proteins can be covalently bound to different hydrogel pads on the same substrate to create multifunctional surfaces useful in emerging bio/proteomic and sensor technologies.

  2. The Electrophoretic Mobility of Proteins near Surfaces

    Science.gov (United States)

    Ramasamy, Perumal; Singh, Avtar; Rafailovich, Miriam; Sokolov, Jonathan

    2004-03-01

    We have attempted to apply the methods developed for surface DNA electrophoresis (1) for proteomics. Droplets of FITC stained Abumin, Poly- L-Lysine, or Casein purchased from Sigma were deposited on glass cover slips. The droplets were then place in contact with a TBE buffer solution contained in a cell molded from PDMS. Pt electrodes were inserted into the cell and a voltage was a applied. The motion of the protein was then imaged with a Leica Confocal microscope as a function of buffer concentration, distance from the surface, and applied voltage. The mobilities were then compared with those of uncharged one micron florescent Polystyrene beads. References: 1)Henzel WJ, Watanabe C, Stults JT., !0 Protein Identification: The Origins of Peptide Mass Fingerprinting. !1 J. American Society for Mass Spectrometry. 14 (September 2003): 931-942 2)Mathesius U, Imin N, Natera SH, Rolfe BG., !0 Proteomics as a functional genomics tool. !1 Methods of Molecular Biology 236: 395-414. *Work supported in part by the NSF-MRSEC program

  3. Applications of Yeast Surface Display for Protein Engineering.

    Science.gov (United States)

    Cherf, Gerald M; Cochran, Jennifer R

    2015-01-01

    The method of displaying recombinant proteins on the surface of Saccharomyces cerevisiae via genetic fusion to an abundant cell wall protein, a technology known as yeast surface display, or simply, yeast display, has become a valuable protein engineering tool for a broad spectrum of biotechnology and biomedical applications. This review focuses on the use of yeast display for engineering protein affinity, stability, and enzymatic activity. Strategies and examples for each protein engineering goal are discussed. Additional applications of yeast display are also briefly presented, including protein epitope mapping, identification of protein-protein interactions, and uses of displayed proteins in industry and medicine. PMID:26060074

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

    OpenAIRE

    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 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 (suspen...

  5. Contributions of adhesive proteins to the cellular and bacterial response to surfaces treated with bioactive polymers: case of poly(sodium styrene sulfonate) grafted titanium surfaces.

    Science.gov (United States)

    Felgueiras, Helena P; Aissa, Ines Ben; Evans, Margaret D M; Migonney, Véronique

    2015-11-01

    The research developed on functionalized model or prosthetic surfaces with bioactive polymers has raised the possibility to modulate and/or control the biological in vitro and in vivo responses to synthetic biomaterials. The mechanisms underlying the bioactivity exhibited by sulfonated groups on surfaces involves both selective adsorption and conformational changes of adsorbed proteins. Indeed, surfaces functionalized by grafting poly(sodium styrene sulfonate) [poly(NaSS)] modulate the cellular and bacterial response by inducing specific interactions with fibronectin (Fn). Once implanted, a biomaterial surface is exposed to a milieu of many proteins that compete for the surface which dictates the subsequent biological response. Once understood, this can be controlled by dictating exposure of active binding sites. In this in vitro study, we report the influence of binary mixtures of proteins [albumin (BSA), Fn and collagen type I (Col I)] adsorbed on poly(NaSS) grafted Ti6Al4V on the adhesion and differentiation of MC3T3-E1 osteoblast-like cells and the adhesion and proliferation of Staphylococcus aureus (S. aureus). Outcomes showed that poly(NaSS) stimulated cell spreading, attachment strength, differentiation and mineralization, whatever the nature of protein provided at the interface compared with ungrafted Ti6Al4V (control). While in competition, Fn and Col I were capable of prevailing over BSA. Fn played an important role in the early interactions of the cells with the surface, while Col I was responsible for increased alkaline phosphatase, calcium and phosphate productions associated with differentiation. Poly(NaSS) grafted surfaces decreased the adhesion of S. aureus and the presence of Fn on these chemically altered surfaces increased bacterial resistance ≈70% compared to the ungrafted Ti6Al4V. Overall, our study showed that poly(NaSS) grafted Ti6Al4V selectively adsorbed proteins (particularly Fn) promoting the adhesion and differentiation of osteoblast

  6. Applications of yeast surface display for protein engineering

    OpenAIRE

    Cherf, Gerald M.; Cochran, Jennifer R.

    2015-01-01

    The method of displaying recombinant proteins on the surface of Saccharomyces cerevisiae via genetic fusion to an abundant cell wall protein, a technology known as yeast surface display, or simply, yeast display, has become a valuable protein engineering tool for a broad spectrum of biotechnology and biomedical applications. This review focuses on the use of yeast display for engineering protein affinity, stability, and enzymatic activity. Strategies and examples for each protein engineering ...

  7. Calreticulin: Roles in Cell-Surface Protein Expression

    Directory of Open Access Journals (Sweden)

    Yue Jiang

    2014-09-01

    Full Text Available In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins.

  8. Targeting Staphylococcus aureus Toxins: A Potential form of Anti-Virulence Therapy

    OpenAIRE

    Cin Kong; Hui-min Neoh; Sheila Nathan

    2016-01-01

    Staphylococcus aureus is an opportunistic pathogen and the leading cause of a wide range of severe clinical infections. The range of diseases reflects the diversity of virulence factors produced by this pathogen. To establish an infection in the host, S. aureus expresses an inclusive set of virulence factors such as toxins, enzymes, adhesins, and other surface proteins that allow the pathogen to survive under extreme conditions and are essential for the bacteria’s ability to spread through ti...

  9. Cell wall sorting of lipoproteins in Staphylococcus aureus.

    OpenAIRE

    Navarre, W W; Daefler, S; Schneewind, O

    1996-01-01

    Many surface proteins are thought to be anchored to the cell wall of gram-positive organisms via their C termini, while the N-terminal domains of these molecules are displayed on the bacterial surface. Cell wall anchoring of surface proteins in Staphylococcus aureus requires both an N-terminal leader peptide and a C-terminal cell wall sorting signal. By fusing the cell wall sorting of protein A to the C terminus of staphylococcal beta-lactamase, we demonstrate here that lipoproteins can also ...

  10. 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.

  11. Two TIR-like domain containing proteins in a newly emerging zoonotic Staphylococcus aureus strain sequence type 398 are potential virulence factors by impacting on the host innate immune response.

    Science.gov (United States)

    Patterson, Nicholas J; Günther, Juliane; Gibson, Amanda J; Offord, Victoria; Coffey, Tracey J; Splitter, Gary; Monk, Ian; Seyfert, Hans-Martin; Werling, Dirk

    2014-01-01

    Staphylococcus aureus, sequence type (ST) 398, is an emerging pathogen and the leading cause of livestock-associated methicillin-resistant S. aureus infections in Europe and North America. This strain is characterized by high promiscuity in terms of host-species and also lacks several traditional S. aureus virulence factors. This does not, however, explain the apparent ease with which it crosses species-barriers. Recently, TIR-domain containing proteins (Tcps) which inhibit the innate immune response were identified in some Gram-negative bacteria. Here we report the presence of two proteins, S. aureus TIR-like Protein 1 (SaTlp1) and S. aureus TIR-like Protein 2 (SaTlp2), expressed by ST398 which contain domain of unknown function 1863 (DUF1863), similar to the Toll/IL-1 receptor (TIR) domain. In contrast to the Tcps in Gram-negative bacteria, our data suggest that SaTlp1 and SaTlp2 increase activation of the transcription factor NF-κB as well as downstream pro-inflammatory cytokines and immune effectors. To assess the role of both proteins as potential virulence factors knock-out mutants were created. These showed a slightly enhanced survival rate in a murine infectious model compared to the wild-type strain at one dose. Our data suggest that both proteins may act as factors contributing to the enhanced ability of ST398 to cross species-barriers. PMID:25538689

  12. 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.

  13. POLY(N-VINYLPYRROLIDONE)-MODIFIED SURFACES REPEL PLASMA PROTEIN ADSORPTION

    Institute of Scientific and Technical Information of China (English)

    Xiao-li Liu; Zhao-qiang Wu; Dan Li; Hong Chen

    2012-01-01

    The present work aimed to study the interaction between plasma proteins and PVP-modified surfaces under more complex protein conditions.In the competitive adsorption of fibrinogen (Fg) and human serum albumin (HSA),the modified surfaces showed preferential adsorption of HSA.In 100% plasma,the amount of Fg adsorbed onto PVP-modified surfaces was as low as 10 ng/cm2,suggesting the excellent protein resistance properties of the modified surfaces.In addition,immunoblots of proteins eluted from the modified surfaces after plasma contact confirmed that PVP-modified surfaces can repel most plasma proteins,especially proteins that play important roles in the process of blood coagulation.

  14. Identification of surface proteins in Enterococcus faecalis V583

    Directory of Open Access Journals (Sweden)

    Eijsink Vincent GH

    2011-03-01

    Full Text Available Abstract Background Surface proteins are a key to a deeper understanding of the behaviour of Gram-positive bacteria interacting with the human gastro-intestinal tract. Such proteins contribute to cell wall synthesis and maintenance and are important for interactions between the bacterial cell and the human host. Since they are exposed and may play roles in pathogenicity, surface proteins are interesting targets for drug design. Results Using methods based on proteolytic "shaving" of bacterial cells and subsequent mass spectrometry-based protein identification, we have identified surface-located proteins in Enterococcus faecalis V583. In total 69 unique proteins were identified, few of which have been identified and characterized previously. 33 of these proteins are predicted to be cytoplasmic, whereas the other 36 are predicted to have surface locations (31 or to be secreted (5. Lipid-anchored proteins were the most dominant among the identified surface proteins. The seemingly most abundant surface proteins included a membrane protein with a potentially shedded extracellular sulfatase domain that could act on the sulfate groups in mucin and a lipid-anchored fumarate reductase that could contribute to generation of reactive oxygen species. Conclusions The present proteome analysis gives an experimental impression of the protein landscape on the cell surface of the pathogenic bacterium E. faecalis. The 36 identified secreted (5 and surface (31 proteins included several proteins involved in cell wall synthesis, pheromone-regulated processes, and transport of solutes, as well as proteins with unknown function. These proteins stand out as interesting targets for further investigation of the interaction between E. faecalis and its environment.

  15. The DUF59 Containing Protein SufT Is Involved in the Maturation of Iron-Sulfur (FeS) Proteins during Conditions of High FeS Cofactor Demand in Staphylococcus aureus.

    Science.gov (United States)

    Mashruwala, Ameya A; Bhatt, Shiven; Poudel, Saroj; Boyd, Eric S; Boyd, Jeffrey M

    2016-08-01

    Proteins containing DUF59 domains have roles in iron-sulfur (FeS) cluster assembly and are widespread throughout Eukarya, Bacteria, and Archaea. However, the function(s) of this domain is unknown. Staphylococcus aureus SufT is composed solely of a DUF59 domain. We noted that sufT is often co-localized with sufBC, which encode for the Suf FeS cluster biosynthetic machinery. Phylogenetic analyses indicated that sufT was recruited to the suf operon, suggesting a role for SufT in FeS cluster assembly. A S. aureus ΔsufT mutant was defective in the assembly of FeS proteins. The DUF59 protein Rv1466 from Mycobacterium tuberculosis partially corrected the phenotypes of a ΔsufT mutant, consistent with a widespread role for DUF59 in FeS protein maturation. SufT was dispensable for FeS protein maturation during conditions that imposed a low cellular demand for FeS cluster assembly. In contrast, the role of SufT was maximal during conditions imposing a high demand for FeS cluster assembly. SufT was not involved in the repair of FeS clusters damaged by reactive oxygen species or in the physical protection of FeS clusters from oxidants. Nfu is a FeS cluster carrier and nfu displayed synergy with sufT. Furthermore, introduction of nfu upon a multicopy plasmid partially corrected the phenotypes of the ΔsufT mutant. Biofilm formation and exoprotein production are critical for S. aureus pathogenesis and vancomycin is a drug of last-resort to treat staphylococcal infections. Defective FeS protein maturation resulted in increased biofilm formation, decreased production of exoproteins, increased resistance to vancomycin, and the appearance of phenotypes consistent with vancomycin-intermediate resistant S. aureus. We propose that SufT, and by extension the DUF59 domain, is an accessory factor that functions in the maturation of FeS proteins. In S. aureus, the involvement of SufT is maximal during conditions of high demand for FeS proteins. PMID:27517714

  16. 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 com

  17. 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

  18. RPE cell surface proteins in normal and dystrophic rats

    International Nuclear Information System (INIS)

    Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE

  19. RPE cell surface proteins in normal and dystrophic rats

    Energy Technology Data Exchange (ETDEWEB)

    Clark, V.M.; Hall, M.O.

    1986-02-01

    Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE.

  20. Characterization of the mechanism of protection mediated by CS-D7, a monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB)

    OpenAIRE

    Gregory ePancari; Hongxia eFan; Sharon eSmith; Amita eJoshi; Robin eHaimbach; Desmond eClark; Yingzhe eLi; Jin eHua; Troy eMcKelvey; Yangsi eOu; James eDrummond; Leslie eCope; Donna eMontgomery; Tessie eMcNeely

    2012-01-01

    We previously reported the development of a human monoclonal antibody (CS-D7, IgG1) with specificity and affinity for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus. CS-D7 mediates opsonophagocytic killing in vitro and protection in a murine sepsis model. In light of recent data indicating that IsdB specific T cells (CD4+, Th17), not Ab, mediate protection after vaccination with IsdB, it is important to investigate the mechanism of protection mediated by CS-D7. Th...

  1. Gamma-cyclodextrin/usnic acid thin film fabricated by MAPLE for improving the resistance of medical surfaces to Staphylococcus aureus colonization

    Science.gov (United States)

    Iordache, Florin; Grumezescu, Valentina; Grumezescu, Alexandru Mihai; Curuţiu, Carmen; Diţu, Lia Mara; Socol, Gabriel; Ficai, Anton; Truşcă, Roxana; Holban, Alina Maria

    2015-05-01

    This study reports on the successful deposition of γ-cyclodextrin/usnic acid (γCD/UA) thin film by Matrix Assisted Pulsed Laser Evaporation (MAPLE) as anti-adherent coating on medical surfaces against microbial colonization. The obtained results demonstrate that these bioactive thin films inhibit Staphylococcus aureus biofilm formation at all stages, starting with their initiation. The antibiofilm effect was constant along the bacterial incubation time. Furthermore, the γCD/UA coatings show a great biocompatibility which means that this material is suitable for the development of modern medical devices with antimicrobial properties.

  2. Increased Expression of Clumping Factor and Fibronectin-Binding Proteins by hemB Mutants of Staphylococcus aureus Expressing Small Colony Variant Phenotypes

    Science.gov (United States)

    Vaudaux, Pierre; Francois, Patrice; Bisognano, Carmelo; Kelley, William L.; Lew, Daniel P.; Schrenzel, Jacques; Proctor, Richard A.; McNamara, Peter J.; Peters, G.; Von Eiff, Christof

    2002-01-01

    Small colony variants (SCVs) of Staphylococcus aureus are slow-growing subpopulations that cause persistent and relapsing infections. The altered phenotype of SCV can arise from defects in menadione or hemin biosynthesis, which disrupt the electron transport chain and decrease ATP concentrations. With SCVs, virulence is altered by a decrease in exotoxin production and susceptibility to various antibiotics, allowing their intracellular survival. The expression of bacterial adhesins by SCVs is poorly documented. We tested fibrinogen- and fibronectin-mediated adhesion of a hemB mutant of S. aureus 8325-4 that is defective for hemin biosynthesis and exhibits a complete SCV phenotype. In this strain, adhesion to fibrinogen and fibronectin was significantly higher than that of its isogenic, normally growing parent and correlated with the increased surface display of these adhesins as assessed by flow cytometry. Real-time quantitative reverse transcription-PCR demonstrated increased expression of clfA and fnb genes by the hemB mutant compared to its isogenic parent. The influence of the hemB mutation on altered adhesin expression was confirmed by showing complete restoration of the wild-type adhesive phenotype in the hemB mutant, either by complementing with intact hemB or by supplementing the growth medium with hemin. Increased surface display of fibrinogen and fibronectin adhesins by the hemB mutation occurred independently from agr, a major regulatory locus of virulence factors in S. aureus. Both agr-positive and agr-lacking hemB mutants were also more efficiently internalized by human embryonic kidney cells than were their isogenic controls, presumably because of increased surface display of their fibronectin adhesins. PMID:12228267

  3. Computational design of protein interactions: designing proteins that neutralize influenza by inhibiting its hemagglutinin surface protein

    Science.gov (United States)

    Fleishman, Sarel

    2012-02-01

    Molecular recognition underlies all life processes. Design of interactions not seen in nature is a test of our understanding of molecular recognition and could unlock the vast potential of subtle control over molecular interaction networks, allowing the design of novel diagnostics and therapeutics for basic and applied research. We developed the first general method for designing protein interactions. The method starts by computing a region of high affinity interactions between dismembered amino acid residues and the target surface and then identifying proteins that can harbor these residues. Designs are tested experimentally for binding the target surface and successful ones are affinity matured using yeast cell surface display. Applied to the conserved stem region of influenza hemagglutinin we designed two unrelated proteins that, following affinity maturation, bound hemagglutinin at subnanomolar dissociation constants. Co-crystal structures of hemagglutinin bound to the two designed binders were within 1Angstrom RMSd of their models, validating the accuracy of the design strategy. One of the designed proteins inhibits the conformational changes that underlie hemagglutinin's cell-invasion functions and blocks virus infectivity in cell culture, suggesting that such proteins may in future serve as diagnostics and antivirals against a wide range of pathogenic influenza strains. We have used this method to obtain experimentally validated binders of several other target proteins, demonstrating the generality of the approach. We discuss the combination of modeling and high-throughput characterization of design variants which has been key to the success of this approach, as well as how we have used the data obtained in this project to enhance our understanding of molecular recognition. References: Science 332:816 JMB, in press Protein Sci 20:753

  4. Crystal structures of Bbp from Staphylococcus aureus reveal the ligand binding mechanism with Fibrinogen α

    OpenAIRE

    Zhang, Xinyue; Wu, Meng; Zhuo, Wei; Gu, Jinke; Zhang, Sensen; Ge, Jingpeng; Yang, Maojun

    2015-01-01

    Bone sialoprotein-binding protein (Bbp), a MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) family protein expressed on the surface of Staphylococcus aureus (S. aureus), mediates adherence to fibrinogen α (Fg α), a component in the extracellular matrix of the host cell and is important for infection and pathogenesis. In this study, we solved the crystal structures of apo-Bbp273−598 and Bbp273−598-Fg α561−575 complex at a resolution of 2.03 Å and 1.45 Å, respective...

  5. Surface passivation for single-molecule protein studies.

    Science.gov (United States)

    Chandradoss, Stanley D; Haagsma, Anna C; Lee, Young Kwang; Hwang, Jae-Ho; Nam, Jwa-Min; Joo, Chirlmin

    2014-04-24

    Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.

  6. Evolutionary analysis of circumsporozoite surface protein and merozoite surface protein-1 (CSP and MSP-1) sequences of malaria parasites

    OpenAIRE

    Tripathi, Vijay; Gupta, Dwijendra

    2011-01-01

    Malaria, one of the world's most common diseases, is caused by the intracellular protozoan parasite known as Plasmodium. In this study, we have determined the evolutionary relationship of two single-copy proteins, circumsporozoite protein (CSP) and merozoite surface protein-1 (MSP-1), among Plasmodium species using various bioinformatics tools and softwares. These two proteins are major blood stage antigens of Plasmodium species. This study demonstrates that the circumsporozoite protein of Pl...

  7. 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

  8. Identification of Major Outer Surface Proteins of Streptococcus agalactiae

    OpenAIRE

    Hughes, Martin J. G.; Moore, Joanne C.; Lane, Jonathan D.; Wilson, Rebecca; Pribul, Philippa K.; Younes, Zabin N.; Dobson, Richard J; Everest, Paul; Reason, Andrew J.; Redfern, Joanne M.; Greer, Fiona M.; Paxton, Thanai; Panico, Maria; Morris, Howard R; Feldman, Robert G.

    2002-01-01

    To identify the major outer surface proteins of Streptococcus agalactiae (group B streptococcus), a proteomic analysis was undertaken. An extract of the outer surface proteins was separated by two-dimensional electrophoresis. The visualized spots were identified through a combination of peptide sequencing and reverse genetic methodologies. Of the 30 major spots identified as S. agalactiae specific, 27 have been identified. Six of these proteins, previously unidentified in S. agalactiae, were ...

  9. VASCo: computation and visualization of annotated protein surface contacts

    Directory of Open Access Journals (Sweden)

    Thallinger Gerhard G

    2009-01-01

    Full Text Available Abstract Background Structural data from crystallographic analyses contain a vast amount of information on protein-protein contacts. Knowledge on protein-protein interactions is essential for understanding many processes in living cells. The methods to investigate these interactions range from genetics to biophysics, crystallography, bioinformatics and computer modeling. Also crystal contact information can be useful to understand biologically relevant protein oligomerisation as they rely in principle on the same physico-chemical interaction forces. Visualization of crystal and biological contact data including different surface properties can help to analyse protein-protein interactions. Results VASCo is a program package for the calculation of protein surface properties and the visualization of annotated surfaces. Special emphasis is laid on protein-protein interactions, which are calculated based on surface point distances. The same approach is used to compare surfaces of two aligned molecules. Molecular properties such as electrostatic potential or hydrophobicity are mapped onto these surface points. Molecular surfaces and the corresponding properties are calculated using well established programs integrated into the package, as well as using custom developed programs. The modular package can easily be extended to include new properties for annotation. The output of the program is most conveniently displayed in PyMOL using a custom-made plug-in. Conclusion VASCo supplements other available protein contact visualisation tools and provides additional information on biological interactions as well as on crystal contacts. The tool provides a unique feature to compare surfaces of two aligned molecules based on point distances and thereby facilitates the visualization and analysis of surface differences.

  10. Yeast surface display for protein engineering and characterization

    OpenAIRE

    Gai, S. Annie; Wittrup, K. Dane

    2007-01-01

    Yeast surface display is being employed to engineer desirable properties into proteins for a broad variety of applications. Labeling with soluble ligands enables rapid and quantitative analysis of yeast-displayed libraries by flow cytometry, while libraries with insoluble or even as-yet-uncharacterized binding targets can be screened through cell-surface selections. In parallel, the utilization of yeast surface display for protein characterization, including in particular the mapping of funct...

  11. New adhesin functions of surface-exposed pneumococcal proteins

    Directory of Open Access Journals (Sweden)

    Vernet Thierry

    2010-07-01

    Full Text Available Abstract Background Streptococcus pneumoniae is a widely distributed commensal Gram-positive bacteria of the upper respiratory tract. Pneumococcal colonization can progress to invasive disease, and thus become lethal, reason why antibiotics and vaccines are designed to limit the dramatic effects of the bacteria in such cases. As a consequence, pneumococcus has developed efficient antibiotic resistance, and the use of vaccines covering a limited number of serotypes such as Pneumovax® and Prevnar® results in the expansion of non-covered serotypes. Pneumococcal surface proteins represent challenging candidates for the development of new therapeutic targets against the bacteria. Despite the number of described virulence factors, we believe that the majority of them remain to be characterized. This is the reason why pneumococcus invasion processes are still largely unknown. Results Availability of genome sequences facilitated the identification of pneumococcal surface proteins bearing characteristic motifs such as choline-binding proteins (Cbp and peptidoglycan binding (LPXTG proteins. We designed a medium throughput approach to systematically test for interactions between these pneumococcal surface proteins and host proteins (extracellular matrix proteins, circulating proteins or immunity related proteins. We cloned, expressed and purified 28 pneumococcal surface proteins. Interactions were tested in a solid phase assay, which led to the identification of 23 protein-protein interactions among which 20 are new. Conclusions We conclude that whether peptidoglycan binding proteins do not appear to be major adhesins, most of the choline-binding proteins interact with host proteins (elastin and C reactive proteins are the major Cbp partners. These newly identified interactions open the way to a better understanding of host-pneumococcal interactions.

  12. Surface Functionalization for Protein and Cell Patterning

    Science.gov (United States)

    Colpo, Pascal; Ruiz, Ana; Ceriotti, Laura; Rossi, François

    The interaction of biological systems with synthetic material surfaces is an important issue for many biological applications such as implanted devices, tissue engineering, cell-based sensors and assays, and more generally biologic studies performed ex vivo. To ensure reliable outcomes, the main challenge resides in the ability to design and develop surfaces or artificial micro-environment that mimic 'natural environment' in interacting with biomolecules and cells without altering their function and phenotype. At this effect, microfabrication, surface chemistry and material science play a pivotal role in the design of advanced in-vitro systems for cell culture applications. In this chapter, we discuss and describe different techniques enabling the control of cell-surface interactions, including the description of some techniques for immobilization of ligands for controlling cell-surface interactions and some methodologies for the creation of well confined cell rich areas.

  13. Decorating microbes : surface display of proteins on Escherichia coli

    NARCIS (Netherlands)

    van Bloois, Edwin; Winter, Remko T.; Kolmar, Harald; Fraaije, Marco W.

    2011-01-01

    Bacterial surface display entails the presentation of recombinant proteins or peptides on the surface of bacterial cells. Escherichia coil is the most frequently used bacterial host for surface display and, as such, a variety of E. coil display systems have been described that primarily promote the

  14. 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

  15. 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

  16. SURF'S UP! – Protein classification by surface comparisons

    Indian Academy of Sciences (India)

    Joanna M Sasin; Adam Godzik; Janusz M Bujnicki

    2007-01-01

    Large-scale genome sequencing and structural genomics projects generate numerous sequences and structures for ‘hypothetical’ proteins without functional characterizations. Detection of homology to experimentally characterized proteins can provide functional clues, but the accuracy of homology-based predictions is limited by the paucity of tools for quantitative comparison of diverging residues responsible for the functional divergence. SURF’S UP! is a web server for analysis of functional relationships in protein families, as inferred from protein surface maps comparison according to the algorithm. It assigns a numerical score to the similarity between patterns of physicochemical features (charge, hydrophobicity) on compared protein surfaces. It allows recognizing clusters of proteins that have similar surfaces, hence presumably similar functions. The server takes as an input a set of protein coordinates and returns files with ``spherical coordinates” of proteins in a PDB format and their graphical presentation, a matrix with values of mutual similarities between the surfaces, and the unrooted tree that represents the clustering of similar surfaces, calculated by the neighbor-joining method. SURF’S UP! facilitates the comparative analysis of physicochemical features of the surface, which are the key determinants of the protein function. By concentrating on coarse surface features, SURF’S UP! can work with models obtained from comparative modelling. Although it is designed to analyse the conservation among homologs, it can also be used to compare surfaces of non-homologous proteins with different three-dimensional folds, as long as a functionally meaningful structural superposition is supplied by the user. Another valuable characteristic of our method is the lack of initial assumptions about the functional features to be compared. SURF’S UP! is freely available for academic researchers at http://asia.genesilico.pl/surfs_up/.

  17. Modulation of Fibronectin Adhesins and Other Virulence Factors in a Teicoplanin-Resistant Derivative of Methicillin-Resistant Staphylococcus aureus

    OpenAIRE

    Renzoni, Adriana; Francois, Patrice; Li, Dongmei; Kelley, William L; Lew, Daniel P.; Vaudaux, Pierre; Schrenzel, Jacques

    2004-01-01

    The impact of glycopeptide resistance on the molecular regulation of Staphylococcus aureus virulence and attachment to host tissues is poorly documented. We compared stable teicoplanin-resistant methicillin-resistant S. aureus (MRSA) strain 14-4 with its teicoplanin-susceptible MRSA parent, strain MRGR3, which exhibits a high degree of virulence in a rat model of chronic foreign body MRSA infection. The levels of fibronectin-mediated adhesion and surface display of fibronectin-binding protein...

  18. Prediction of Protein Structure Using Surface Accessibility Data.

    Science.gov (United States)

    Hartlmüller, Christoph; Göbl, Christoph; Madl, Tobias

    2016-09-19

    An approach to the de novo structure prediction of proteins is described that relies on surface accessibility data from NMR paramagnetic relaxation enhancements by a soluble paramagnetic compound (sPRE). This method exploits the distance-to-surface information encoded in the sPRE data in the chemical shift-based CS-Rosetta de novo structure prediction framework to generate reliable structural models. For several proteins, it is demonstrated that surface accessibility data is an excellent measure of the correct protein fold in the early stages of the computational folding algorithm and significantly improves accuracy and convergence of the standard Rosetta structure prediction approach. PMID:27560616

  19. Hydration dynamics near a model protein surface

    International Nuclear Information System (INIS)

    The evolution of water dynamics from dilute to very high concentration solutions of a prototypical hydrophobic amino acid with its polar backbone, N-acetyl-leucine-methylamide (NALMA), is studied by quasi-elastic neutron scattering and molecular dynamics simulation for both the completely deuterated and completely hydrogenated leucine monomer. We observe several unexpected features in the dynamics of these biological solutions under ambient conditions. The NALMA dynamics shows evidence of de Gennes narrowing, an indication of coherent long timescale structural relaxation dynamics. The translational water dynamics are analyzed in a first approximation with a jump diffusion model. At the highest solute concentrations, the hydration water dynamics is significantly suppressed and characterized by a long residential time and a slow diffusion coefficient. The analysis of the more dilute concentration solutions takes into account the results of the 2.0M solution as a model of the first hydration shell. Subtracting the first hydration layer based on the 2.0M spectra, the translational diffusion dynamics is still suppressed, although the rotational relaxation time and residential time are converged to bulk-water values. Molecular dynamics analysis shows spatially heterogeneous dynamics at high concentration that becomes homogeneous at more dilute concentrations. We discuss the hydration dynamics results of this model protein system in the context of glassy systems, protein function, and protein-protein interfaces

  20. Arginine Inhibits Adsorption of Proteins on Polystyrene Surface

    Science.gov (United States)

    Shikiya, Yui; Tomita, Shunsuke; Arakawa, Tsutomu; Shiraki, Kentaro

    2013-01-01

    Nonspecific adsorption of protein on solid surfaces causes a reduction of concentration as well as enzyme inactivation during purification and storage. However, there are no versatile inhibitors of the adsorption between proteins and solid surfaces at low concentrations. Therefore, we examined additives for the prevention of protein adsorption on polystyrene particles (PS particles) as a commonly-used material for vessels such as disposable test tubes and microtubes. A protein solution was mixed with PS particles, and then adsorption of protein was monitored by the concentration and activity of protein in the supernatant after centrifugation. Five different proteins bound to PS particles through electrostatic, hydrophobic, and aromatic interactions, causing a decrease in protein concentration and loss of enzyme activity in the supernatant. Among the additives, including arginine hydrochloride (Arg), lysine hydrochloride, guanidine hydrochloride, NaCl, glycine, and glucose, Arg was most effective in preventing the binding of proteins to PS particles as well as activity loss. Moreover, even after the mixing of protein and PS particles, the addition of Arg caused desorption of the bound protein from PS particles. This study demonstrated a new function of Arg, which expands the potential for application of Arg to proteins. PMID:23967100

  1. The ability of S.aureus to form biofilm on the Ti-6Al-7Nb scaffolds produced by Selective Laser Melting and subjected to the different types of surface modifications.

    Science.gov (United States)

    Szymczyk, Patrycja; Junka, Adam; Ziółkowski, Grzegorz; Smutnicka, Danuta; Bartoszewicz, Marzenna; Chlebus, Edward

    2013-01-01

    The Gram-positive coccus, Staphylococcus aureus, is the leading etiologic agent of limb and life-threatening biofilm-related infections in the patients following the orthopaedic implantations. The aim of the present paper is to estimate the ability of S. aureus to form biofilm on titanium alloy (Ti-6Al-7Nb) scaffolds produced by Selective Laser Melting (SLM) and subjected to the different types of surface modifications, including ultrasonic cleaning and chemical polishing. The results obtained indicate significantly the decreased ability of S.aureus to form biofilm on the surface of scaffolds subjected to the chemical polishing in comparison to the scaffolds cleaned ultrasonically. The data provided can be useful for future applications of the SLM technology in production of Ti-6Al-7Nb medical implants.

  2. Expression of surface hydrophobic proteins by Candida albicans in vivo.

    OpenAIRE

    Glee, P M; Sundstrom, P; Hazen, K C

    1995-01-01

    Candida albicans modulates cell surface hydrophobicity during growth and morphogenesis in vitro. To determine if surface hydrophobicity is expressed during pathogenesis, we generated a polyclonal antiserum against yeast hydrophobic proteins. The antiserum was then used for indirect immunofluorescence analysis of tissues from mice colonized and chronically infected with C. albicans. Results demonstrated that yeast hydrophobic proteins are exposed on fungal cells present in host tissues. The po...

  3. Controlled release of proteins from polymer-modified surfaces

    OpenAIRE

    Fang, Fang; Szleifer, I.

    2006-01-01

    The ability to control the rate of adsorption and desorption of proteins from surfaces is studied by using a molecular theory. We show how changing the chemical structure and charge of short linear and branched grafted polymers to an electrode surface can be used to promote fast adsorption of charged proteins on a time scale of seconds and control the desorption in a time scale ranging from milliseconds to hours. The optimal controlled release is found from the interplay of electrostatic attr...

  4. Two TIR-like domain containing proteins in a newly emerging zoonotic Staphyloccoccus aureus strain sequence type 398 are potential virulence factors by impacting on the host innate immune response

    Directory of Open Access Journals (Sweden)

    Nicholas J Patterson

    2014-12-01

    Full Text Available Staphylococcus aureus, sequence type (ST 398, is an emerging pathogen and the leading cause of livestock-associated methicillin-resistant Staphylococcus aureus infections in Europe and North America. This strain is characterised by high promiscuity in terms of host species and also lacks several traditional S. aureus virulence factors. This does not however explain the apparent ease with which it crosses species-barriers. Recently, TIR-domain containing proteins (Tcps inhibitng the innate immune response were identified in some Gram-negative bacteria. Here we report the presence of two proteins, S. aureus TIR-like Protein 1 (SaTlp1 and S. aureus TIR-like Protein 2 (SaTlp2, expressed by ST398 which contain Domain of Unknown Function 1863 (DUF1863, similar to the Toll/IL-1 receptor (TIR domain. In contrast to the Tcps in Gram-negative bacteria, our data suggest that SaTlp1 and SaTlp2 increase activation of the transcription factor NF-κB as well as downstream pro-inflammatory cytokines and immune effectors. To assess the role of both proteins as potential virulence factors knock-out mutants were created. These showed the potential for a slightly increased survival rate in a murine infectious model compared to the wild-type strain at one dose, but the data did not reach level of significance. Our data suggest that both proteins may act as factors contributing to the enhanced ability of ST398 to cross species-barriers.

  5. Contamination of environmental surfaces by methicillin-resistant Staphylococcus aureus (MRSA) in rooms of inpatients with MRSA-positive body sites.

    Science.gov (United States)

    Kurashige, E Jessica Ohashi; Oie, Shigeharu; Furukawa, H

    2016-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) can contaminate environmental surfaces that are frequently touched by the hands of patients with MRSA colonization/infection. There have been many studies in which the presence or absence of MRSA contamination was determined but no studies in which MRSA contamination levels were also evaluated in detail. We evaluated MRSA contamination of environmental surfaces (overbed tables, bed side rails, and curtains) in the rooms of inpatients from whom MRSA was isolated via clinical specimens. We examined the curtains within 7-14 days after they had been newly hung. The environmental surfaces were wiped using gauze (molded gauze for wiping of surface bacteria; 100% cotton, 4cm×8cm) moistened with sterile physiological saline. The MRSA contamination rate and mean counts (range) were 25.0% (6/24 samples) and 30.6 (0-255)colony-forming units (cfu)/100cm(2), respectively, for the overbed tables and 31.6% (6/19 samples) and 159.5 (0-1620)cfu/100cm(2), respectively, for the bed side rails. No MRSA was detected in 24 curtain samples. The rate of MRSA contamination of environmental surfaces was high for the overbed tables and bed side rails but low for the curtains. Therefore, at least until the 14th day of use, frequent disinfection of curtains may be not necessary.

  6. Conferring Thermostability to Mesophilic Proteins through Optimized Electrostatic Surfaces

    OpenAIRE

    Torrez, Michael; Schultehenrich, Michael; Livesay, Dennis R.

    2003-01-01

    Recently, there have been several experimental reports of proteins displaying appreciable stability gains through mutation of one or two amino acid residues. Here, we employ a simple theoretical model to quickly screen mutant structures for increased thermostability through optimization of the protein's electrostatic surface. Our results are able to reproduce the experimental observation that elimination of like-charge repulsions and creation of opposite-charge attractions on the protein surf...

  7. 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.

  8. Methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii on computer interface surfaces of hospital wards and association with clinical isolates

    Directory of Open Access Journals (Sweden)

    Ma Ling

    2009-10-01

    Full Text Available Abstract Background Computer keyboards and mice are potential reservoirs of nosocomial pathogens, but routine disinfection for non-water-proof computer devices is a problem. With better hand hygiene compliance of health-care workers (HCWs, the impact of these potential sources of contamination on clinical infection needs to be clarified. Methods This study was conducted in a 1600-bed medical center of southern Taiwan with 47 wards and 282 computers. With education and monitoring program of hand hygiene for HCWs, the average compliance rate was 74% before our surveillance. We investigated the association of methicillin-resistant Staphylococcus aureus (MRSA, Pseudomonas aeruginosa and Acinetobacter baumannii, three leading hospital-acquired pathogens, from ward computer keyboards, mice and from clinical isolates in non-outbreak period by pulsed field gel electrophoresis and antibiogram. Results Our results revealed a 17.4% (49/282 contamination rate of these computer devices by S. aureus, Acinetobacter spp. or Pseudomonas spp. The contamination rates of MRSA and A. baumannii in the ward computers were 1.1% and 4.3%, respectively. No P. aeruginosa was isolated. All isolates from computers and clinical specimens at the same ward showed different pulsotypes. However, A. baumannii isolates on two ward computers had the same pulsotype. Conclusion With good hand hygiene compliance, we found relatively low contamination rates of MRSA, P. aeruginosa and A. baumannii on ward computer interface, and without further contribution to nosocomial infection. Our results suggested no necessity of routine culture surveillance in non-outbreak situation.

  9. Levels of Antibody against 11 Staphylococcus aureus Antigens in a Healthy Population ▿

    OpenAIRE

    Colque-Navarro, Patricia; Jacobsson, Gunnar; Andersson, Rune; Flock, Jan-Ingmar; Möllby, Roland

    2010-01-01

    Serum samples from 151 healthy individuals aged from 15 to 89 years were investigated by enzyme-linked immunosorbent assay (ELISA) for IgG levels against 11 different purified antigens from Staphylococcus aureus. Surface antigens, such as teichoic acid, clumping factors A and B, and bone sialoprotein binding protein, and extracellular proteins, such as alpha-toxin, lipase, enterotoxin A, toxic shock syndrome toxin, scalded-skin syndrome toxin, fibrinogen binding protein, and extracellular adh...

  10. Competitive protein adsorption to polymer surface from human serum

    DEFF Research Database (Denmark)

    Holmberg, Maria; Jensen, Karin Bagger Stibius; Larsen, Niels Bent;

    2008-01-01

    and on the presence and concentration of other proteins in bulk solutions during adsorption. Generally, lower albumin and IgG adsorption was observed on the modified and more hydrophilic polymer surfaces, but otherwise the modified and unmodified polymer surfaces showed the same adsorption characteristics....

  11. Identification and characterization of Vibrio cholerae surface proteins by radioiodination

    International Nuclear Information System (INIS)

    Whole cells and isolated outer membrane from Vibrio cholerae (Classical, Inaba) were radiolabeled with Iodogen or Iodo-beads as catalyst. Radiolabeling of whole cells was shown to be surface specific by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis of whole cells and cell fractions. Surface-labeled whole cells regularly showed 16 distinguishable protein species, of which nine were found in radiolabeled outer membrane preparations obtained by a lithium chloride- lithium acetate procedure. Eight of these proteins were found in outer membranes prepared by sucrose density gradient centrifugation and Triton X-100 extraction of radiolabeled whole cells. The mobility of several proteins was shown to be affected by temperature, and the major protein species exposed on the cell surface was shown to consist of at least two different peptides

  12. Comparison of multi-drug resistant environmental methicillin-resistant Staphylococcus aureus [MRSA] isolated from recreational beaches and high touch surfaces in built environments

    Directory of Open Access Journals (Sweden)

    Marilyn C Roberts

    2013-04-01

    Full Text Available Over the last decade community-acquired methicillin-resistant Staphylococcus aureus [MRSA] has emerged as a major cause of disease in the general population with no health care exposure or known classical risk factors for MRSA infections. The potential community reservoirs have not been well defined though certain strains such as ST398 and USA300 have been well studied in some settings. MRSA has been isolated from recreational beaches, high-touch surfaces in homes, universities and other community environmental surfaces. However, in most cases the strains were not characterized to determine if they are related to community-acquired or hospital-acquired clinical strains. We compared 55 environmental MRSA from 805 samples including sand, fresh and marine water samples from local marine and fresh water recreational beaches (n=296, high touch surfaces on the University of Washington campus (n=294, surfaces in UW undergraduate housing (n=85, and the local community (n=130. Eleven USA300, representing 20% of the isolates, were found on the UW campus surfaces, student housing surfaces and on the community surfaces but not in the recreational beach samples from the Northwest USA. Similarly, the predominant animal ST133 was found in the recreational beach samples but not in the high touch surface samples. All USA300 isolates were multi-drug resistant carrying 2-6 different antibiotic resistance genes coding for kanamycin, macrolides and/or macrolides-lincosamides-streptogramin B and tetracycline, with the majority [72%] carrying 4-6 different antibiotic resistance genes. A surprising 98% of the 55 MRSA isolates were resistant to other classes of antibiotics and most likely represent reservoirs for these genes in the environment.

  13. 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

  14. A mass spectrometric-derived cell surface protein atlas.

    Directory of Open Access Journals (Sweden)

    Damaris Bausch-Fluck

    Full Text Available Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa. The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.

  15. A mass spectrometric-derived cell surface protein atlas.

    Science.gov (United States)

    Bausch-Fluck, Damaris; Hofmann, Andreas; Bock, Thomas; Frei, Andreas P; Cerciello, Ferdinando; Jacobs, Andrea; Moest, Hansjoerg; Omasits, Ulrich; Gundry, Rebekah L; Yoon, Charles; Schiess, Ralph; Schmidt, Alexander; Mirkowska, Paulina; Härtlová, Anetta; Van Eyk, Jennifer E; Bourquin, Jean-Pierre; Aebersold, Ruedi; Boheler, Kenneth R; Zandstra, Peter; Wollscheid, Bernd

    2015-01-01

    Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome) of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC) technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA) providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa). The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments. PMID:25894527

  16. The surface modification of stainless steel and the correlation between the surface properties and protein adsorption.

    Science.gov (United States)

    Kang, Chan-Koo; Lee, Yoon-Sik

    2007-07-01

    Protein adsorption on a biomaterial surface is of great importance as it usually induces unfavorable biological cascades, with the result that much surface modification research has had to be performed in an effort to prevent this. In this study, we developed surface modification methods for stainless steel, which is a representative metal for biomedical device. The stainless steels were first smoothened to different extents by electropolishing, in order to obtain a rough or smooth surface. On these two kinds of substrates, we introduced epoxide groups to the metal surface by silanization with 3-glycidoxypropyltrimethoxysilane (GPTS). Then, various polymers such as poly(ethylene glycol) (PEG), poly(tetrahydrofuran glycol) (PTG), poly(propylene glycol) (PPG) and poly(dimethylsiloxane) (PDMS) were grafted on the silanized stainless steels. Each surface modification step was confirmed by various analytical methods. Contact angle measurement revealed that the surface hydrophilicity was controllable by polymer grafting. Root-mean-square (RMS) data of atomic force microscopy showed that surface roughness was dramatically changed by electropolishing. Based on these results, the correlation between surface properties and protein adsorption was investigated. In the protein adsorption study, we observed that all of the polymer-grafted stainless steels exhibited lower protein adsorption, when compared with bare stainless steel. Moreover, a hydrophilic and smooth surface was found to be the best of choice for decreasing the protein adsorption. PMID:17277988

  17. Protein function annotation by local binding site surface similarity.

    Science.gov (United States)

    Spitzer, Russell; Cleves, Ann E; Varela, Rocco; Jain, Ajay N

    2014-04-01

    Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex-PSIM, a previously reported surface-based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against ∼60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that predated query protein biochemical annotation for five out of the eight query proteins. A panel of 12 currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the poorly characterized proteins.

  18. Surface plasmon resonance imaging for parallelized detection of protein biomarkers

    Science.gov (United States)

    Piliarik, Marek; Párová, Lucie; Vaisocherová, Hana; Homola, Jiří

    2009-05-01

    We report a novel high-throughput surface plasmon resonance (SPR) biosensor for rapid and parallelized detection of protein biomarkers. The biosensor is based on a high-performance SPR imaging sensor with polarization contrast and internal referencing which yields a considerably higher sensitivity and resolution than conventional SPR imaging systems (refractive index resolution 2 × 10-7 RIU). We combined the SPR imaging biosensor with microspotting to create an array of antibodies. DNA-directed protein immobilization was utilized for the spatially resolved attachment of antibodies. Using Human Chorionic Gonadotropin (hCG) as model protein biomarker, we demonstrated the potential for simultaneous detection of proteins in up to 100 channels.

  19. Protein Engineering and Selection Using Yeast Surface Display.

    Science.gov (United States)

    Angelini, Alessandro; Chen, Tiffany F; de Picciotto, Seymour; Yang, Nicole J; Tzeng, Alice; Santos, Michael S; Van Deventer, James A; Traxlmayr, Michael W; Wittrup, K Dane

    2015-01-01

    Yeast surface display is a powerful technology for engineering a broad range of protein scaffolds. This protocol describes the process for de novo isolation of protein binders from large combinatorial libraries displayed on yeast by using magnetic bead separation followed by flow cytometry-based selection. The biophysical properties of isolated single clones are subsequently characterized, and desired properties are further enhanced through successive rounds of mutagenesis and flow cytometry selections, resulting in protein binders with increased stability, affinity, and specificity for target proteins of interest. PMID:26060067

  20. Adhesion and biofilm formation by Staphylococcus aureus from food processing plants as affected by growth medium, surface type and incubation temperature

    Directory of Open Access Journals (Sweden)

    Heloísa Maria Ângelo Jerônimo

    2012-12-01

    Full Text Available This study assessed the effect of different growth media [BHI broth, BHI broth plus glucose (10 g/100 mL and BHI broth plus NaCl (5 g/100 mL] and incubation temperatures (28 or 37 ºC on the adherence, detachment and biofilm formation on polypropylene and stainless steel surfaces (2 x 2 cm coupons for a prolonged period (24-72 h by some strains of Staphylococcus aureus (S3, S28 and S54 from food processing plants. The efficacy of the sanitizers sodium hypochlorite (250 mg/mL and peracetic acid (30 mg/mL in reducing the number of viable bacterial cells in a preformed biofilm was also evaluated. S. aureus strains adhered in highest numbers in BHI broth, regardless of the type of surface or incubation temperature. Cell detachment from surfaces revealed high persistence over the incubation period. The number of cells needed for biofilm formation was noted in all experimental systems after 3 days. Peracetic acid and sodium hypochlorite were not efficient in completely removing the cells of S. aureus adhered onto polypropylene and stainless steel surfaces. From these results, the assayed strains revealed high capacities to adhere and form biofilms on polypropylene and stainless steel surfaces under the different growth conditions, and the cells in biofilm matrixes were resistant to total removal when exposed to the sanitizers sodium hypochlorite and peracetic acid.Este estudo teve como objetivo avaliar o efeito de diferentes meios de crescimento [caldo BHI, caldo BHI adicionado de glucose (10 g/100 mL e caldo BHI adicionado de NaCl (5 g/100 mL] e temperaturas de incubação (28 e 37 ºC sobre a adesão, separação e formação de biofilme sobre superfícies (2 x 2 cm de polipropileno e aço inoxidável durante longo tempo de incubação (24-72 h por parte de cepas de Staphylococcus aureus (S3, S58 e S54 isoladas de plantas de processamento de alimentos. Também foi avaliada a eficácia dos sanitizantes hipoclorito de sódio (250 mg/mL e ácido perac

  1. Staphylococcus saprophyticus surface-associated protein (Ssp) is associated with lifespan reduction in Caenorhabditis elegans.

    Science.gov (United States)

    Szabados, Florian; Mohner, Amelie; Kleine, Britta; Gatermann, Sören G

    2013-10-01

    Staphylococcal lipases have been proposed as pathogenicity factors. In Staphylococcus saprophyticus the surface-associated protein (Ssp) has been previously characterized as a cell wall-associated true lipase. A S. saprophyticus Δssp::ermB mutant has been described as less virulent in an in vivo model of urinary tract infection compared with its wild-type. This is the first report showing that S. saprophyticus induced a lifespan reduction in Caenorhabditis elegans similar to that of S. aureus RN4220. In two S. saprophyticus Δssp::ermB mutants lifespan reduction in C. elegans was partly abolished. In order to attribute virulence to the lipase activity itself and distinguish this phenomenon from the presence of the Ssp-protein, the conserved active site of the lipase was modified by site-directed ligase-independent mutagenesis and lipase activity-deficient mutants were constructed. These results indicate that the Ssp is associated with pathogenicity in C. elegans and one could speculate that the lipase activity itself is responsible for this virulence.

  2. Protein Adsorption to Surface Chemistry and Crystal Structure Modification of Titanium Surfaces

    Directory of Open Access Journals (Sweden)

    Ryo Jimbo

    2010-07-01

    Full Text Available Objectives: To observe the early adsorption of extracellular matrix and blood plasma proteins to magnesium-incorporated titanium oxide surfaces, which has shown superior bone response in animal models.Material and Methods: Commercially pure titanium discs were blasted with titanium dioxide (TiO2 particles (control, and for the test group, TiO2 blasted discs were further processed with a micro-arc oxidation method (test. Surface morphology was investigated by scanning electron microscopy, surface topography by optic interferometry, characterization by X-ray photoelectron spectroscopy (XPS, and by X-ray diffraction (XRD analysis. The adsorption of 3 different proteins (fibronectin, albumin, and collagen type I was investigated by an immunoblotting technique.Results: The test surface showed a porous structure, whereas the control surface showed a typical TiO2 blasted structure. XPS data revealed magnesium-incorporation to the anodic oxide film of the surface. There was no difference in surface roughness between the control and test surfaces. For the protein adsorption test, the amount of albumin was significantly higher on the control surface whereas the amount of fibronectin was significantly higher on the test surface. Although there was no significant difference, the test surface had a tendency to adsorb more collagen type I.Conclusions: The magnesium-incorporated anodized surface showed significantly higher fibronectin adsorption and lower albumin adsorption than the blasted surface. These results may be one of the reasons for the excellent bone response previously observed in animal studies.

  3. Surface (S)-layer proteins of Deinococcus radiodurans and their utility as vehicles for surface localization of functional proteins.

    Science.gov (United States)

    Misra, Chitra Seetharam; Basu, Bhakti; Apte, Shree Kumar

    2015-12-01

    The radiation resistant bacterium, Deinococcus radiodurans contains two major surface (S)-layer proteins, Hpi and SlpA. The Hpi protein was shown to (a) undergo specific in vivo cleavage, and (b) closely associate with the SlpA protein. Using a non-specific acid phosphatase from Salmonella enterica serovar Typhi, PhoN as a reporter, the Surface Layer Homology (SLH) domain of SlpA was shown to bind deinococcal peptidoglycan-containing cell wall sacculi. The association of SlpA with Hpi on one side and peptidoglycan on the other, localizes this protein in the 'interstitial' layer of the deinoccocal cell wall. Gene chimeras of hpi-phoN and slh-phoN were constructed to test efficacy of S-layer proteins, as vehicles for cell surface localization in D. radiodurans. The Hpi-PhoN protein localized exclusively in the membrane fraction, and displayed cell-based phosphatase activity in vivo. The SLH-PhoN, which localized to both cytosolic and membrane fractions, displayed in vitro activity but no cell-based in vivo activity. Hpi, therefore, emerged as an efficient surface localizing protein and can be exploited for suitable applications of this superbug. PMID:26450150

  4. Surface (S)-layer proteins of Deinococcus radiodurans and their utility as vehicles for surface localization of functional proteins.

    Science.gov (United States)

    Misra, Chitra Seetharam; Basu, Bhakti; Apte, Shree Kumar

    2015-12-01

    The radiation resistant bacterium, Deinococcus radiodurans contains two major surface (S)-layer proteins, Hpi and SlpA. The Hpi protein was shown to (a) undergo specific in vivo cleavage, and (b) closely associate with the SlpA protein. Using a non-specific acid phosphatase from Salmonella enterica serovar Typhi, PhoN as a reporter, the Surface Layer Homology (SLH) domain of SlpA was shown to bind deinococcal peptidoglycan-containing cell wall sacculi. The association of SlpA with Hpi on one side and peptidoglycan on the other, localizes this protein in the 'interstitial' layer of the deinoccocal cell wall. Gene chimeras of hpi-phoN and slh-phoN were constructed to test efficacy of S-layer proteins, as vehicles for cell surface localization in D. radiodurans. The Hpi-PhoN protein localized exclusively in the membrane fraction, and displayed cell-based phosphatase activity in vivo. The SLH-PhoN, which localized to both cytosolic and membrane fractions, displayed in vitro activity but no cell-based in vivo activity. Hpi, therefore, emerged as an efficient surface localizing protein and can be exploited for suitable applications of this superbug.

  5. Methicillin-resistant Staphylococcus aureus on surfaces of an Intensive Care Unit Staphylococcus aureus resistente a la meticilina en superfícies de una Unidad de Cuidados Intensivos Staphylococcus aureus resistente à meticilina em superfícies de uma Unidade de Terapia Intensiva

    Directory of Open Access Journals (Sweden)

    Adriano Menis Ferreira

    2011-01-01

    Full Text Available OBJECTIVE: To evaluate the presence of methicillin-resistant Staphylococcus aureus (MRSA in areas close to patients in a General Intensive Care Unit. METHODS: This is a cross-sectional study, in which microbiological samples were collected from five surfaces (left / right bed siderails, bed crank, table, buttons on the infusion pump, and cotton gowns from each of ten patient rooms, totaling 63 samples. To collect samples, the Petri FilmTM Staph Express Count System 3M TM was used to screen for methicillin resistance, with the Mueller-Hinton agar supplemented with 4% sodium chloride and 6 µg / ml of oxacillin. Descriptive analysis was conducted to determine the frequency (n and percentage (% of contamination of environmental surfaces. RESULTS: Of 48 samples positive for Staphylococcus aureus, 29 (60.4% were resistant to methicillin. The incidence on the siderails and bed cranks, table, buttons on the infusion pumps and aprons were, respectively, 55.5%, 57.1%, 57.1%, 60.0% and 75.0%. CONCLUSION: The results suggest that the surfaces around the patient constitute a major threat, as they represent secondary reservoirs of MRSA.OBJETIVO: Evaluar la presencia de Staphylococcus aureus resistente a la meticilina (MRSA en superficies cercanas a los pacientes internados en una Unidad de Cuidados Intensivos General. MÉTODOS: Se trata de un estudio transversal, en el cual se recolectaron muestras microbiológicas de cinco superficies (enrejados derecho/izquierdo, manivela de la cama, mesa, botones de la bomba de infusión y mandiles de algodón de cada diez unidades de pacientes, totalizando 63 muestras. Para la recolección, se utilizaron placas Petri FilmTM Staph Express Count System 3M TM y para el triaje de resistencia a la meticilina, el agar Mueller-Hinton adicionado del 4% de cloruro de sodio y 6 µg/ml oxacilina. Se emplearon análisis descriptivos para determinar la frecuencia (n y el porcentaje (% de contaminación de las superficies ambientales

  6. Methicillin-Resistant Staphylococcus aureus (MRSA Contamination in Bedside Surfaces of a Hospital Ward and the Potential Effectiveness of Enhanced Disinfection with an Antimicrobial Polymer Surfactant

    Directory of Open Access Journals (Sweden)

    John W. M. Yuen

    2015-03-01

    Full Text Available The aim in this study was to assess the effectiveness of a quaternary ammonium chloride (QAC surfactant in reducing surface staphylococcal contamination in a routinely operating medical ward occupied by patients who had tested positive for methicillin-resistant Staphylococcus aureus (MRSA. The QAC being tested is an antibacterial film that is sprayed onto a surface and can remain active for up to 8 h. A field experimental study was designed with the QAC plus daily hypochlorite cleaning as the experimental group and hypochlorite cleaning alone as the control group. The method of swabbing on moistened surfaces was used for sampling. It was found that 83% and 77% of the bedside surfaces of MRSA-positive and MRSA-negative patients respectively were contaminated with staphylococci at 08:00 hours, and that the staphylococcal concentrations increased by 80% at 1200 h over a 4-hour period with routine ward and clinical activities. Irrespective of the MRSA status of the patients, high-touch surfaces around the bed-units within the studied medical ward were heavily contaminated (ranged 1 to 276 cfu/cm2 amongst the sites with positive culture with staphylococcal bacteria including MRSA, despite the implementation of daily hypochlorite wiping. However, the contamination rate dropped significantly from 78% to 11% after the application of the QAC polymer. In the experimental group, the mean staphylococcal concentration of bedside surfaces was significantly (p < 0.0001 reduced from 4.4 ± 8.7 cfu/cm2 at 08:00 hours to 0.07 ± 0.26 cfu/cm2 at 12:00 hours by the QAC polymer. The results of this study support the view that, in addition to hypochlorite wiping, the tested QAC surfactant is a potential environmental decontamination strategy for preventing the transmission of clinically important pathogens in medical wards.

  7. Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

    Science.gov (United States)

    Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

    2015-10-01

    Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

  8. Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

    Science.gov (United States)

    Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

    2015-10-01

    Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters. PMID:26371748

  9. Interaction of Serum Proteins with Surface of Hemodialysis Fiber Membranes

    Science.gov (United States)

    Afrin, Rehana; Shirako, Yuji; Kishimoto, Kikuo; Ikai, Atsushi

    2012-08-01

    The poly(vinyl pyrrolidone)-covered hydrophilic surface of hollow-fiber membranes (fiber membrane, hereafter) for hemodialysis was mechanically probed using modified tips on an atomic force microscope (AFM) with covalent crosslinkers and several types of serum protein. The retraction part of many of the force extension (F-E) curves obtained with AFM tips coated with serum albumin had a long and smooth extension up to 200-300 nm indicating forced elongation of poly(vinyl pyrrolidone) chains. When fibrinogen-coated tips were used, long extension F-E curves up to 500 nm with multiple peaks were obtained in addition to smooth curves most likely reflecting the unfolding of fibrinogen molecules. The results indicated that individual polymer chains had a significant affinity toward serum proteins. The adhesion frequency of tips coated with serum proteins was lower on the poly(vinyl pyrrolidone) surface than on the uncoated hydrophobic polysulfone surface.

  10. Comparison of surface and hydrogel-based protein microchips.

    Science.gov (United States)

    Zubtsov, D A; Savvateeva, E N; Rubina, A Yu; Pan'kov, S V; Konovalova, E V; Moiseeva, O V; Chechetkin, V R; Zasedatelev, A S

    2007-09-15

    Protein microchips are designed for high-throughput evaluation of the concentrations and activities of various proteins. The rapid advance in microchip technology and a wide variety of existing techniques pose the problem of unified approach to the assessment and comparison of different platforms. Here we compare the characteristics of protein microchips developed for quantitative immunoassay with those of antibodies immobilized on glass surfaces and in hemispherical gel pads. Spotting concentrations of antibodies used for manufacturing of microchips of both types and concentrations of antigen in analyte solution were identical. We compared the efficiency of antibody immobilization, the intensity of fluorescence signals for both direct and sandwich-type immunoassays, and the reaction-diffusion kinetics of the formation of antibody-antigen complexes for surface and gel-based microchips. Our results demonstrate higher capacity and sensitivity for the hydrogel-based protein microchips, while fluorescence saturation kinetics for the two types of microarrays was comparable.

  11. Protein sequences bound to mineral surfaces persist into deep time

    Science.gov (United States)

    Demarchi, Beatrice; Hall, Shaun; Roncal-Herrero, Teresa; Freeman, Colin L; Woolley, Jos; Crisp, Molly K; Wilson, Julie; Fotakis, Anna; Fischer, Roman; Kessler, Benedikt M; Rakownikow Jersie-Christensen, Rosa; Olsen, Jesper V; Haile, James; Thomas, Jessica; Marean, Curtis W; Parkington, John; Presslee, Samantha; Lee-Thorp, Julia; Ditchfield, Peter; Hamilton, Jacqueline F; Ward, Martyn W; Wang, Chunting Michelle; Shaw, Marvin D; Harrison, Terry; Domínguez-Rodrigo, Manuel; MacPhee, Ross DE; Kwekason, Amandus; Ecker, Michaela; Kolska Horwitz, Liora; Chazan, Michael; Kröger, Roland; Thomas-Oates, Jane; Harding, John H; Cappellini, Enrico; Penkman, Kirsty; Collins, Matthew J

    2016-01-01

    Proteins persist longer in the fossil record than DNA, but the longevity, survival mechanisms and substrates remain contested. Here, we demonstrate the role of mineral binding in preserving the protein sequence in ostrich (Struthionidae) eggshell, including from the palaeontological sites of Laetoli (3.8 Ma) and Olduvai Gorge (1.3 Ma) in Tanzania. By tracking protein diagenesis back in time we find consistent patterns of preservation, demonstrating authenticity of the surviving sequences. Molecular dynamics simulations of struthiocalcin-1 and -2, the dominant proteins within the eggshell, reveal that distinct domains bind to the mineral surface. It is the domain with the strongest calculated binding energy to the calcite surface that is selectively preserved. Thermal age calculations demonstrate that the Laetoli and Olduvai peptides are 50 times older than any previously authenticated sequence (equivalent to ~16 Ma at a constant 10°C). DOI: http://dx.doi.org/10.7554/eLife.17092.001 PMID:27668515

  12. 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

  13. Antigenicity and Immunogenicity of Plasmodium vivax Merozoite Surface Protein-3

    OpenAIRE

    Amanda R Bitencourt; Elaine C Vicentin; Jimenez, Maria C.; Ricardo Ricci; Leite, Juliana A.; Fabio T Costa; Luis C Ferreira; Bruce Russell; François Nosten; Laurent Rénia; Galinski, Mary R.; Barnwell, John W.; Rodrigues, Mauricio M; Soares, Irene S

    2013-01-01

    A recent clinical trial in African children demonstrated the potential utility of merozoite surface protein (MSP)-3 as a vaccine against Plasmodium falciparum malaria. The present study evaluated the use of Plasmodium vivax MSP-3 (PvMSP-3) as a target antigen in vaccine formulations against malaria caused by P. vivax. Recombinant proteins representing MSP-3α and MSP-3β of P. vivax were expressed as soluble histidine-tagged bacterial fusions. Antigenicity during natural infection was evaluated...

  14. Selectivity by small-molecule inhibitors of protein interactions can be driven by protein surface fluctuations.

    Directory of Open Access Journals (Sweden)

    David K Johnson

    2015-02-01

    Full Text Available Small-molecules that inhibit interactions between specific pairs of proteins have long represented a promising avenue for therapeutic intervention in a variety of settings. Structural studies have shown that in many cases, the inhibitor-bound protein adopts a conformation that is distinct from its unbound and its protein-bound conformations. This plasticity of the protein surface presents a major challenge in predicting which members of a protein family will be inhibited by a given ligand. Here, we use biased simulations of Bcl-2-family proteins to generate ensembles of low-energy conformations that contain surface pockets suitable for small molecule binding. We find that the resulting conformational ensembles include surface pockets that mimic those observed in inhibitor-bound crystal structures. Next, we find that the ensembles generated using different members of this protein family are overlapping but distinct, and that the activity of a given compound against a particular family member (ligand selectivity can be predicted from whether the corresponding ensemble samples a complementary surface pocket. Finally, we find that each ensemble includes certain surface pockets that are not shared by any other family member: while no inhibitors have yet been identified to take advantage of these pockets, we expect that chemical scaffolds complementing these "distinct" pockets will prove highly selective for their targets. The opportunity to achieve target selectivity within a protein family by exploiting differences in surface fluctuations represents a new paradigm that may facilitate design of family-selective small-molecule inhibitors of protein-protein interactions.

  15. Lack of Involvement of Fenton Chemistry in Death of Methicillin-Resistant and Methicillin-Sensitive Strains of Staphylococcus aureus and Destruction of Their Genomes on Wet or Dry Copper Alloy Surfaces

    Science.gov (United States)

    2016-01-01

    The pandemic of hospital-acquired infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has declined, but the evolution of strains with enhanced virulence and toxins and the increase of community-associated infections are still a threat. In previous studies, 107 MRSA bacteria applied as simulated droplet contamination were killed on copper and brass surfaces within 90 min. However, contamination of surfaces is often via finger tips and dries rapidly, and it may be overlooked by cleaning regimes (unlike visible droplets). In this new study, a 5-log reduction of a hardy epidemic strain of MRSA (epidemic methicillin-resistant S. aureus 16 [EMRSA-16]) was observed following 10 min of contact with copper, and a 4-log reduction was observed on copper nickel and cartridge brass alloys in 15 min. A methicillin-sensitive S. aureus (MSSA) strain from an osteomyelitis patient was killed on copper surfaces in 15 min, and 4-log and 3-log reductions occurred within 20 min of contact with copper nickel and cartridge brass, respectively. Bacterial respiration was compromised on copper surfaces, and superoxide was generated as part of the killing mechanism. In addition, destruction of genomic DNA occurs on copper and brass surfaces, allaying concerns about horizontal gene transfer and copper resistance. Incorporation of copper alloy biocidal surfaces may help to reduce the spread of this dangerous pathogen. PMID:26826226

  16. Hepatitis B virus large surface protein: function and fame

    OpenAIRE

    Churin, Yuri; Roderfeld, Martin; Roeb, Elke

    2015-01-01

    Chronic infection with hepatitis B virus (HBV) is the leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. HBV life cycle begins with viral attachment to hepatocytes, mediated by the large HBV surface protein (LHBs). Identification of the sodium-taurocholate cotransporting polypeptide (NTCP) as a HBV receptor has revealed a suitable target for viral entry inhibition. Analysis of serum hepatitis B surface antigen (HBsAg) level is a non-invasive diagnostic parameter that imp...

  17. Conformal nanopatterning of extracellular matrix proteins onto topographically complex surfaces.

    Science.gov (United States)

    Sun, Yan; Jallerat, Quentin; Szymanski, John M; Feinberg, Adam W

    2015-02-01

    Our Patterning on Topography (PoT) printing technique enables fibronectin, laminin and other proteins to be applied to biomaterial surfaces in complex geometries that are inaccessible using traditional soft lithography techniques. Engineering combinatorial surfaces that integrate topographical and biochemical micropatterns enhances control of the biotic-abiotic interface. Here, we used this method to understand cardiomyocyte response to competing physical and chemical cues in the microenvironment.

  18. Quantifying protein-protein interactions in the ubiquitin pathway by surface plasmon resonance

    DEFF Research Database (Denmark)

    Hartmann-Petersen, Rasmus; Gordon, Colin

    2005-01-01

    The commercial availability of instruments, such as Biacore, that are capable of monitoring surface plasmon resonance (SPR) has greatly simplified the quantification of protein-protein interactions. Already, this technique has been used for some studies of the ubiquitin-proteasome system. Here we...

  19. 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.

  20. 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.

  1. 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...

  2. Binding of collagen to Staphylococcus aureus Cowan 1.

    OpenAIRE

    Speziale, P; Raucci, G; Visai, L.; Switalski, L M; Timpl, R; Höök, M

    1986-01-01

    Collagen binds to a receptor protein present on the surfaces of Staphylococcus aureus cells. Binding of 125I-labeled type II collagen to its bacterial receptor is reversible, and Scatchard plot analysis indicates the presence of one class of receptor that occurs on an average of 3 X 10(4) copies per cell and binds type II collagen with a Kd of 10(-7) M. Studies on the specificity of collagen cell binding indicate that the receptor does not recognize noncollagenous proteins but binds all of th...

  3. Sputter deposited bioceramic coatings: surface characterisation and initial protein adsorption studies using surface-MALDI-MS

    DEFF Research Database (Denmark)

    Boyd, A. R.; Burke, G. A.; Duffy, H.;

    2011-01-01

    Protein adsorption onto calcium phosphate (Ca–P) bioceramics utilised in hard tissue implant applications has been highlighted as one of the key events that influences the subsequent biological response, in vivo. This work reports on the use of surface-matrix assisted laser desorption ionisation...... to a combination of growth factors and lipoproteins present in serum. From the data obtained here it is evident that surface-MALDI-MS has significant utility as a tool for studying the dynamic nature of protein adsorption onto the surfaces of bioceramic coatings, which most likely plays a significant role...

  4. Trichomonas vaginalis surface proteins: a view from the genome

    DEFF Research Database (Denmark)

    Hirt, R. P.; Noel, C. J.; Sicheritz-Pontén, Thomas;

    2007-01-01

    Surface proteins of mucosal microbial pathogens play multiple and essential roles in initiating and sustaining the colonization of the heavily defended mucosa. The protist Trichomonas vaginalis is one of the most common human sexually transmitted pathogens that colonize the urogenital mucosa...

  5. Factor H-related proteins determine complement-activating surfaces.

    Science.gov (United States)

    Józsi, Mihály; Tortajada, Agustin; Uzonyi, Barbara; Goicoechea de Jorge, Elena; Rodríguez de Córdoba, Santiago

    2015-06-01

    Complement factor H-related proteins (FHRs) are strongly associated with different diseases involving complement dysregulation, which suggests a major role for these proteins regulating complement activation. Because FHRs are evolutionarily and structurally related to complement inhibitor factor H (FH), the initial assumption was that the FHRs are also negative complement regulators. Whereas weak complement inhibiting activities were originally reported for these molecules, recent developments indicate that FHRs may enhance complement activation, with important implications for the role of these proteins in health and disease. We review these findings here, and propose that FHRs represent a complex set of surface recognition molecules that, by competing with FH, provide improved discrimination of self and non-self surfaces and play a central role in determining appropriate activation of the complement pathway.

  6. Neutrophil surface adhesion molecule and toll like receptor dynamics in crossbred cows suffering from Staphylococcus aureus subclinical and clinical mastitis

    Directory of Open Access Journals (Sweden)

    Dilip Kumar Swain

    2016-06-01

    Conclusion: Host elicits stage specific expression of surface adhesion molecules and TLR2 and TLR4 as dynamic host innate immune response against Staphylococcal mastitis. [J Adv Vet Anim Res 2016; 3(2.000: 99-105

  7. Architectural repertoire of ligand-binding pockets on protein surfaces.

    Science.gov (United States)

    Weisel, Martin; Kriegl, Jan M; Schneider, Gisbert

    2010-03-01

    Knowledge of the three-dimensional structure of ligand binding sites in proteins provides valuable information for computer-assisted drug design. We present a method for the automated extraction and classification of ligand binding site topologies, in which protein surface cavities are represented as branched frameworks. The procedure employs a growing neural gas approach for pocket topology assignment and pocket framework generation. We assessed the structural diversity of 623 known ligand binding site topologies based on framework cluster analysis. At a resolution of 5 A only 23 structurally distinct topology groups were formed; this suggests an overall limited structural diversity of ligand-accommodating protein cavities. Higher resolution allowed for identification of protein-family specific pocket features. Pocket frameworks highlight potentially preferred modes of ligand-receptor interactions and will help facilitate the identification of druggable subpockets suitable for ligand affinity and selectivity optimization. PMID:20069621

  8. Proteins of 30 and 36 kilodaltons, membrane constituents of the Staphylococcus aureus L form, induce production of tumor necrosis factor alpha and activate the human immunodeficiency virus type 1 long terminal repeat.

    OpenAIRE

    Akashi, A; Ono, S.; Kuwano, K.; Arai, S

    1996-01-01

    We have previously demonstrated that the membrane of the Staphylococcus aureus L form induced tumor necrosis factor alpha (TNF-alpha) from murine macrophages. In this study, we purified two proteins which induce TNF-alpha production from a human monocytic cell line, THP-1, and murine macrophages. These molecules were purified from delipidated membranes by deoxycholic acid extraction, two-step anion-exchange chromatography, and preparative electrophoresis. Sodium dodecyl sulfate-polyacrylamide...

  9. Organic bioelectronics probing conformational changes in surface confined proteins.

    Science.gov (United States)

    Macchia, Eleonora; Alberga, Domenico; Manoli, Kyriaki; Mangiatordi, Giuseppe F; Magliulo, Maria; Palazzo, Gerardo; Giordano, Francesco; Lattanzi, Gianluca; Torsi, Luisa

    2016-01-01

    The study of proteins confined on a surface has attracted a great deal of attention due to its relevance in the development of bio-systems for laboratory and clinical settings. In this respect, organic bio-electronic platforms can be used as tools to achieve a deeper understanding of the processes involving protein interfaces. In this work, biotin-binding proteins have been integrated in two different organic thin-film transistor (TFT) configurations to separately address the changes occurring in the protein-ligand complex morphology and dipole moment. This has been achieved by decoupling the output current change upon binding, taken as the transducing signal, into its component figures of merit. In particular, the threshold voltage is related to the protein dipole moment, while the field-effect mobility is associated with conformational changes occurring in the proteins of the layer when ligand binding occurs. Molecular Dynamics simulations on the whole avidin tetramer in presence and absence of ligands were carried out, to evaluate how the tight interactions with the ligand affect the protein dipole moment and the conformation of the loops surrounding the binding pocket. These simulations allow assembling a rather complete picture of the studied interaction processes and support the interpretation of the experimental results. PMID:27312768

  10. Organic bioelectronics probing conformational changes in surface confined proteins

    Science.gov (United States)

    Macchia, Eleonora; Alberga, Domenico; Manoli, Kyriaki; Mangiatordi, Giuseppe F.; Magliulo, Maria; Palazzo, Gerardo; Giordano, Francesco; Lattanzi, Gianluca; Torsi, Luisa

    2016-06-01

    The study of proteins confined on a surface has attracted a great deal of attention due to its relevance in the development of bio-systems for laboratory and clinical settings. In this respect, organic bio-electronic platforms can be used as tools to achieve a deeper understanding of the processes involving protein interfaces. In this work, biotin-binding proteins have been integrated in two different organic thin-film transistor (TFT) configurations to separately address the changes occurring in the protein-ligand complex morphology and dipole moment. This has been achieved by decoupling the output current change upon binding, taken as the transducing signal, into its component figures of merit. In particular, the threshold voltage is related to the protein dipole moment, while the field-effect mobility is associated with conformational changes occurring in the proteins of the layer when ligand binding occurs. Molecular Dynamics simulations on the whole avidin tetramer in presence and absence of ligands were carried out, to evaluate how the tight interactions with the ligand affect the protein dipole moment and the conformation of the loops surrounding the binding pocket. These simulations allow assembling a rather complete picture of the studied interaction processes and support the interpretation of the experimental results.

  11. Antigenic characterization of dimorphic surface protein in Mycobacterium tuberculosis.

    Science.gov (United States)

    Matsuba, Takashi; Siddiqi, Umme Ruman; Hattori, Toshio; Nakajima, Chie; Fujii, Jun; Suzuki, Yasuhiko

    2016-05-01

    The Mycobacterium tuberculosis Rv0679c protein is a surface protein that contributes to host cell invasion. We previously showed that a single nucleotide transition of the Rv0679c gene leads to a single amino acid substitution from asparagine to lysine at codon 142 in the Beijing genotype family. In this study, we examined the immunological effect of this substitution. Several recombinant proteins were expressed in Escherichia coli and Mycobacterium smegmatis and characterized with antisera and two monoclonal antibodies named 5D4-C2 and 8G10-H2. A significant reduction of antibody binding was detected by enzyme-linked immunosorbent assay (ELISA) and western blot analysis in the Lys142-type protein. This reduction of 8G10-H2 binding was more significant, with the disappearance of a signal in the proteins expressed by recombinant mycobacteria in western blot analysis. In addition, epitope mapping analysis of the recombinant proteins showed a linear epitope by 5D4-C2 and a discontinuous epitope by 8G10-H2. The antibody recognizing the conformational epitope detected only mycobacterial Asn142-type recombinant protein. Our results suggest that a single amino acid substitution of Rv0679c has potency for antigenic change in Beijing genotype strains. PMID:27190237

  12. 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 ... risk. This sheet talks about whether exposure to staphylococcus aureus may increase the risk for birth defects ...

  13. Surface display of proteins by Gram-negative bacterial autotransporters

    Directory of Open Access Journals (Sweden)

    Mourez Michael

    2006-06-01

    Full Text Available Abstract Expressing proteins of interest as fusions to proteins of the bacterial envelope is a powerful technique with many biotechnological and medical applications. Autotransporters have recently emerged as a good tool for bacterial surface display. These proteins are composed of an N-terminal signal peptide, followed by a passenger domain and a translocator domain that mediates the outer membrane translocation of the passenger. The natural passenger domain of autotransporters can be replaced by heterologous proteins that become displayed at the bacterial surface by the translocator domain. The simplicity and versatility of this system has made it very attractive and it has been used to display functional enzymes, vaccine antigens as well as polypeptides libraries. The recent advances in the study of the translocation mechanism of autotransporters have raised several controversial issues with implications for their use as display systems. These issues include the requirement for the displayed polypeptides to remain in a translocation-competent state in the periplasm, the requirement for specific signal sequences and "autochaperone" domains, and the influence of the genetic background of the expression host strain. It is therefore important to better understand the mechanism of translocation of autotransporters in order to employ them to their full potential. This review will focus on the recent advances in the study of the translocation mechanism of autotransporters and describe practical considerations regarding their use for bacterial surface display.

  14. Characterization of the mechanism of protection mediated by CS-D7, a monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB

    Directory of Open Access Journals (Sweden)

    Gregory ePancari

    2012-03-01

    Full Text Available We previously reported the development of a human monoclonal antibody (CS-D7, IgG1 with specificity and affinity for the iron regulated surface determinant B (IsdB of Staphylococcus aureus. CS-D7 mediates opsonophagocytic killing in vitro and protection in a murine sepsis model. In light of recent data indicating that IsdB specific T cells (CD4+, Th17, not Ab, mediate protection after vaccination with IsdB, it is important to investigate the mechanism of protection mediated by CS-D7. The mAb was examined to determine if it blocked heme binding to IsdB in vitro. The mAb was not found to have heme blocking activity, nor did it prevent bacterial growth under in vivo conditions, in an implanted growth chamber. To assess the role of the mAb Fc a point mutation was introduced at aa 297 (CS-D7●N297A. This point mutation removes Fc effector functions. In vitro analysis of the mutein confirmed that it lacked measurable binding to FcγR, and that it did not fix complement. The mutein had dramatically reduced in vitro opsonic OP activity compared to CS-D7. Nonetheless, the mutein conferred protection equivalent to the wild type mAb in the murine sepsis model. Both wild type and mutein mAbs were efficacious in FcγR deletion mice (including both FcγRII-/- mice and FcγRIII-/- mice, indicating that these receptors were not essential for mAb mediated protection in vivo. Protection mediated by CS-D7 was lost in Balb/c mice depleted of C3 with cobra venom factor (CFV, was lost in mice depleted of superoxide dismutase (SOD in P47phox deletion mice, and was absent in SCID mice. Enhanced clearance of S. aureus in the liver of CS-D7 treated mice and enhanced production of INF-γ, but not of IL17, may play a role in the mechanism of protection mediated by the mAb. CS-D7 apparently mediates survival in challenged mice through a mechanism involving complement, phagocytes, and lymphocytes, but which does not depend on interaction with FcγR, or on blocking heme

  15. 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.

  16. Die subklinische Staphylococcus aureus-Mastitis - Sanierung eines hessischen Milcherzeugerbetriebes und epidemiologische Untersuchungen mittels Staphylokokken-Protein A (spa)-Typisierung

    OpenAIRE

    Sauerwald, Claudia

    2013-01-01

    In der vorliegenden Studie wurde die Sanierung einer durch S. aureus verursachten Eutergesundheitsstörung in einem hessischen Milchviehbestand mittels klassischer Sanierungsmaßnahmen über einen Zeitraum von 18 Monaten begleitet. Durch konsequente Einhaltung der Sanierungsmaßnahmen nach dem Fünf-Punkte-Plan und die räumliche Trennung der Herde in eine S. aureus-positive und -negative Gruppe sank die S. aureus-Prävalenz im Betrieb innerhalb von 15 Monaten von 30% auf

  17. 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

  18. Protein Adsorption to Surface Chemistry and Crystal Structure Modification of Titanium Surfaces

    OpenAIRE

    Ryo Jimbo; Mikael Ivarsson; Anita Koskela; Young-Taeg Sul; Johansson, Carina B.

    2010-01-01

    ABSTRACT Objectives To observe the early adsorption of extracellular matrix and blood plasma proteins to magnesium-incorporated titanium oxide surfaces, which has shown superior bone response in animal models. Material and Methods Commercially pure titanium discs were blasted with titanium dioxide (TiO2) particles (control), and for the test group, TiO2 blasted discs were further processed with a micro-arc oxidation method (test). Surface morphology was investigated by scanning electron micro...

  19. Sputter deposited bioceramic coatings: surface characterisation and initial protein adsorption studies using surface-MALDI-MS.

    Science.gov (United States)

    Boyd, A R; Burke, G A; Duffy, H; Holmberg, M; O' Kane, C; Meenan, B J; Kingshott, P

    2011-01-01

    Protein adsorption onto calcium phosphate (Ca-P) bioceramics utilised in hard tissue implant applications has been highlighted as one of the key events that influences the subsequent biological response, in vivo. This work reports on the use of surface-matrix assisted laser desorption ionisation mass spectrometry (Surface-MALDI-MS) as a technique for the direct detection of foetal bovine serum (FBS) proteins adsorbed to hybrid calcium phosphate/titanium dioxide surfaces produced by a novel radio frequency (RF) magnetron sputtering method incorporating in situ annealing between 500°C and 700°C during deposition. XRD and XPS analysis indicated that the coatings produced at 700°C were hybrid in nature, with the presence of Ca-P and titanium dioxide clearly observed in the outer surface layer. In addition to this, the Ca/P ratio was seen to increase with increasing annealing temperature, with values of between 2.0 and 2.26 obtained for the 700°C samples. After exposure to FBS solution, surface-MALDI-MS indicated that there were significant differences in the protein patterns as shown by unique peaks detected at masses below 23.1 kDa for the different surfaces. These adsorbates were assigned to a combination of growth factors and lipoproteins present in serum. From the data obtained here it is evident that surface-MALDI-MS has significant utility as a tool for studying the dynamic nature of protein adsorption onto the surfaces of bioceramic coatings, which most likely plays a significant role in subsequent bioactivity of the materials.

  20. 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. PMID:27599942

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Nanometer polymer surface features: the influence on surface energy, protein adsorption and endothelial cell adhesion

    Science.gov (United States)

    Carpenter, Joseph; Khang, Dongwoo; Webster, Thomas J.

    2008-12-01

    Current small diameter (vascular graft materials exhibit poor long-term patency due to thrombosis and intimal hyperplasia. Tissue engineered solutions have yielded functional vascular tissue, but some require an eight-week in vitro culture period prior to implantation—too long for immediate clinical bedside applications. Previous in vitro studies have shown that nanostructured poly(lactic-co-glycolic acid) (PLGA) surfaces elevated endothelial cell adhesion, proliferation, and extracellular matrix synthesis when compared to nanosmooth surfaces. Nonetheless, these studies failed to address the importance of lateral and vertical surface feature dimensionality coupled with surface free energy; nor did such studies elicit an optimum specific surface feature size for promoting endothelial cell adhesion. In this study, a series of highly ordered nanometer to submicron structured PLGA surfaces of identical chemistry were created using a technique employing polystyrene nanobeads and poly(dimethylsiloxane) (PDMS) molds. Results demonstrated increased endothelial cell adhesion on PLGA surfaces with vertical surface features of size less than 18.87 nm but greater than 0 nm due to increased surface energy and subsequently protein (fibronectin and collagen type IV) adsorption. Furthermore, this study provided evidence that the vertical dimension of nanometer surface features, rather than the lateral dimension, is largely responsible for these increases. In this manner, this study provides key design parameters that may promote vascular graft efficacy.

  6. 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.

  7. Formation of biofilms by Staphylococcus aureus on stainless steel and glass surfaces and its resistance to some selected chemical sanitizers Formação de biofilme por Staphylococcus aureus na superfície de aço inoxidável e vidro e sua resistência a alguns sanificantes químicos

    Directory of Open Access Journals (Sweden)

    Simone Cristina Marques

    2007-09-01

    Full Text Available The objectives of this work were to verify the capability of Staphylococcus aureus of forming bio-film on stainless steel and glass surfaces; to evaluate the efficiency of sodium dichloroisocyanurate, hydrogen peroxide and peracetic acid in inactivating Staphylococcus aureus cells adhered onto these surfaces; and to visualize biofilm development by scanning electron microscopy before and after sanitizer treatment. The surfaces studied consisted of 10x20mm chips immersed in Petri dishes containing BHI broth inoculated with S. aureus ATCC 25923. Biofilm formation was observed after 15-day incubation, when the cells were removed using the swab technique, followed by Baird Parker agar plating. Also, the efficiency of the chemical sanitizers on the chip surfaces was tested and the non-removed cells were counted on the Baird-Parker agar. After biofilm formation and use of sanitizers, the chips were respectively observed by scanning electronic microscopy following a pre-existing protocol. The obtained results showed biofilm formation on both surfaces, with bacterial count in the order of 10(7 CFU/cm² on and 10(8 CFU/cm² on stainless steel and glass surfaces, respectively. Peracetic acid was the most efficient in removing adhered cells, presenting 5.26 and 4.5 decimal reduction for adhered cells on stainless steel and glass surfaces, respectively.Os objetivos deste trabalho foram verificar a capacidade de Staphylococcus aureus formar biofilme nas superfícies de aço inoxidável e vidro, avaliar a eficiência do dicloroisocianurato de sódio, peróxido de hidrogênio e ácido peracético na inativação de células de S. aureus aderidas e visualização por microscopia eletrônica de varredura, o desenvolvimento antes e depois do tratamento das superfícies com os sanificantes. As superfícies foram cupons 10x200mm imersos em placas de Petri contendo caldo BHI inoculado com cultura de Staphylococcus aureus ATCC 25923. A formação de biofilme foi

  8. Preventing protein adsorption from a range of surfaces using an aqueous fish protein extract

    DEFF Research Database (Denmark)

    Pillai, Saju; Arpanaei, Ayyoob; Meyer, Rikke L.;

    2009-01-01

    We utilize an aqueous extract of fish proteins (FPs) as a coating for minimizing the adsorption of fibrinogen (Fg) and human serum albumin (HSA). The surfaces include stainless steel (SS), gold (Au), silicon dioxide (SiO2), and poly(styrene) (PS). The adsorption processes (kinetics and adsorbed...

  9. Characterization of the Eimeria maxima sporozoite surface protein IMP1.

    Science.gov (United States)

    Jenkins, M C; Fetterer, R; Miska, K; Tuo, W; Kwok, O; Dubey, J P

    2015-07-30

    The purpose of this study was to characterize Eimeria maxima immune-mapped protein 1 (IMP1) that is hypothesized to play a role in eliciting protective immunity against E. maxima infection in chickens. RT-PCR analysis of RNA from unsporulated and sporulating E. maxima oocysts revealed highest transcription levels at 6-12h of sporulation with a considerable downregulation thereafter. Alignment of IMP1 coding sequence from Houghton, Weybridge, and APU-1 strains of E. maxima revealed single nucleotide polymorphisms that in some instances led to amino acid changes in the encoded protein sequence. The E. maxima (APU-1) IMP1 cDNA sequence was cloned and expressed in 2 different polyHis Escherichia coli expression vectors. Regardless of expression vector, recombinant E. maxima IMP1 (rEmaxIMP1) was fairly unstable in non-denaturing buffer, which is consistent with stability analysis of the primary amino acid sequence. Antisera specific for rEmaxIMP1 identified a single 72 kDa protein or a 61 kDa protein by non-reducing or reducing SDS-PAGE/immunoblotting. Immunofluorescence staining with anti-rEmaxIMP1, revealed intense surface staining of E. maxima sporozoites, with negligible staining of merozoite stages. Immuno-histochemical staining of E. maxima-infected chicken intestinal tissue revealed staining of E. maxima developmental stages in the lamnia propia and crypts at both 24 and 48 h post-infection, and negligible staining thereafter. The expression of IMP1 during early stages of in vivo development and its location on the sporozoite surface may explain in part the immunoprotective effect of this protein against E. maxima infection. PMID:26012860

  10. Biointerface: protein enhanced stem cells binding to implant surface.

    Science.gov (United States)

    Chrzanowski, W; Kondyurin, A; Lee, Jae Ho; Lord, Megan S; Bilek, M M M; Kim, Hae-Won

    2012-09-01

    The number of metallic implantable devices placed every year is estimated at 3.7 million. This number has been steadily increasing over last decades at a rate of around 8 %. In spite of the many successes of the devices the implantation of biomaterial into tissues almost universally leads to the development of an avascular sac, which consists of fibrous tissue around the device and walls off the implant from the body. This reaction can be detrimental to the function of implant, reduces its lifetime, and necessitates repeated surgery. Clearly, to reduce the number of revision surgeries and improve long-term implant function it is necessary to enhance device integration by modulating cell adhesion and function. In this paper we have demonstrated that it is possible to enhance stem cell attachment using engineered biointerfaces. To create this functional interface, samples were coated with polymer (as a precursor) and then ion implanted to create a reactive interface that aids the binding of biomolecules--fibronectin. Both AFM and XPS analyses confirmed the presence of protein layers on the samples. The amount of protein was significant greater for the ion implanted surfaces and was not disrupted upon washing with detergent, hence the formation of strong bonds with the interface was confirmed. While, for non ion implanted surfaces, a decrease of protein was observed after washing with detergent. Finally, the number of stem cells attached to the surface was enhanced for ion implanted surfaces. The studies presented confirm that the developed bionterface with immobilised fibronectin is an effective means to modulate stem cell attachment. PMID:22714559

  11. Biological properties of Lactobacillus surface proteins 

    Directory of Open Access Journals (Sweden)

    Barbara Buda

    2013-04-01

    Full Text Available Lactobacillus, a genus of Gram-positive bacteria, includes many strains of probiotic microflora. Probiotics, by definition, are living microorganisms that exert beneficial effects on the host organism. The morphology and physiology of the Lactobacillus bacterial genus are described. The structure of the cell wall of Gram-positive bacteria is discussed. The surface S-layer of Lactobacillus composed of proteins (SLP with low molecular mass is presented. Cell surface proteins participating in the regulation of growth and survival of the intestinal epithelium cells are characterized. The influence of stress factors such as increased temperature, pH, and enzymes of gastric and pancreatic juice on SLP expression is described. The ability of binding of heavy metal ions by S-layer proteins is discussed. The characteristics of these structures, including the ability to adhere to epithelial cells, and the inhibition of invasion of pathogenic microflora of type Shigella, Salmonella, Escherichia coli and Clostridium and their toxins, are presented. 

  12. 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.

  13. Hepatitis B virus large surface protein: function and fame.

    Science.gov (United States)

    Churin, Yuri; Roderfeld, Martin; Roeb, Elke

    2015-02-01

    Chronic infection with hepatitis B virus (HBV) is the leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. HBV life cycle begins with viral attachment to hepatocytes, mediated by the large HBV surface protein (LHBs). Identification of the sodium-taurocholate cotransporting polypeptide (NTCP) as a HBV receptor has revealed a suitable target for viral entry inhibition. Analysis of serum hepatitis B surface antigen (HBsAg) level is a non-invasive diagnostic parameter that improves HBV treatment opportunities. Furthermore, HBsAg plays an important role in manipulation of host immune response by HBV. However, observations in patients with chronic hepatitis B under conditions of immune suppression and in transgenic mouse models of HBV infection suggest, that in absence of adaptive immune responses cellular mechanisms induced by HBV may also lead to the development of liver diseases. Thus, the multifaceted pathological aspects of HBsAg predetermine the design of new therapeutical options modulating associated biological implications. PMID:25713800

  14. Surface peptide mapping of protein I and protein III of four strains of Neisseria gonorrhoeae

    Energy Technology Data Exchange (ETDEWEB)

    Judd, R.C.

    1982-08-01

    Whole cells and isolated outer membranes (OMs) of four strains of gonococci were surface radioiodinated with either lactoperoxidase or Iodogen (Pierce Chemical Co., Rockford, Ill.). These preparations were solubilized in sodium dodecyl sulfate and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Surface-radioiodinated protein I (PI) and PIII bands were excised from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and digested with alpha-chymotrypsin, and the resultant /sup 125/I-peptide fragments were resolved by high-voltage electrophoresis and thin-layer chromatography (i.e., surface peptide mapping). Radioemitting peptidic fragments were visualized by autoradiography. Results demonstrated that the PI molecule of each gonococcal strain studied had unique iodinatable peptides exposed on the surface of whole cells and OMs, whereas PIIIs appeared to have the same portion of the molecule exposed on the surface of bacteria or OMs, regardless of the gonococcal strain from which they were isolated. Many more radiolabeled peptides were seen in surface peptide maps of PIs from radiolabeled OMs than in those from radioiodinated whole cells, whereas different peptidic fragments were seen in the surface peptide maps of PIIIs from radiolabeled OMs than were seen in those from radiolabeled whole cells. These data suggest that PI may contribute strain-specific antigenic determinants and PIII may contribute cross-reactive determinants and that the surface exposure of PI and PIII is different in isolated OMs than in the OM of intact gonococci.

  15. 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.

  16. 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.

  17. 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.

  18. Antigenicity and immunogenicity of Plasmodium vivax merozoite surface protein-3.

    Science.gov (United States)

    Bitencourt, Amanda R; Vicentin, Elaine C; Jimenez, Maria C; Ricci, Ricardo; Leite, Juliana A; Costa, Fabio T; Ferreira, Luis C; Russell, Bruce; Nosten, François; Rénia, Laurent; Galinski, Mary R; Barnwell, John W; Rodrigues, Mauricio M; Soares, Irene S

    2013-01-01

    A recent clinical trial in African children demonstrated the potential utility of merozoite surface protein (MSP)-3 as a vaccine against Plasmodium falciparum malaria. The present study evaluated the use of Plasmodium vivax MSP-3 (PvMSP-3) as a target antigen in vaccine formulations against malaria caused by P. vivax. Recombinant proteins representing MSP-3α and MSP-3β of P. vivax were expressed as soluble histidine-tagged bacterial fusions. Antigenicity during natural infection was evaluated by detecting specific antibodies using sera from individuals living in endemic areas of Brazil. A large proportion of infected individuals presented IgG antibodies to PvMSP-3α (68.2%) and at least 1 recombinant protein representing PvMSP-3β (79.1%). In spite of the large responder frequency, reactivity to both antigens was significantly lower than was observed for the immunodominant epitope present on the 19-kDa C-terminal region of PvMSP-1. Immunogenicity of the recombinant proteins was studied in mice in the absence or presence of different adjuvant formulations. PvMSP-3β, but not PvMSP-3α, induced a TLR4-independent humoral immune response in the absence of any adjuvant formulation. The immunogenicity of the recombinant antigens were also tested in formulations containing different adjuvants (Alum, Salmonella enterica flagellin, CpG, Quil A,TiterMax® and incomplete Freunds adjuvant) and combinations of two adjuvants (Alum plus flagellin, and CpG plus flagellin). Recombinant PvMSP-3α and PvMSP-3β elicited higher antibody titers capable of recognizing P. vivax-infected erythrocytes harvested from malaria patients. Our results confirm that P. vivax MSP-3 antigens are immunogenic during natural infection, and the corresponding recombinant proteins may be useful in elucidating their vaccine potential. PMID:23457498

  19. Antigenicity and immunogenicity of Plasmodium vivax merozoite surface protein-3.

    Directory of Open Access Journals (Sweden)

    Amanda R Bitencourt

    Full Text Available A recent clinical trial in African children demonstrated the potential utility of merozoite surface protein (MSP-3 as a vaccine against Plasmodium falciparum malaria. The present study evaluated the use of Plasmodium vivax MSP-3 (PvMSP-3 as a target antigen in vaccine formulations against malaria caused by P. vivax. Recombinant proteins representing MSP-3α and MSP-3β of P. vivax were expressed as soluble histidine-tagged bacterial fusions. Antigenicity during natural infection was evaluated by detecting specific antibodies using sera from individuals living in endemic areas of Brazil. A large proportion of infected individuals presented IgG antibodies to PvMSP-3α (68.2% and at least 1 recombinant protein representing PvMSP-3β (79.1%. In spite of the large responder frequency, reactivity to both antigens was significantly lower than was observed for the immunodominant epitope present on the 19-kDa C-terminal region of PvMSP-1. Immunogenicity of the recombinant proteins was studied in mice in the absence or presence of different adjuvant formulations. PvMSP-3β, but not PvMSP-3α, induced a TLR4-independent humoral immune response in the absence of any adjuvant formulation. The immunogenicity of the recombinant antigens were also tested in formulations containing different adjuvants (Alum, Salmonella enterica flagellin, CpG, Quil A,TiterMax® and incomplete Freunds adjuvant and combinations of two adjuvants (Alum plus flagellin, and CpG plus flagellin. Recombinant PvMSP-3α and PvMSP-3β elicited higher antibody titers capable of recognizing P. vivax-infected erythrocytes harvested from malaria patients. Our results confirm that P. vivax MSP-3 antigens are immunogenic during natural infection, and the corresponding recombinant proteins may be useful in elucidating their vaccine potential.

  20. Anti-biofilm formation of a novel stainless steel against Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Nan, Li; Yang, Ke [Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016 (China); Ren, Guogang, E-mail: g.g.ren@herts.ac.uk [University of Hertfordshire, Hatfield AL10 9AB (United Kingdom)

    2015-06-01

    Staphylococcus aureus (S. aureus) is a bacterium frequently found proliferating on metal surfaces such as stainless steels used in healthcare and food processing facilities. Past research has shown that a novel Cu-bearing 304 type stainless steel (304CuSS) exhibits excellent antibacterial ability (i.e. against S. aureus) in a short time period (24 h.). This work was dedicated to investigate the 304CuSS's inhibition ability towards the S. aureus biofilm formation for an extended period of 7 days after incubation. It was found that the antibacterial rate of the 304CuSS against sessile bacterial cells reached over 99.9% in comparison with the 304SS. The thickness and sizes of the biofilms on the 304SS surfaces increased markedly with period of contact, and thus expected higher risk of bio-contamination, indicated by the changes of surface free energy between biofilm and the steel surfaces. The results demonstrated that the 304CuSS exhibited strong inhibition on the growth and adherence of the biofilms. The surface free energy of the 304CuSS after contact with sessile bacterial cells was much lower than that of the 304SS towards the same culture times. The continuously dissolved Cu{sup 2+} ions well demonstrated the dissolution ability of Cu-rich precipitates after exposure to S. aureus solution, from 3.1 ppm (2 days) to 4.5 ppm (7 days). For this to occur, a hypothesis mechanism might be established for 304CuSS in which the Cu{sup 2+} ions were released from Cu-rich phases that bond with extracellular polymeric substances (EPS) of the microorganisms. And these inhibited the activities of cell protein/enzymes and effectively prevented planktonic bacterial cells attaching to the 304CuSS metal surface.

  1. Anti-biofilm formation of a novel stainless steel against Staphylococcus aureus

    International Nuclear Information System (INIS)

    Staphylococcus aureus (S. aureus) is a bacterium frequently found proliferating on metal surfaces such as stainless steels used in healthcare and food processing facilities. Past research has shown that a novel Cu-bearing 304 type stainless steel (304CuSS) exhibits excellent antibacterial ability (i.e. against S. aureus) in a short time period (24 h.). This work was dedicated to investigate the 304CuSS's inhibition ability towards the S. aureus biofilm formation for an extended period of 7 days after incubation. It was found that the antibacterial rate of the 304CuSS against sessile bacterial cells reached over 99.9% in comparison with the 304SS. The thickness and sizes of the biofilms on the 304SS surfaces increased markedly with period of contact, and thus expected higher risk of bio-contamination, indicated by the changes of surface free energy between biofilm and the steel surfaces. The results demonstrated that the 304CuSS exhibited strong inhibition on the growth and adherence of the biofilms. The surface free energy of the 304CuSS after contact with sessile bacterial cells was much lower than that of the 304SS towards the same culture times. The continuously dissolved Cu2+ ions well demonstrated the dissolution ability of Cu-rich precipitates after exposure to S. aureus solution, from 3.1 ppm (2 days) to 4.5 ppm (7 days). For this to occur, a hypothesis mechanism might be established for 304CuSS in which the Cu2+ ions were released from Cu-rich phases that bond with extracellular polymeric substances (EPS) of the microorganisms. And these inhibited the activities of cell protein/enzymes and effectively prevented planktonic bacterial cells attaching to the 304CuSS metal surface

  2. Molecular biology of Chlamydia pneumoniae surface proteins and their role in immunopathogenicity

    DEFF Research Database (Denmark)

    Christiansen, Gunna; Boesen, Thomas; Hjernø, Karin;

    1999-01-01

    present on the surface of the bacteria, we analyzed what components are present on the C pneumoniae surface. We identified a family of proteins, the GGAI or Omp4-15 proteins, of which at least 3 are present on the surface of C pneumoniae. We immunized rabbits with recombinant GGAI proteins and used...

  3. Surface Analyses and Immune Reactivities of Major Cell Wall-Associated Proteins of Group A Streptococcus

    OpenAIRE

    Cole, Jason N; Ramirez, Ruben D.; Currie, Bart J.; Cordwell, Stuart J.; Djordjevic, Steven P.; Mark J Walker

    2005-01-01

    A proteomic analysis was undertaken to identify cell wall-associated proteins of Streptococcus pyogenes. Seventy-four distinct cell wall-associated proteins were identified, 66 of which were novel. Thirty-three proteins were immunoreactive with pooled S. pyogenes-reactive human antisera. Biotinylation of the GAS cell surface identified 23 cell wall-associated proteins that are surface exposed.

  4. Proteomics and glycoproteomics of pluripotent stem-cell surface proteins.

    Science.gov (United States)

    Sun, Bingyun

    2015-03-01

    Pluripotent stem cells are a unique cell type with promising potential in regenerative and personalized medicine. Yet the difficulty to understand and coax their seemingly stochastic differentiation and spontaneous self-renewal have largely limited their clinical applications. A call has been made by numerous researchers for a better characterization of surface proteins on these cells, in search of biomarkers that can dictate developmental stages and lineage specifications, and can help formulate mechanistic insight of stem-cell fate choices. In the past two decades, proteomics has gained significant recognition in profiling surface proteins at high throughput. This review will summarize the impact of these studies on stem-cell biology, and discuss the used proteomic techniques. A systematic comparison of all the techniques and their results is also attempted here to help reveal pros, cons, and the complementarity of the existing methods. This awareness should assist in selecting suitable strategies for stem-cell related research, and shed light on technical improvements that can be explored in the future. PMID:25211708

  5. Proteomics and glycoproteomics of pluripotent stem-cell surface proteins.

    Science.gov (United States)

    Sun, Bingyun

    2015-03-01

    Pluripotent stem cells are a unique cell type with promising potential in regenerative and personalized medicine. Yet the difficulty to understand and coax their seemingly stochastic differentiation and spontaneous self-renewal have largely limited their clinical applications. A call has been made by numerous researchers for a better characterization of surface proteins on these cells, in search of biomarkers that can dictate developmental stages and lineage specifications, and can help formulate mechanistic insight of stem-cell fate choices. In the past two decades, proteomics has gained significant recognition in profiling surface proteins at high throughput. This review will summarize the impact of these studies on stem-cell biology, and discuss the used proteomic techniques. A systematic comparison of all the techniques and their results is also attempted here to help reveal pros, cons, and the complementarity of the existing methods. This awareness should assist in selecting suitable strategies for stem-cell related research, and shed light on technical improvements that can be explored in the future.

  6. Surface proteins of bacteria of the genus Bifidobacterium 

    Directory of Open Access Journals (Sweden)

    Ewa Dylus

    2013-05-01

    Full Text Available Beneficial effects due to the presence of probiotic bacteria of the genus Bifidobacterium in the human intestinal tract are still an interesting object of study. So far activities have been confirmed of bifidobacteria in stimulation of the host immune system, stimulation of tumor cell apoptosis, improvement of bowel motility, alleviation of symptoms of lactose intolerance, cholesterol lowering capacity, prevention and treatment of diarrhea and irritable bowel syndrome, alleviation of allergy or atopic dermatitis, maintenance of homeostasis of the intestine, and stimulation of the development of normal intestinal microflora in infants. A multitude of therapeutic properties encourages researchers to investigate the possibility of using the potential of Bifidobacterium in the prevention and treatment of other conditions such as rheumatoid arthritis and depression. Although it is known that the beneficial effects are due to intestinal mucosal colonization by these bacteria, the cell components responsible for the colonization are still not determined. In addition to the beneficial effects of probiotic administration, there were also negative effects including sepsis. Therefore research has been directed to identify specific components of Bifidobacterium responsible for probiotic effects. Currently researchers are focused on identifying, isolating and evaluating the properties of surface proteins that are probably involved in the adhesion of bacterial cells to the intestinal epithelium, improving colonization. This paper is an overview of current knowledge on Bifidobacterium surface proteins. The ways of transport and anchoring proteins in Gram-positive bacterial cells, the assembly of cell wall, and a description of the genus Bifidobacterium are presented.

  7. Surface Proteins of Streptococcus agalactiae and Related Proteins in Other Bacterial Pathogens.

    OpenAIRE

    Lindahl, Gunnar; Stålhammar-Carlemalm, Margaretha; Areschoug, Thomas

    2005-01-01

    Streptococcus agalactiae (group B Streptococcus) is the major cause of invasive bacterial disease, including meningitis, in the neonatal period. Although prophylactic measures have contributed to a substantial reduction in the number of infections, development of a vaccine remains an important goal. While much work in this field has focused on the S. agalactiae polysaccharide capsule, which is an important virulence factor that elicits protective immunity, surface proteins have received incre...

  8. 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...

  9. Antimicrobial Peptide P60.4Ac-Containing Creams and Gel for Eradication of Methicillin-Resistant Staphylococcus aureus from Cultured Skin and Airway Epithelial Surfaces.

    Science.gov (United States)

    Haisma, Elisabeth M; Göblyös, Anikó; Ravensbergen, Bep; Adriaans, Alwin E; Cordfunke, Robert A; Schrumpf, Jasmijn; Limpens, Ronald W A L; Schimmel, Kirsten J M; den Hartigh, Jan; Hiemstra, Pieter S; Drijfhout, Jan Wouter; El Ghalbzouri, Abdoelwaheb; Nibbering, Peter H

    2016-07-01

    We previously found the LL-37-derived peptide P60.4Ac to be effective against methicillin-resistant Staphylococcus aureus (MRSA) on human epidermal models (EMs). The goal of this study was to identify the preferred carrier for this peptide for topical application on skin and mucosal surfaces. We prepared P60.4Ac in three formulations, i.e., a water-in-oil cream with lanolin (Softisan 649), an oil-in-water cream with polyethylene glycol hexadecyl ether (Cetomacrogol), and a hydroxypropyl methylcellulose (hypromellose) 4000 gel. We tested the antimicrobial efficacy of the peptide in these formulations against mupirocin-resistant and -sensitive MRSA strains on EMs and bronchial epithelial models (BEMs). The cytotoxic effects of formulated P60.4Ac on these models were determined using histology and WST-1 and lactate dehydrogenase assays. Moreover, we assessed the stability of the peptide in these formulations with storage for up to 3 months. Killing of MRSA by P60.4Ac in the two creams was less effective than that by P60.4Ac in the hypromellose gel. In agreement with those findings, P60.4Ac in the hypromellose gel was highly effective in eradicating the two MRSA strains from EMs. We found that even 0.1% (wt/wt) P60.4Ac in the hypromellose gel killed >99% of the viable planktonic bacteria and >85% of the biofilm-associated bacteria on EMs. Hypromellose gels containing 0.1% and 0.5% (wt/wt) P60.4Ac effectively reduced the numbers of viable MRSA cells from BEMs by >90%. No cytotoxic effects of P60.4Ac in the hypromellose gel with up to 2% (wt/wt) P60.4Ac on keratinocytes in EMs and in the hypromellose gel with up to 0.5% (wt/wt) P60.4Ac on epithelial cells in BEMs were observed. High-performance liquid chromatography analysis showed that P60.4Ac was stable in the Softisan cream and the hypromellose gel but not in the Cetomacrogol cream. We conclude that P60.4Ac formulated in hypromellose gel is both stable and highly effective in eradicating MRSA from colonized EMs and

  10. 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.

  11. Existence of a new protein component with the same function as the LukF component of leukocidin or gamma-hemolysin and its gene in Staphylococcus aureus P83.

    Science.gov (United States)

    Choorit, W; Kaneko, J; Muramoto, K; Kamio, Y

    1995-01-01

    Staphylococcal toxins, leukocidin and gamma-hemolysin, consist of two protein components, i.e. LukF and LukS for leukocodin and H gamma I and H gamma II for gamma-hemolysin. From a culture fluid of Staphylococcus aureus strain P83, a new protein component of leukocidin or gamma-hemolysin which was designated as LukM was isolated. This component showed the same biological activity as that of LukF component for leukocidin or H gamma I component of gamma-hemolysin in combination with LukS or H gamma II. However, the LukM component cross-reacted with the anti-LukS antibodies but not with the anti-LukF antibodies. On the basis of chemical analysis of the LukM component and the cloning and nucleotide sequencing of the lukM gene of S. aureus P83, we have demonstrated that LukM is an entirely new protein component of leukocidin or gamma-hemolysin. The deduced amino acid sequence of LukM from the lukM gene showed 66.7% and 67% identity to that of LukS and H gamma II, respectively. However, the amino acid sequence of LukM and LukF showed only 29% homology.

  12. Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model

    Science.gov (United States)

    Lemmens-den Toom, N. A.; Willemse, J.; Koning, R. A.; Demmers, J. A. A.; Dekkers, D. H. W.; Rijkers, E.; El Ghalbzouri, A.; Nibbering, P. H.; van Wamel, W.

    2016-01-01

    Background & 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 alpha-toxin, during biofilm formation of five different (methicillin resistant) S. aureus strains on Leiden human epidermal models (LEMs) and polystyrene surfaces (PS) using a competitive Luminex-based assay. Results All five S. aureus strains formed biofilms on PS, whereas only three out of five strains formed biofilms on LEMs. Out of the 52 tested proteins, six functionally diverse proteins (ClfB, glucosaminidase, IsdA, IsaA, SACOL0688 and nuclease) were detected in biofilms of all strains on both PS and LEMs. At the same time, four toxins (alpha-toxin, gamma-hemolysin B and leukocidins D and E), two immune modulators (formyl peptide receptor-like inhibitory protein and Staphylococcal superantigen-like protein 1), and two other proteins (lipase and LytM) were detectable in biofilms by all five S. aureus strains on LEMs, but not on PS. In contrast, fibronectin-binding protein B (FnbpB) was detectable in biofilms by all S. aureus biofilms on PS, but not on LEMs. These data were largely confirmed by the results from proteomic and transcriptomic analyses and in case of alpha-toxin additionally by GFP-reporter technology. Conclusion Functionally diverse virulence factors of (methicillin-resistant) S. aureus are present during biofilm formation on LEMs and PS. These results could aid in identifying novel targets for future treatment strategies against biofilm-associated infections. PMID:26741798

  13. Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model.

    Directory of Open Access Journals (Sweden)

    P M den Reijer

    Full Text Available The ability of Staphylococcus aureus to successfully colonize (abiotic 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 alpha-toxin, during biofilm formation of five different (methicillin resistant S. aureus strains on Leiden human epidermal models (LEMs and polystyrene surfaces (PS using a competitive Luminex-based assay.All five S. aureus strains formed biofilms on PS, whereas only three out of five strains formed biofilms on LEMs. Out of the 52 tested proteins, six functionally diverse proteins (ClfB, glucosaminidase, IsdA, IsaA, SACOL0688 and nuclease were detected in biofilms of all strains on both PS and LEMs. At the same time, four toxins (alpha-toxin, gamma-hemolysin B and leukocidins D and E, two immune modulators (formyl peptide receptor-like inhibitory protein and Staphylococcal superantigen-like protein 1, and two other proteins (lipase and LytM were detectable in biofilms by all five S. aureus strains on LEMs, but not on PS. In contrast, fibronectin-binding protein B (FnbpB was detectable in biofilms by all S. aureus biofilms on PS, but not on LEMs. These data were largely confirmed by the results from proteomic and transcriptomic analyses and in case of alpha-toxin additionally by GFP-reporter technology.Functionally diverse virulence factors of (methicillin-resistant S. aureus are present during biofilm formation on LEMs and PS. These results could aid in identifying novel targets for future treatment strategies against biofilm-associated infections.

  14. Surface-layer protein from Caulobacter crescentus: expression, purification and X-ray crystallographic analysis.

    Science.gov (United States)

    Jones, Michael D; Chan, Anson C K; Nomellini, John F; Murphy, Michael E P; Smit, John

    2016-09-01

    Protein surface layers are self-assembling, paracrystalline lattices on the surface of many prokaryotes. Surface-layer proteins have not benefited from widespread structural analysis owing to their resistance to crystallization. Here, the successful expression of a truncated version of RsaA, the surface-layer protein from Caulobacter crescentus, from a Caulobacter protein-expression system is reported. The purification, crystallization and initial X-ray diffraction analysis of the truncated RsaA, the largest surface-layer protein studied to date and the first from a Gram-negative bacterium, are also reported. PMID:27599857

  15. Adhesive polypeptides of Staphylococcus aureus identified using a novel secretion library technique in Escherichia coli

    Directory of Open Access Journals (Sweden)

    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

  16. 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.

  17. Nanoporous titanium surfaces for sustained elution of proteins and antibiotics.

    Directory of Open Access Journals (Sweden)

    Amirhossein Ketabchi

    Full Text Available Current medically relevant metals for prosthetic reconstructions enjoy a relatively good success rate, but their performance drops significantly in patients with compromised health status, and post-surgical infections still remain an important challenge. To address these problems, different nanotechnology-based strategies have been exploited to create implantable metals with an enhanced bioactivity and antibacterial capacities. Among these, oxidative nanopatterning has emerged as a very effective approach to engender nanoporous surfaces that stimulate and guide the activity of adhering cells. The resulting nanoporosity is also attractive because it offers nanoconfined volumes that can be exploited to load bioactive compounds and modulate their release over time. Such extended elution is needed since a single exposure to growth factors and/or antibiotics, for instance, may not be adequate to further sustain bone regeneration and/or to counteract bacterial colonization. In this article, we assessed the capacities of nanoporous titanium surfaces generated by oxidative nanopatterning to provide controlled and sustained elution of proteins and antibiotic molecules. To this end, we have selected bovine serum albumin (BSA and vancomycin to reflect commonly used compounds, and investigated their adsorption and elution by Fourier-transform infrared (FT-IR and ultraviolet-visible (UV-VIS spectroscopy. Our results demonstrate that while the elution of albumin is not significantly affected by the nanoporosity, in the case of vancomycin, nanoporous surfaces provided an extended release. These findings were successively correlated to the establishment of interactions with the surface and physical-entrapment effects exerted by the nanopores, ultimately highlighting their synergistic contribution to the release profiles and thus their importance in the design of nanostructured eluting platforms for applications in medicine.

  18. Effectiveness of cleaning and sanitizing procedures in controlling the adherence of Pseudomonas fluorescens, Salmonella Enteritidis, and Staphylococcus aureus to domestic kitchen surfaces Eficiência dos procedimentos de limpeza e de sanitização no controle da adesão de Pseudomonas fluorescens, Salmonella Enteritidis e Staphylococcus aureus em superfícies usadas em cozinhas domésticas

    Directory of Open Access Journals (Sweden)

    Iara Dias Silva

    2010-03-01

    Full Text Available The effectiveness of cleaning and sanitizing procedures in controlling Staphylococcus aureus, Salmonella Enteritidis, and Pseudomonasfluorescens adhered to granite and stainless steel was evaluated. There was no significant difference (p > 0.05 in the adherence of pure cultures of these microorganisms to stainless steel. The numbers of P. fluorescens and S. Enteritidis adhered to granite were greater (p 0.05 between the surfaces. However, a significant difference was observed (p A eficiência dos procedimentos de limpeza e sanitização no controle de Staphylococcus aureus, Salmonella Enteritidis e Pseudomonasfluorescens aderidas em granito e aço inoxidável foi avaliada. Não houve diferença significativa (p > 0,05 na adesão destes microrganismos quando em cultura pura, em aço inoxidável. O número de células aderidas de P. fluorescens e S. Enteritidis foi maior (p 0,05 entre as superfícies. Entretanto, observou-se uma diferença (p < 0,05 entre as soluções sanitizantes utilizadas. Hipoclorito de sódio e ácido peracético apresentaram maior ação bactericida (p < 0,05 que o composto de amônia quaternária. Observou-se que S. aureus apresentou menor resistência à ação desses sanitizantes. Os resultados mostram a importância da adequada realização dos procedimentos de limpeza e sanitização para evitar a adesão bacteriana e formação de biofilme.

  19. 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...

  20. SURFACE MODIFICATION OF TITANIUM FILMS WITH SODIUM ION IMPLANTATION: SURFACE PROPERTIES AND PROTEIN ADSORPTION

    Institute of Scientific and Technical Information of China (English)

    K. Y. Cai

    2007-01-01

    Sodium implanted titanium films with different ion doses were characterized to correlate their ion implantation parameters. Native titanium films and ion implanted titanium films were characterized with combined techniques of X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and light microscopy (LM). The surface presented increased sodium concentration on treated titanium films with ion dose increasing, except for the group with the highest ion dose of 4× 1017 ions/cm2. XPS depth profiling displayed that sodium entered titanium film around 25-50 nm depth depending on its implantation ion dose. AFM characterization showed that sodium ion implantation treatment changed the surface morphology from a relatively smooth titanium film to rough surfaces corresponding to different implantation doses.After sodium implantation, implanted titanium films presented big particles with island structure morphology. The surface morphology and particle growth displayed the corresponding trend.Fibrinogen adsorption on these titanium films was performed to correlate with the surface properties of treated titanium films. The results show that protein adsorption on ion-implanted samples with dose of 2 × 1017 and 4 × 1017 are statistically higher (p < 0. 01) than samples treated with dose of 5×1016 and 1 ×1017, as well as the control samples.

  1. Development of a strategy for the identification of surface proteins in the pathogenic microsporidian Nosema bombycis.

    Science.gov (United States)

    Zhao, Weixi; Hao, Youjin; Wang, Linglin; Zhou, Zeyang; Li, Zhi

    2015-06-01

    Parasite-host interactions mediated by cell surface proteins have been implicated as a critical step in infections caused by the microsporidian Nosema bombycis. Such cell surface proteins are considered as promising diagnostic markers and targets for drug development. However, little research has specifically addressed surface proteome identification in microsporidia due to technical barriers. Here, a combined strategy was developed to separate and identify the surface proteins of N. bombycis. Briefly, following (1) biotinylation of the spore surface, (2) extraction of total proteins with an optimized method and (3) streptavidin affinity purification of biotinylated proteins, 22 proteins were identified based on LC-MS/MS analysis. Among them, 5 proteins were confirmed to be localized on the surface of N. bombycis. A total of 8 proteins were identified as hypothetical extracellular proteins, whereas 7 other hypothetical proteins had no available function annotation. Furthermore, a protein with a molecular weight of 18·5 kDa was localized on the spore surface by western blotting and immunofluorescence analysis, even though it was predicted to be a nuclear protein by bioinformatics. Collectively, our work provides an effective strategy for isolating microsporidian surface protein components for both drug target identification and further diagnostic research on microsporidian disease control. PMID:25811320

  2. Lacritin and other autophagy associated proteins in ocular surface health.

    Science.gov (United States)

    Karnati, Roy; Talla, Venu; Peterson, Katherine; Laurie, Gordon W

    2016-03-01

    Advantage may be taken of macroautophagy ('autophagy') to promote ocular health. Autophagy continually captures aged or damaged cellular material for lysosomal degradation and recyling. When autophagic flux is chronically elevated, or alternatively deficient, health suffers. Chronic elevation of flux and stress are the consequence of inflammatory cytokines or of dry eye tears but not normal tears invitro. Exogenous tear protein lacritin transiently accelerates flux to restore homeostasis invitro and corneal health invivo, and yet the monomeric active form of lacritin appears to be selectively deficient in dry eye. Tissue transglutaminase-dependent cross-linking of monomer decreases monomer quantity and monomer affinity for coreceptor syndecan-1 thereby abrogating activity. Tissue transglutaminase is elevated in dry eye. Mutation of arylsulfatase A, arylsulfatase B, ceroid-lipofuscinosis neuronal 3, mucolipin, or Niemann-Pick disease type C1 respectively underlie several diseases of apparently insufficient autophagic flux that affect the eye, including: metachromatic leukodystrophy, mucopolysaccharidosis type VI, juvenile-onset Batten disease, mucolipidosis IV, and Niemann-Pick type C associated with myelin sheath destruction of corneal sensory and ciliary nerves and of the optic nerve; corneal clouding, ocular hypertension, glaucoma and optic nerve atrophy; accumulation of 'ceroid-lipofuscin' in surface conjunctival cells, and in ganglion and neuronal cells; decreased visual acuity and retinal dystrophy; and neurodegeneration. For some, enzyme or gene replacement, or substrate reduction, therapy is proving to be successful. Here we discuss examples of restoring ocular surface homeostasis through alteration of autophagy, with particular attention to lacritin. PMID:26318608

  3. Lacritin and other autophagy associated proteins in ocular surface health.

    Science.gov (United States)

    Karnati, Roy; Talla, Venu; Peterson, Katherine; Laurie, Gordon W

    2016-03-01

    Advantage may be taken of macroautophagy ('autophagy') to promote ocular health. Autophagy continually captures aged or damaged cellular material for lysosomal degradation and recyling. When autophagic flux is chronically elevated, or alternatively deficient, health suffers. Chronic elevation of flux and stress are the consequence of inflammatory cytokines or of dry eye tears but not normal tears invitro. Exogenous tear protein lacritin transiently accelerates flux to restore homeostasis invitro and corneal health invivo, and yet the monomeric active form of lacritin appears to be selectively deficient in dry eye. Tissue transglutaminase-dependent cross-linking of monomer decreases monomer quantity and monomer affinity for coreceptor syndecan-1 thereby abrogating activity. Tissue transglutaminase is elevated in dry eye. Mutation of arylsulfatase A, arylsulfatase B, ceroid-lipofuscinosis neuronal 3, mucolipin, or Niemann-Pick disease type C1 respectively underlie several diseases of apparently insufficient autophagic flux that affect the eye, including: metachromatic leukodystrophy, mucopolysaccharidosis type VI, juvenile-onset Batten disease, mucolipidosis IV, and Niemann-Pick type C associated with myelin sheath destruction of corneal sensory and ciliary nerves and of the optic nerve; corneal clouding, ocular hypertension, glaucoma and optic nerve atrophy; accumulation of 'ceroid-lipofuscin' in surface conjunctival cells, and in ganglion and neuronal cells; decreased visual acuity and retinal dystrophy; and neurodegeneration. For some, enzyme or gene replacement, or substrate reduction, therapy is proving to be successful. Here we discuss examples of restoring ocular surface homeostasis through alteration of autophagy, with particular attention to lacritin.

  4. Anaplasma marginale major surface protein 1a directs cell surface display of tick BM95 immunogenic peptides on Escherichia coli.

    Science.gov (United States)

    Canales, Mario; Almazán, Consuelo; Pérez de la Lastra, José M; de la Fuente, José

    2008-07-31

    The surface display of heterologous proteins on live Escherichia coli using anchoring motifs from outer membranes proteins has impacted on many areas of biochemistry, molecular biology and biotechnology. The Anaplasma marginale major surface protein 1a (MSP1a) contains N-terminal surface-exposed repeated peptides (28-289 amino acids) that are involved in pathogen interaction with host cell receptors and is surface-displayed when the recombinant protein is expressed in E. coli. Therefore, it was predicted that MSP1a would surface display on E. coli peptides inserted in the N-terminal repeats region of the protein. The Rhipicephalus (Boophilus) microplus BM86 and BM95 glycoproteins are homologous proteins that protect cattle against tick infestations. In this study, we demonstrated that a recombinant protein comprising tick BM95 immunogenic peptides fused to the A. marginale MSP1a N-terminal region is displayed on the E. coli surface and is recognized by anti-BM86 and anti-MSP1a antibodies. This system provides a novel approach to the surface display of heterologous antigenic proteins on live E. coli and suggests the possibility to use the recombinant bacteria for immunization studies against cattle tick infestations. PMID:18582976

  5. 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.

  6. Effect of Biofilm Growth on Expression of Surface Proteins of Actinomyces naeslundii Genospecies 2

    OpenAIRE

    Paddick, James S.; Brailsford, Susan R; Rao, Susmitha; Soares, Renata F.; Kidd, Edwina A. M.; Beighton, David; Homer, Karen A.

    2006-01-01

    The predominant surface proteins of biofilm and planktonic Actinomyces naeslundii, a primary colonizer of the tooth surface, were examined. Seventy-nine proteins (the products of 52 genes) were identified in biofilm cells, and 30 of these, including adhesins, chaperones, and stress-response proteins, were significantly up-regulated relative to planktonic cells.

  7. Characterization of the surface of protein-adsorbed dental materials by wetting and streaming potential measurements

    NARCIS (Netherlands)

    Matsumura, H; Kawasaki, K; Okumura, N; Kambara, M; Norde, W

    2003-01-01

    In this study we have elucidated the water-wettability and the electrokinetic surface potential of protein-covered dental materials. The proteins used here as typical proteins were human serum albumin and lysozyme from hen's egg. The wettability (hydrophobicity/hydrophilicity) and the surface potent

  8. Characterization of the surface of protein-adsorbed dental materials by wetting and streaming potential measurements

    NARCIS (Netherlands)

    Matsumura, H.; Kawasaki, K.; Okumura, N.; Kambara, M.; Norde, W.

    2003-01-01

    In this study we have elucidated the water-wettability and the electrokinetic surface potential of protein-covered dental materials. The proteins used here as typical proteins were human serum albumin and lysozyme from hen*s egg. The wettability (hydrophobicity/hydrophilicity) and the surface potent

  9. Surface Display of Domain Ⅲ of Japanese Encephalitis Virus E Protein on Salmonella Typhimurium by Using an Ice Nucleation Protein

    Institute of Scientific and Technical Information of China (English)

    Jian-lin Dou; Tao Jing; Jing-jing Fan; Zhi-ming Yuan

    2011-01-01

    A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection.Due to its ubiquitous ability to invade host cells,Salmonella typhimurium might be a good candidate for displaying viral antigens.We demonstrated the surface display of domain III of Japanese encephalitis virus E protein and the enhanced green fluorescent protein on S.typhimurium BRD509 using the ice nucleation protein.The effects of the motif in the ice nucleation protein on the effective display of integral protein were also investigated.The results showed that display motifs in the protein can target integral foreign protein on the surface of S.typhimurium BRD509.Moreover,recombinant strains with surface displayed viral proteins retained their invasiveness,suggesting that the recombinant S.typhimurium can be used as live vaccine vector for eliciting complete immunogenicity.The data may yield better understanding of the mechanism by which ice nucleation protein displays foreign proteins in the Salmonella strain.

  10. Cell surface hydrophobicity is conveyed by S-layer proteins - A study in recombinant lactobacilli

    NARCIS (Netherlands)

    Mei, H.C. van der; Belt-Gritter, B. van de; Pouwels, P.H.; Martinez, B.; Busscher, H.J.

    2003-01-01

    Cell surface hydrophobicity is one of the most important factors controlling adhesion of microorganisms to surfaces. In this paper, cell surface properties of lactobacilli and recombinant lactobacilli with and without a surface layer protein (SLP) associated with cell surface hydrophobicity were det

  11. 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.

  12. Protein-based biofilm matrices in Staphylococci

    Directory of Open Access Journals (Sweden)

    Pietro eSpeziale

    2014-12-01

    Full Text Available Staphylococcus aureus and Staphylococcus epidermidis are the most important etiological agents of biofilm associated-infections on indwelling medical devices. Biofilm infections may also develop independently of indwelling devices, e.g. in native valve endocarditis, bone tissue and open wounds. After attachment to tissue or indwelling medical devices that have been conditioned with host plasma proteins, staphylococcal biofilms grow and produce a specific environment which provides the conditions for cell-cell interaction and formation of multicellular communities. Bacteria living in biofilms express a variety of macromolecules, including exopolysaccharides, proteins, extracellular eDNA and other polymers. The S. aureus surface protein C and G (SasC and SasG, clumping factor B (ClfB, serine aspartate repeat protein (SdrC, the biofilm-associated protein (Bap and the fibronectin/fibrinogen-binding proteins (FnBPA and FnBPB are individually implicated in biofilm matrix formation. In S. epidermidis, a protein named accumulation-associated protein (Aap contributes to both the primary attachment phase and the establishment of intercellular connections by forming fibrils on the cell surface. In S. epidermidis proteinaceous biofilm formation can also be mediated by the extracellular matrix binding protein (Embp and S. epidermidis surface protein C (SesC. Additionally, multifunctional proteins such as extracellular adherence protein (Eap and extracellular matrix protein binding protein (Emp of S. aureus and the iron-regulated surface determinant protein C (IsdC of S. lugdunensis can promote biofilm formation in iron-depleted conditions. This multitude of proteins intervene at different stages of biofilm formation with certain proteins contributing to biofilm accumulation and others mediating primary attachment to surfaces. This review examines the contribution of proteins to biofilm formation in staphylococci. The potential to develop vaccines to prevent

  13. The sortase A substrates FnbpA, FnbpB, ClfA and ClfB antagonize colony spreading of Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Eleni Tsompanidou

    Full Text Available Staphylococcus aureus is an important human pathogen that is renowned both for its rapid transmission within hospitals and the community, and for the formation of antibiotic resistant biofilms on medical implants. Recently, it was shown that S. aureus is able to spread over wet surfaces. This motility phenomenon is promoted by the surfactant properties of secreted phenol-soluble modulins (PSMs, which are also known to inhibit biofilm formation. The aim of the present studies was to determine whether any cell surface-associated S. aureus proteins have an impact on colony spreading. To this end, we analyzed the spreading capabilities of strains lacking non-essential components of the protein export and sorting machinery. Interestingly, our analyses reveal that the absence of sortase A (SrtA causes a hyper-spreading phenotype. SrtA is responsible for covalent anchoring of various proteins to the staphylococcal cell wall. Accordingly, we show that the hyper-spreading phenotype of srtA mutant cells is an indirect effect that relates to the sortase substrates FnbpA, FnbpB, ClfA and ClfB. These surface-exposed staphylococcal proteins are known to promote biofilm formation, and cell-cell interactions. The hyper-spreading phenotype of srtA mutant staphylococcal cells was subsequently validated in Staphylococcus epidermidis. We conclude that cell wall-associated factors that promote a sessile lifestyle of S. aureus and S. epidermidis antagonize the colony spreading motility of these bacteria.

  14. 3D structural analysis of proteins using electrostatic surfaces based on image segmentation

    Science.gov (United States)

    Vlachakis, Dimitrios; Champeris Tsaniras, Spyridon; Tsiliki, Georgia; Megalooikonomou, Vasileios; Kossida, Sophia

    2016-01-01

    Herein, we present a novel strategy to analyse and characterize proteins using protein molecular electro-static surfaces. Our approach starts by calculating a series of distinct molecular surfaces for each protein that are subsequently flattened out, thus reducing 3D information noise. RGB images are appropriately scaled by means of standard image processing techniques whilst retaining the weight information of each protein’s molecular electrostatic surface. Then homogeneous areas in the protein surface are estimated based on unsupervised clustering of the 3D images, while performing similarity searches. This is a computationally fast approach, which efficiently highlights interesting structural areas among a group of proteins. Multiple protein electrostatic surfaces can be combined together and in conjunction with their processed images, they can provide the starting material for protein structural similarity and molecular docking experiments.

  15. Facile Photoimmobilization of Proteins onto Low-Binding PEG-Coated Polymer Surfaces

    DEFF Research Database (Denmark)

    Larsen, Esben Kjær Unmack; Mikkelsen, Morten Bo Lindholm; Larsen, Niels Bent

    2014-01-01

    Immobilization of proteins onto polymer surfaces usually requires specific reactive functional groups. Here, we show an easy one-step method to conjugate protein covalently onto almost any polymer surface, including low protein-binding poly(ethylene glycol) (PEG), without the requirement for the...... surface areas, showing ng/mL sensitivity to a cytokine antigen target. Moreover, spatially patterned attachment of fluorescently labeled protein onto the low-binding PEG-coated surface was achieved with a projection lithography system that enabled the creation of micrometer-sized protein features....... presence of specific functional groups. Several types of proteins, including alkaline phosphatase, bovine serum albumin, and polyclonal antibodies, were photoimmobilized onto a PEG-coated polymer surface using a water-soluble benzophenone as photosensitizer. Protein functionality after immobilization was...

  16. Adsorption of a model protein, the GroEL chaperonin, on surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Leung, Carl; Palmer, Richard E [Nanoscale Physics Research Laboratory, School of Physics and Astronomy, University of Birmingham, Edgbaston, Birmingham B15 2TT (United Kingdom)], E-mail: carl.leung@kcl.ac.uk

    2008-09-03

    Understanding and controlling protein adsorption on surfaces is fundamental to many biological processes ranging from cell adhesion to the fabrication of protein biochips. In general, proteins need to retain their 3D conformation to perform their intended functions. However, when they are presented with a solid surface, complex interactions ranging from weak non-covalent binding to strong covalent bonding may occur, which can potentially induce conformational changes within the adsorbed protein. To investigate the surface adsorption process and its effects on a model protein, the chaperonin GroEL, we have applied contact mode atomic force microscopy, in buffer solution to probe the interactions between single proteins and surfaces in real space. We will discuss the adsorption of GroEL molecules on planar surfaces (mica, graphite and gold) and specifically tailored nanostructured surfaces, which present structural features on the size scale of individual biological molecules. (topical review)

  17. Adsorption of a model protein, the GroEL chaperonin, on surfaces

    International Nuclear Information System (INIS)

    Understanding and controlling protein adsorption on surfaces is fundamental to many biological processes ranging from cell adhesion to the fabrication of protein biochips. In general, proteins need to retain their 3D conformation to perform their intended functions. However, when they are presented with a solid surface, complex interactions ranging from weak non-covalent binding to strong covalent bonding may occur, which can potentially induce conformational changes within the adsorbed protein. To investigate the surface adsorption process and its effects on a model protein, the chaperonin GroEL, we have applied contact mode atomic force microscopy, in buffer solution to probe the interactions between single proteins and surfaces in real space. We will discuss the adsorption of GroEL molecules on planar surfaces (mica, graphite and gold) and specifically tailored nanostructured surfaces, which present structural features on the size scale of individual biological molecules. (topical review)

  18. Wolbachia surface protein induces innate immune responses in mosquito cells

    Directory of Open Access Journals (Sweden)

    Pinto Sofia B

    2012-01-01

    Full Text Available Abstract Background Wolbachia endosymbiotic bacteria are capable of inducing chronic upregulation of insect immune genes in some situations and this phenotype may influence the transmission of important insect-borne pathogens. However the molecules involved in these interactions have not been characterized. Results Here we show that recombinant Wolbachia Surface Protein (WSP stimulates increased transcription of immune genes in mosquito cells derived from the mosquito Anopheles gambiae, which is naturally uninfected with Wolbachia; at least two of the upregulated genes, TEP1 and APL1, are known to be important in Plasmodium killing in this species. When cells from Aedes albopictus, which is naturally Wolbachia-infected, were challenged with WSP lower levels of upregulation were observed than for the An. gambiae cells. Conclusions We have found that WSP is a strong immune elicitor in a naturally Wolbachia-uninfected mosquito species (Anopheles gambiae while a milder elicitor in a naturally-infected species (Aedes albopictus. Since the WSP of a mosquito non-native (nematode Wolbachia strain was used, these data suggest that there is a generalized tolerance to WSP in Ae. albopictus.

  19. Potassium Uptake Modulates Staphylococcus aureus Metabolism.

    Science.gov (United States)

    Gries, Casey M; Sadykov, Marat R; Bulock, Logan L; Chaudhari, Sujata S; Thomas, Vinai C; Bose, Jeffrey L; Bayles, Kenneth W

    2016-01-01

    As a leading cause of community-associated and nosocomial infections, Staphylococcus aureus requires sophisticated mechanisms that function to maintain cellular homeostasis in response to its exposure to changing environmental conditions. The adaptation to stress and maintenance of homeostasis depend largely on membrane activity, including supporting electrochemical gradients and synthesis of ATP. This is largely achieved through potassium (K(+)) transport, which plays an essential role in maintaining chemiosmotic homeostasis, affects antimicrobial resistance, and contributes to fitness in vivo. Here, we report that S. aureus Ktr-mediated K(+) uptake is necessary for maintaining cytoplasmic pH and the establishment of a proton motive force. Metabolite analyses revealed that K(+) deficiency affects both metabolic and energy states of S. aureus by impairing oxidative phosphorylation and directing carbon flux toward substrate-level phosphorylation. Taken together, these results underline the importance of K(+) uptake in maintaining essential components of S. aureus metabolism. IMPORTANCE Previous studies describing mechanisms for K(+) uptake in S. aureus revealed that the Ktr-mediated K(+) transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K(+) uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K(+) uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K(+) uptake in establishing efficient carbon utilization. PMID:27340697

  20. Regulation of Macrophage Recognition through the Interplay of Nanoparticle Surface Functionality and Protein Corona.

    Science.gov (United States)

    Saha, Krishnendu; Rahimi, Mehran; Yazdani, Mahdieh; Kim, Sung Tae; Moyano, Daniel F; Hou, Singyuk; Das, Ridhha; Mout, Rubul; Rezaee, Farhad; Mahmoudi, Morteza; Rotello, Vincent M

    2016-04-26

    Using a family of cationic gold nanoparticles (NPs) with similar size and charge, we demonstrate that proper surface engineering can control the nature and identity of protein corona in physiological serum conditions. The protein coronas were highly dependent on the hydrophobicity and arrangement of chemical motifs on NP surface. The NPs were uptaken in macrophages in a corona-dependent manner, predominantly through recognition of specific complement proteins in the NP corona. Taken together, this study shows that surface functionality can be used to tune the protein corona formed on NP surface, dictating the interaction of NPs with macrophages.

  1. Photoswitchable method for the ordered attachment of proteins to surfaces

    Science.gov (United States)

    Camarero, Julio A.; DeYoreo, James J.; Kwon, Youngeun

    2011-07-05

    Described herein is a method for the attachment of proteins to any solid support with control over the orientation of the attachment. The method is extremely efficient, not requiring the previous purification of the protein to be attached, and can be activated by UV-light. Spatially addressable arrays of multiple protein components can be generated by using standard photolithographic techniques.

  2. A novel co-culture model of murine K12 osteosarcoma cells and S. aureus on common orthopedic implant materials: 'the race to the surface' studied in vitro.

    Science.gov (United States)

    McConda, David B; Karnes, Jonathan M; Hamza, Therwa; Lindsey, Brock A

    2016-07-01

    Infection is a major cause of orthopedic implant failure. There are few studies assessing both tissue cell and bacterial adherence on common orthopedic implant materials in a co-culture environment. An in vitro co-culture model was created using K12 osteosarcoma cells and Staphylococcus aureus in a medium incubated over metal disks for 48 h. The results showed that, in the presence of S. aureus, there were fewer osteosarcoma cells attached to the disks for all substrata tested. There were significantly more osteosarcoma cells adhering to the cobalt chrome than the stainless steel and titanium disks. Overall, in the presence of osteosarcoma cells, there were more bacteria adhering to the disks for all the substrata tested, with significantly more bacteria adhering to the stainless steel disks compared to cobalt chrome and titanium disks. Scanning electron microscopy verified that osteosarcoma cells and bacteria were adherent to the metal disks after incubation for 48 h. Furthermore, the observation that more bacteria were in the co-culture than in the control sample suggests that the osteosarcoma cells serve as a nutrient source for the bacteria. Future models assessing the interaction of osteogenic cells with bacteria on a substratum would be improved if the model accounted for the role of the immune system in secondary bone healing. PMID:27142312

  3. Cloning and surface expression in Escherichia coli of a structural gene encoding a surface protein of Haemophilus influenzae type b.

    OpenAIRE

    Holmans, P L; Loftus, T A; Hansen, E J

    1985-01-01

    Recombinant DNA technology was used to clone a gene coding for a surface protein of Haemophilus influenzae type b (Hib) into Escherichia coli. Chromosomal DNA from a clinical isolate of Hib was cleaved with EcoRI and ligated into plasmid vectors containing three different translational reading frames. E. coli carrying recombinant plasmids were screened in a colony blot-radioimmunoassay system by using murine monoclonal antibodies (mabs) directed against cell surface-exposed proteins of Hib. m...

  4. The role of surfactant proteins in DPPC enrichment of surface films.

    OpenAIRE

    Veldhuizen, E J; Batenburg, J.J.; van Golde, L M; Haagsman, H.P.

    2000-01-01

    A pressure-driven captive bubble surfactometer was used to determine the role of surfactant proteins in refinement of the surface film. The advantage of this apparatus is that surface films can be spread at the interface of an air bubble with a different lipid/protein composition than the subphase vesicles. Using different combinations of subphase vesicles and spread surface films a clear correlation between dipalmitoylphosphatidylcholine (DPPC) content and minimum surface tension was observe...

  5. Surface adhesion of fusion proteins containing the hydrophobins HFBI and HFBII from Trichoderma reesei

    OpenAIRE

    Linder, Markus; Szilvay, Geza R.; Nakari-Setälä, Tiina; Söderlund, Hans; Penttilä, Merja

    2002-01-01

    Hydrophobins are surface-active proteins produced by filamentous fungi, where they seem to be ubiquitous. They have a variety of roles in fungal physiology related to surface phenomena, such as adhesion, formation of surface layers, and lowering of surface tension. Hydrophobins can be divided into two classes based on the hydropathy profile of their primary sequence. We have studied the adhesion behavior of two Trichoderma reesei class II hydrophobins, HFBI and HFBII, as isolated proteins and...

  6. Scanning electron microscopy study of protein immobilized on SIO2 Sol-gel surfaces

    Directory of Open Access Journals (Sweden)

    Assis O.B.G.

    2003-01-01

    Full Text Available Uniform attachment of enzymes to solid surfaces is essential in the development of bio and optical sensor devices. Immobilization by adsorption according to hydrophilic or hydrophobic nature is dependent on the charges and defects of the support surfaces. Sol-gel SiO2 densified glass surfaces, frequently used as supports for protein immobilization, are evaluated via scanning electron microscopy. The model protein is globular enzyme lysozyme, deposited by adsorption on functionalized surfaces. Formation of a protein layer is confirmed by FTIR spectroscopy, and the SEM images suggest discontinuous adsorption in areas where cracks predominate on the glass surface.

  7. Enhanced protein retention on poly(caprolactone) via surface initiated polymerization of acrylamide

    Science.gov (United States)

    Ma, Yuhao; Cai, Mengtan; He, Liu; Luo, Xianglin

    2016-01-01

    To enhance the biocompatibility or extend the biomedical application of poly(caprolactone) (PCL), protein retention on PCL surface is often required. In this study, poly(acrylamide) (PAAm) brushes were grown from PCL surface via surface-initiated atom transfer radical polymerization (SI-ATRP) and served as a protein-capturing platform. Grafted PAAm was densely packed on surface and exhibited superior protein retention ability. Captured protein was found to be resistant to washing under detergent environment. Furthermore, protein structure after being captured was investigated by circular dichroism (CD) spectroscopy, and the CD spectra verified that secondary structure of captured proteins was maintained, indicating no denaturation of protein happened for retention process.

  8. Sampling the conformation of protein surface residues for flexible protein docking

    Directory of Open Access Journals (Sweden)

    Amenta Nina

    2010-11-01

    Full Text Available Abstract Background The problem of determining the physical conformation of a protein dimer, given the structures of the two interacting proteins in their unbound state, is a difficult one. The location of the docking interface is determined largely by geometric complementarity, but finding complementary geometry is complicated by the flexibility of the backbone and side-chains of both proteins. We seek to generate candidates for docking that approximate the bound state well, even in cases where there is backbone and/or side-chain difference from unbound to bound states. Results We divide the surfaces of each protein into local patches and describe the effect of side-chain flexibility on each patch by sampling the space of conformations of its side-chains. Likely positions of individual side-chains are given by a rotamer library; this library is used to derive a sample of possible mutual conformations within the patch. We enforce broad coverage of torsion space. We control the size of the sample by using energy criteria to eliminate unlikely configurations, and by clustering similar configurations, resulting in 50 candidates for a patch, a manageable number for docking. Conclusions Using a database of protein dimers for which the bound and unbound structures of the monomers are known, we show that from the unbound patch we are able to generate candidates for docking that approximate the bound structure. In patches where backbone change is small (within 1 Å RMSD of bound, we are able to account for flexibility and generate candidates that are good approximations of the bound state (82% are within 1 Å and 98% are within 1.4 Å RMSD of the bound conformation. We also find that even in cases of moderate backbone flexibility our candidates are able to capture some of the overall shape change. Overall, in 650 of 700 test patches we produce a candidate that is either within 1 Å RMSD of the bound conformation or is closer to the bound state than the

  9. Flagellin and outer surface proteins from Borrelia burgdorferi are not glycosylated

    OpenAIRE

    Štěrba, Ján

    2012-01-01

    Glycosylation of four proteins from Borrelia burgdorferi s.s. was investigated ? flagellins FlaA, FlaB, and outer surface proteins OspA and OspB. Glycosylation of these four proteins was not proved by any of the used techniques. However, other glycan-staining positive proteins were present in the borrelia samples. These proteins were suggested to originate in the culture medium.

  10. Protein analysis in dissolved organic matter: what free proteins from soil leachate and surface water can tell us a perspective

    Science.gov (United States)

    Schulze, W.

    2004-12-01

    Mass spectrometry based analysis of proteins is widely used to study cellular processes in model organisms. However, it has not yet routinely been applied in environmental research. Based on observations that protein can readily be detected as a component of dissolved organic matter (DOM), this article gives an example about the possible use of protein analysis in ecology and environmental sciences focusing on different terrestrial ecosystems. At this stage, there are two areas of interest: (1) the identification of phylogenetic groups contributing to the DOM protein pool, and (2) identification of the organismic origin of specific enzymes that are important for ecosystem processes. In this paper, mass spectrometric protein analysis was applied to identify proteins from DOM and organism-free surface water samples derived from different environments. It is concluded, that mass spectrometric protein analysis is capable of distinguishing phylogenetic origin of proteins from leachates of different soil horizons, and from various sources of terrestrial surface water. Current limitation is imposed by the limited knowledge of complete genomes of soil organisms. The protein analysis allows to relate protein presence to biogeochemical processes, and to identify the source organisms for specific active enzymes. Further applications, such as in pollution research are conceivable. In summary, the analysis of proteins opens a new area of research between the fields of microbiology and biogeochemistry.

  11. 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.

  12. Correlation between fundamental binding forces and clinical prognosis of Staphylococcus aureus infections of medical implants

    Energy Technology Data Exchange (ETDEWEB)

    Yongsunthon, Ruchirej; Fowler, Vance; Lower, Brian H.; Vellano, Francis P.; Alexander, Emily; Reller, L. Barth; Corey, G. Ralph; Lower, Steven

    2007-02-01

    Implanted medical devices (e.g., prosthetic heart valves, permanent pacemakers) significantly improve the quality of life for many humans. However, a common clinical observation is that such devices become colonized with potentially life-threatening Staphylococcus aureus biofilms, which are difficult to combat with host defenses or antibiotics. This study attempts to draw a correlation between the clinical outcome of patients with implanted cardiac devices and the fundamental binding forces ultimately responsible for the initiation of an S. aureus biofilm in-situ. Atomic force microscopy was used to measure forces between a fibronectin-coated probe (simulating a prosthetic implant) and 15 different strains of S. aureus isolated from either patients with infected cardiac devices (invasive population) or healthy human subjects (control population). The fibronectin-coated probe was repeatedly brought into and out of contact with a bacterium’s surface, “fishing” for a reaction with the cell’s fibronectin-binding proteins. More than 40,000 force profiles were measured on 5-10 different cells for each of the 15 clinical strains. A unique force-signature was observed for a binding event between the fibronectin-coated probe and the bacteria. When grouped by the frequency of this force-signature, there was a strong distinction (p=0.01) between the invasive and control populations of S. aureus. This discovery suggests that biofilm forming bacteria may be classified according to their “force taxonomy”, which could have a positive effect on health care as it bridges the long-standing disconnect between macroscopic, clinical investigations and nanometer-scale forces ultimately responsible for a bond between S. aureus and the surface of a prosthetic device.

  13. Pre-absorbed immunoproteomics: a novel method for the detection of Streptococcus suis surface proteins.

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    Full Text Available Streptococcus suis serotype 2 (SS2 is a zoonotic pathogen that can cause infections in pigs and humans. Bacterial surface proteins are often investigated as potential vaccine candidates and biomarkers of virulence. In this study, a novel method for identifying bacterial surface proteins is presented, which combines immunoproteomic and immunoserologic techniques. Critical to the success of this new method is an improved procedure for generating two-dimensional electrophoresis gel profiles of S. suis proteins. The S. suis surface proteins identified in this study include muramidase-released protein precursor (MRP and an ABC transporter protein, while MRP is thought to be one of the main virulence factors in SS2 located on the bacterial surface. Herein, we demonstrate that the ABC transporter protein can bind to HEp-2 cells, which strongly suggests that this protein is located on the bacterial cell surface and may be involved in pathogenesis. An immunofluorescence assay confirmed that the ABC transporter is localized to the bacterial outer surface. This new method may prove to be a useful tool for identifying surface proteins, and aid in the development of new vaccine subunits and disease diagnostics.

  14. Proteomic analysis of cell surface-associated proteins from probiotic Lactobacillus plantarum

    DEFF Research Database (Denmark)

    Beck, Hans Christian; Madsen, Søren M; Glenting, Jacob;

    2009-01-01

    In the present study, we used a proteomic approach to identify surface-associated proteins from the probiotic bacterium Lactobacillus plantarum 299v. Proteins were extracted from the cell surface using a mild wash in phosphate buffer and analysed by sodium dodecyl sulphate-polyacrylamide gel...

  15. Effect of mechanical denaturation on surface free energy of protein powders.

    Science.gov (United States)

    Mohammad, Mohammad Amin; Grimsey, Ian M; Forbes, Robert T; Blagbrough, Ian S; Conway, Barbara R

    2016-10-01

    Globular proteins are important both as therapeutic agents and excipients. However, their fragile native conformations can be denatured during pharmaceutical processing, which leads to modification of the surface energy of their powders and hence their performance. Lyophilized powders of hen egg-white lysozyme and β-galactosidase from Aspergillus oryzae were used as models to study the effects of mechanical denaturation on the surface energies of basic and acidic protein powders, respectively. Their mechanical denaturation upon milling was confirmed by the absence of their thermal unfolding transition phases and by the changes in their secondary and tertiary structures. Inverse gas chromatography detected differences between both unprocessed protein powders and the changes induced by their mechanical denaturation. The surfaces of the acidic and basic protein powders were relatively basic, however the surface acidity of β-galactosidase was higher than that of lysozyme. Also, the surface of β-galactosidase powder had a higher dispersive energy compared to lysozyme. The mechanical denaturation decreased the dispersive energy and the basicity of the surfaces of both protein powders. The amino acid composition and molecular conformation of the proteins explained the surface energy data measured by inverse gas chromatography. The biological activity of mechanically denatured protein powders can either be reversible (lysozyme) or irreversible (β-galactosidase) upon hydration. Our surface data can be exploited to understand and predict the performance of protein powders within pharmaceutical dosage forms. PMID:27434157

  16. Protein sequences bound to mineral surfaces persist into deep time

    DEFF Research Database (Denmark)

    Demarchi, Beatrice; Hall, Shaun; Roncal-Herrero, Teresa;

    2016-01-01

    Proteins persist longer in the fossil record than DNA, but the longevity, survival mechanisms and substrates remain contested. Here, we demonstrate the role of mineral binding in preserving the protein sequence in ostrich (Struthionidae) eggshell, including from the palaeontological sites of Laet...

  17. Straightforward protein immobilization on Sylgard 184 PDMS microarray surface.

    Science.gov (United States)

    Heyries, Kevin A; Marquette, Christophe A; Blum, Loïc J

    2007-04-10

    In this work, a straightforward technique for protein immobilization on Sylgard 184 is described. The method consists of a direct transfer of dried protein/salt solutions to the PDMS interface during the polymer curing. Such non-conventional treatment of proteins was found to have no major negative consequence on their integrity. The mechanisms of this direct immobilization were investigated using a lysine modified dextran molecule as a model. Clear experimental results suggested that both chemical bounding and molding effect were implicated. As a proof of concept study, three different proteins were immobilized on a single microarray (Arachis hypogaea lectin, rabbit IgG, and human IgG) and used as antigens for capture of chemiluminescent immunoassays. The proteins were shown to be easily recognized by their specific antibodies, giving antibody detection limits in the fmol range.

  18. Influence of carvacrol and thymol on the physiological attributes, enterotoxin production and surface characteristics of Staphylococcus aureus strains isolated from foods

    Directory of Open Access Journals (Sweden)

    E.L. Souza

    2013-01-01

    Full Text Available This study evaluated the influence of the phenolic compounds carvacrol (CAR and thymol (THY on some physiological characteristics and on the modulation of the secretion of some staphylococcal virulence factors, that is, coagulase and enterotoxin. This study also investigated possible mechanisms for the establishment of the anti-staphylococcal activity of these compounds. Sublethal concentrations (0.3 and 0.15 µL/mL of CAR and THY inhibited the activity of the enzymes coagulase and lipase and led to a decrease in salt tolerance. At the tested sublethal concentrations, both CAR and THY led to a total suppression of enterotoxin production. The loss of a 260-nm-absorbing material and an efflux of potassium ions occurred immediately after the addition of CAR and THY at 0.6 and 1.2 µL/mL and increased up to 120 min of exposure. Electron microscopy of cells exposed to CAR and THY (0.6 µL/mL revealed that individual cells appeared to be deformed, with projections of cellular material. The observations of leakage of cellular material and an altered cell surface suggest that gross damage to a cell's cytoplasmic membrane, which results in a disruption in protein secretion, could be responsible for the anti-staphylococcal properties of CAR and THY.

  19. Characterization of methicillin-resistant Staphylococcus aureus Sequence Type 398

    DEFF Research Database (Denmark)

    Christiansen, Mette Theilgaard

    Staphylococcus aureus is an opportunistic pathogen that colonizes the nares and skin surfaces of several animal species, including man. S. aureus can cause a wide variety of infections ranging from superficial soft tissue and skin infections to severe and deadly systemic infections. Traditionally S....... aureus and methicillin-resistant Staphylococcus aureus (MRSA) have been associated with hospitals, but during the past decades MRSA has emerged in the community and now a new branch of MRSA has been found in association with livestock (LA-MRSA). A specific lineage (multilocus sequence type 398 (ST398...... for LA-MRSA ST398 survival on porcine skin and nasal epithelium ex vivo were identified. These genes could represent targets for de-colonization, which could help prevent further spread and adaption of LA-MRSA ST398. Manuscript III describes the construction of the S. aureus VirulenceFinder database...

  20. Surface characterization of proteins using multi-fractal property of heat-denatured aggregates

    Science.gov (United States)

    Lahiri, Tapobrata; Mishra, Hrishikesh; Sarkar, Subrata; Misra, Krishna

    2008-01-01

    Multi-fractal property of heat-denatured protein aggregates (HDPA) is characteristic of its individual form. The visual similarity between digitally generated microscopic images of HDPA with that of surface-image of its individual X-ray structures in protein databank (PDB) displayed using Visual Molecular Dynamics (VMD) viewer is the basis of the study. We deigned experiments to view the fractal nature of proteins at different aggregate scales. Intensity based multi-fractal dimensions (ILMFD) extracted from various planes of digital microscopic images of protein aggregates were used to characterize HDPA into different classes. Moreover, the ILMFD parameters extracted from aggregates show similar classification pattern to digital images of protein surface displayed by VMD viewer using PDB entry. We discuss the use of irregular patterns of heat-denatured aggregate proteins to understand various surface properties in native proteins. PMID:18795110

  1. Staphylococcus aureus Transcriptome Architecture

    DEFF Research Database (Denmark)

    Mäder, Ulrike; Nicolas, Pierre; Depke, Maren;

    2016-01-01

    Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S....... aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according...... to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19...

  2. Expression and Immunogenicity of Recombinant Immunoreactive Surface Protein 2 of Anaplasma phagocytophilum

    OpenAIRE

    Yu, Qiang; Chen, Chuang-Fu; Chen, Qiang; Zhang, Li-Juan

    2012-01-01

    Human granulocytic anaplasmosis (HGA), caused by Anaplasma phagocytophilum, is an emerging tick-borne zoonotic disease throughout the world. The first HGA cases in China were documented in 2008, and the greatest challenge posed by the disease is rapid and accurate diagnosis during the acute phage of illness. In this study, we successfully cloned and expressed an A. phagocytophilum immunoreactive surface protein (major surface protein 2 [MSP2]) and demonstrated that this recombinant protein ha...

  3. In Silico Study of Variable Surface Proteins in Plasmodium Species: Perspectives in Drug Design.

    Science.gov (United States)

    Yadav, Manoj Kumar; Swati, D

    2016-09-01

    The variable surface proteins expressed by P. falciparum and P. vivax are transported to the surface of infected erythrocyte and are exposed to the host immune system. The possibility of using variable surface proteins as a common drug target has been analyzed in both the Plasmodium species. Sequence analysis of variable surface proteins showed a low-level conservation within as well as between the species. Amino acid composition analysis revealed higher frequency of hydrophilic amino acids as compared with that of hydrophobic residues. In order to gain more insight into their diverse functional role, the three-dimensional structure was predicted using comparative modeling approach. These models were evaluated and validated by checking stereochemistry of underlying amino acids. Structural alignment of variable surface proteins by superimposing them shows less conservation. Due to differences at sequence as well as structural level, the variable surface proteins are expected to show difference in their degree of invasiveness. These differences were also cross-examined by evolutionary study, and the results obtained were in accordance with the aforesaid study. The existence of structural differences noticed in the present study showed that the variable surface proteins could not be used as a common drug target in both the malarial species. Therefore, species-specific strategy may be followed for drug targeting against variable surface proteins of P. falciparum and P. vivax. PMID:26253721

  4. In-cell thermodynamics and a new role for protein surfaces.

    Science.gov (United States)

    Smith, Austin E; Zhou, Larry Z; Gorensek, Annelise H; Senske, Michael; Pielak, Gary J

    2016-02-16

    There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge-charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.

  5. Structural determinants for protein adsorption/non-adsorption to silica surface

    International Nuclear Information System (INIS)

    The understanding of the mechanisms involved in the interaction of proteins with inorganic surfaces is of major interest in both fundamental research and applications such as nano-technology. However, despite intense research, the mechanisms and the structural determinants of protein/surface interactions are still unclear. We developed a strategy consisting in identifying, in a mixture of hundreds of soluble proteins, those proteins that are adsorbed on the surface and those that are not. If the two protein subsets are large enough, their statistical comparative analysis must reveal the physicochemical determinants relevant for adsorption versus non-adsorption. This methodology was tested with silica nanoparticles. We found that the adsorbed proteins contain a higher number of charged amino acids, particularly arginine, which is consistent with involvement of this basic amino acid in electrostatic interactions with silica. The analysis also identified a marked bias toward low aromatic amino acid content (phenylalanine, tryptophan, tyrosine and histidine) in adsorbed proteins. Structural analyses and molecular dynamics simulations of proteins from the two groups indicate that non-adsorbed proteins have twice as many p-p interactions and higher structural rigidity. The data are consistent with the notion that adsorption is correlated with the flexibility of the protein and with its ability to spread on the surface. Our findings led us to propose a refined model of protein adsorption. (authors)

  6. Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid

    International Nuclear Information System (INIS)

    Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface 125I-labeled proteins with a series of monoclonal antibodies (MAbs). Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with [35S] methionine, 14C-amino acids, or [3H] palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a large p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11

  7. Noninvasive noble metal nanoparticle arrays for surface-enhanced Raman spectroscopy of proteins

    Science.gov (United States)

    Inya-Agha, Obianuju; Forster, Robert J.; Keyes, Tia E.

    2007-02-01

    Noble metal nanoparticles arrays are well established substrates for surface enhanced Raman spectroscopy (SERS). Their ability to enhance optical fields is based on the interaction of their surface valence electrons with incident electromagnetic radiation. In the array configuration, noble metal nanoparticles have been used to produce SER spectral enhancements of up to 10 8 orders of magnitude, making them useful for the trace analysis of physiologically relevant analytes such as proteins and peptides. Electrostatic interactions between proteins and metal surfaces result in the preferential adsorption of positively charged protein domains onto metal surfaces. This preferential interaction has the effect of disrupting the native conformation of the protein fold, with a concomitant loss of protein function. A major historic advantage of Raman microspectroscopy has been is its non-invasive nature; protein denaturation on the metal surfaces required for SER spectroscopy renders it a much more invasive technique. Further, part of the analytical power of Raman spectroscopy lies in its use as a secondary conformation probe. The protein structural loss which occurs on the metal surface results in secondary conformation readings which are not true to the actual native state of the analyte. This work presents a method for chemical fabrication of noble metal SERS arrays with surface immobilized layers which can protect protein native conformation without excessively mitigating the electromagnetic enhancements of spectra. Peptide analytes are used as model systems for proteins. Raman spectra of alpha lactalbumin on surfaces and when immobilized on these novel arrays are compared. We discuss the ability of the surface layer to protect protein structure whilst improving signal intensity.

  8. Selective labelling of cell-surface proteins using CyDye DIGE Fluor minimal dyes.

    Science.gov (United States)

    Hagner-McWhirter, Asa; Winkvist, Maria; Bourin, Stephanie; Marouga, Rita

    2008-01-01

    Surface proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan DIGE technology and analysis of protein expression changes using DeCyder 2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

  9. Staphylococcus aureus intestinal colonization is associated with increased frequency of S. aureus on skin of hospitalized patients

    Directory of Open Access Journals (Sweden)

    Donskey Curtis J

    2007-09-01

    Full Text Available Abstract Background Intestinal colonization by Staphylococcus aureus among hospitalized patients has been associated with increased risk of staphylococcal infection and could potentially contribute to transmission. We hypothesized that S. aureus intestinal colonization is associated with increased frequency of S. aureus on patients' skin and nearby environmental surfaces. Methods Selected inpatients were cultured weekly for S. aureus from stool, nares, skin (groin and axilla, and environmental surfaces (bed rail and bedside table. Investigator's hands were cultured after contacting the patients' skin and the environmental surfaces. Results Of 71 subjects, 32 (45.1% had negative nares and stool cultures, 23 (32.4% had positive nares and stool cultures, 13 (18.3% were nares carriers only, and 3 (4.2% were stool carriers only. Of the 39 patients with S. aureus carriage, 30 (76.9% had methicillin-resistant isolates. In comparison to nares colonization only, nares and intestinal colonization was associated with increased frequency of positive skin cultures (41% versus 77%; p = 0.001 and trends toward increased environmental contamination (45% versus 62%; p = 0.188 and acquisition on investigator's hands (36% versus 60%; p = 0.057. Patients with negative nares and stool cultures had low frequency of S. aureus on skin and the environment (4.8% and 11.3%, respectively. Conclusion We found that hospitalized patients with S. aureus nares and/or stool carriage frequently had S. aureus on their skin and on nearby environmental surfaces. S. aureus intestinal colonization was associated with increased frequency of positive skin cultures, which could potentially facilitate staphylococcal infections and nosocomial transmission.

  10. Silica as a Matrix for Encapsulating Proteins: Surface Effects on Protein Structure Assessed by Circular Dichroism Spectroscopy

    Directory of Open Access Journals (Sweden)

    Genet H. Zemede

    2012-08-01

    Full Text Available The encapsulation of biomolecules in solid materials that retain the native properties of the molecule is a desired feature for the development of biosensors and biocatalysts. In the current study, protein entrapment in silica-based materials is explored using the sol-gel technique. This work surveys the effects of silica confinement on the structure of several model polypeptides, including apomyoglobin, copper-zinc superoxide dismutase, polyglutamine, polylysine, and type I antifreeze protein. Changes in the secondary structure of each protein following encapsulation are monitored by circular dichroism spectroscopy. In many cases, silica confinement reduces the fraction of properly-folded protein relative to solution, but addition of a secondary solute or modification of the silica surface leads to an increase in structure. Refinement of the glass surface by addition of a monosubstituted alkoxysilane during sol-gel processing is shown to be a valuable tool for testing the effects of surface chemistry on protein structure. Because silica entrapment prevents protein aggregation by isolating individual protein molecules in the pores of the glass material, one may monitor aggregation-prone polypeptides under solvent conditions that are prohibited in solution, as demonstrated with polyglutamine and a disease-related variant of superoxide dismutase.

  11. 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.

  12. Identification of Posttranslational Modification-Dependent Protein Interactions Using Yeast Surface Displayed Human Proteome Libraries.

    Science.gov (United States)

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    The identification of proteins that interact specifically with posttranslational modifications such as phosphorylation is often necessary to understand cellular signaling pathways. Numerous methods for identifying proteins that interact with posttranslational modifications have been utilized, including affinity-based purification and analysis, protein microarrays, phage display, and tethered catalysis. Although these techniques have been used successfully, each has limitations. Recently, yeast surface-displayed human proteome libraries have been utilized to identify protein fragments with affinity for various target molecules, including phosphorylated peptides. When coupled with fluorescently activated cell sorting and high throughput methods for the analysis of selection outputs, yeast surface-displayed human proteome libraries can rapidly and efficiently identify protein fragments with affinity for any soluble ligand that can be fluorescently detected, including posttranslational modifications. In this review we compare the use of yeast surface display libraries to other methods for the identification of interactions between proteins and posttranslational modifications and discuss future applications of the technology. PMID:26060076

  13. Surface derivatization strategy for combinatorial analysis of cell response to mixtures of protein domains.

    Science.gov (United States)

    Chiang, Chunyi; Karuri, Stella W; Kshatriya, Pradnya P; Schwartz, Jeffrey; Schwarzbauer, Jean E; Karuri, Nancy W

    2012-01-10

    We report a robust strategy for conjugating mixtures of two or more protein domains to nonfouling polyurethane surfaces. In our strategy, the carbamate groups of polyurethane are reacted with zirconium alkoxide from the vapor phase to give a surface-bound oxide that serves as a chemical layer that can be used to bond organics to the polymer substrate. A hydroxyalkylphosphonate monolayer was synthesized on this layer, which was then used to covalently bind primary amine groups in protein domains using chloroformate-derived cross-linking. The effectiveness of this synthesis strategy was gauged by using an ELISA to measure competitive, covalent bonding of cell-binding (III(9-10)) and fibronectin-binding (III(1-2)) domains of the cell adhesion protein fibronectin. Cell adhesion, spreading, and fibronectin matrix assembly were examined on surfaces conjugated with single domains, a 1:1 surface mixture of III(1-2) and III(9-10), and a recombinant protein "duplex" containing both domains in one fusion protein. The mixture performed as well as or better than the other surfaces in these assays. Our surface activation strategy is amenable to a wide range of polymer substrates and free amino group-containing protein fragments. As such, this technique may be used to create biologically specific materials through the immobilization of specific protein groups or mixtures thereof on a substrate surface.

  14. New developments for the site-specific attachment of protein to surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A

    2005-05-12

    Protein immobilization on surfaces is of great importance in numerous applications in biology and biophysics. The key for the success of all these applications relies on the immobilization technique employed to attach the protein to the corresponding surface. Protein immobilization can be based on covalent or noncovalent interaction of the molecule with the surface. Noncovalent interactions include hydrophobic interactions, hydrogen bonding, van der Waals forces, electrostatic forces, or physical adsorption. However, since these interactions are weak, the molecules can get denatured or dislodged, thus causing loss of signal. They also result in random attachment of the protein to the surface. Site-specific covalent attachment of proteins onto surfaces, on the other hand, leads to molecules being arranged in a definite, orderly fashion and uses spacers and linkers to help minimize steric hindrances between the protein surface. This work reviews in detail some of the methods most commonly used as well as the latest developments for the site-specific covalent attachment of protein to solid surfaces.

  15. Multidimensional profiling of cell surface proteins and nuclear markers

    Energy Technology Data Exchange (ETDEWEB)

    Han, Ju; Chang, Hang; Andarawewa, Kumari; Yaswen, Paul; Helen Barcellos-Hoff, Mary; Parvin, Bahram

    2009-01-30

    Cell membrane proteins play an important role in tissue architecture and cell-cell communication. We hypothesize that segmentation and multidimensional characterization of the distribution of cell membrane proteins, on a cell-by-cell basis, enable improved classification of treatment groups and identify important characteristics that can otherwise be hidden. We have developed a series of computational steps to (i) delineate cell membrane protein signals and associate them with a specific nucleus; (ii) compute a coupled representation of the multiplexed DNA content with membrane proteins; (iii) rank computed features associated with such a multidimensional representation; (iv) visualize selected features for comparative evaluation through heatmaps; and (v) discriminate between treatment groups in an optimal fashion. The novelty of our method is in the segmentation of the membrane signal and the multidimensional representation of phenotypic signature on a cell-by-cell basis. To test the utility of this method, the proposed computational steps were applied to images of cells that have been irradiated with different radiation qualities in the presence and absence of other small molecules. These samples are labeled for their DNA content and E-cadherin membrane proteins. We demonstrate that multidimensional representations of cell-by-cell phenotypes improve predictive and visualization capabilities among different treatment groups, and identify hidden variables.

  16. Protein immobilization capacity and covalent binding coverage of pulsed plasma polymer surfaces

    International Nuclear Information System (INIS)

    Three carbon surfaces were deposited using pulsed plasma enhanced chemical vapour deposition method: a low and a high nitrogen-containing plasma polymer surfaces and a diamond-like carbon surface. The surfaces were analysed using both X-ray photoelectron spectroscopy (XPS) technique and the enzyme-linked immunosorbent assay (ELISA) method combining with sodium dodecyl sulphate (SDS) cleaning to investigate the capacity and covalent binding of the immobilized proteins. A good correlation was found on quantification of remaining protein after SDS cleaning using the ELISA method and the XPS technique. All surfaces had similar initial capacity of protein attachment but with large different resistance to SDS cleaning. The analysis showed that the high nitrogen-containing plasma polymer was the best biocompatible material due to its highest resistance to SDS cleaning, i.e. with the highest quantity (∼80%) of proteins bound covalently.

  17. Lipidation Effect on Surface Adsorption and Associated Fibrillation of the Model Protein Insulin.

    Science.gov (United States)

    Hedegaard, Sofie Fogh; Cárdenas, Marité; Barker, Robert; Jorgensen, Lene; van de Weert, Marco

    2016-07-19

    Lipidation of proteins is used in the pharmaceutical field to increase the therapeutic efficacy of proteins. In this study, we investigate the effect of a 14-carbon fatty acid modification on the adsorption behavior of human insulin to a hydrophobic solid surface and the subsequent fibrillation development under highly acidic conditions and elevated temperature by comparing to the fibrillation of human insulin. At these stressed conditions, the lipid modification accelerates the rate of fibrillation in bulk solution. With the use of several complementary surface-sensitive techniques, including quartz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM), and neutron reflectivity (NR), we show that there are two levels of structurally different protein organization at a hydrophobic surface for both human insulin and the lipidated analogue: a dense protein layer formed within minutes on the surface and a diffuse outer layer of fibrillar structures which took hours to form. The two layers may only be weakly connected, and proteins from both layers are able to desorb from the surface. The lipid modification increases the protein surface coverage and the thickness of both layer organizations. Upon lipidation not only the fibrillation extent but also the morphology of the fibrillar structures changes from fibril clusters on the surface to a more homogeneous network of fibrils covering the entire hydrophobic surface. PMID:27348237

  18. Association of lipids with integral membrane surface proteins of Mycoplasma hyorhinis

    International Nuclear Information System (INIS)

    Triton X-114 (TX-114)-phase fractionation was used to identify and characterize integral membrane surface proteins of the wall-less procaryote Mycoplasma hyorhinis GDL. Phase fractionation of mycoplasmas followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed selective partitioning of approximately 30 [35S]methionine-labeled intrinsic membrane proteins into the TX-114 phase. Similar analysis of [3H]palmitate-labeled cells showed that approximately 20 proteins of this organism were associated with lipid, all of which also efficiently partitioned as integral membrane components into the detergent phase. Immunoblotting and immunoprecipitation of TX-114-phase proteins from 125I-surface-labeled cells with four monoclonal antibodies to distinct surface epitopes of M. hyorhinis identified surface proteins p120, p70, p42, and p23 as intrinsic membrane components. Immunoprecipitation of [3H]palmitate-labeled TX-114-phase proteins further established that surface proteins p120, p70, and p23 (a molecule that mediates complement-dependent mycoplasmacidal monoclonal antibody activity) were among the lipid-associated proteins of this organism. Two of these proteins, p120 and p123, were acidic (pI less than or equal to 4.5), as shown by two-dimensional isoelectric focusing. This study established that M. hyorhinis contains an abundance of integral membrane proteins tightly associated with lipids and that many of these proteins are exposed at the external surface of the single limiting plasma membrane. Monoclonal antibodies are reported that will allow detailed analysis of the structure and processing of lipid-associated mycoplasma proteins

  19. Role of Monocytes in Experimental Staphylococcus aureus Endocarditis

    OpenAIRE

    Veltrop, Marcel H. A. M.; Bancsi, Maurice J. L. M. F.; Bertina, Rogier M.; Thompson, Jan

    2000-01-01

    In the pathogenesis of bacterial endocarditis (BE), the clotting system plays a cardinal role in the formation and maintenance of the endocardial vegetations. The extrinsic pathway is involved in the activation of the coagulation pathway with tissue factor (TF) as the key protein. Staphylococcus aureus is a frequently isolated bacterium from patients with BE. We therefore investigated whether S. aureus can induce TF activity (TFA) on fibrin-adherent monocytes, used as an in vitro model of BE....

  20. Evaluation of Protein Adsorption on Chitosan Surfaces with Reflectometry Interference Spectroscopy

    Directory of Open Access Journals (Sweden)

    Chao Qun Ma

    2001-10-01

    Full Text Available Using a biomedical sensor setup RIfS we have investigated the kinetic behavior of human albumin (Alb, human fibrinogen (Fib, and human immunoglobulin G (IgG adsorbed onto surfaces of chitosan. Polystyrene (PS was used as the control material in this study. The optical thickness of three kinds of proteins measured by RIfS was related to their molecular dimensions and potential orientations on a film surface. According to the operation principle of RIfS and the molecular dimensions of three kinds of proteins, the adsorbed layers of proteins onto the surface of chitosan and PS was calculated by using a newly introduced equation. The microstructure of the chitosan and polystyrene film and the surfaces with adsorbed proteins were imaged by atomic force microscopy (AFM. With AFM analyses the lateral distribution of the protein molecules on surfaces have been recognized. The results show that the number of adsorbed layers of the three proteins on the surface of chitosan are 0.635 for Alb, 0.158 for Fib and 0.0967 for IgG, and of polystyrene are: 0.577 for IgG, 0.399 for Fib, 0.336 for Alb. This study confirmed that RIfS is a useful tool for the analysis of plasma proteins adsorbed on a surface of biomaterials. Results show that at first on the surface of chitosan film much more Alb than Fib was adsorbed which demonstrated that chitosan has a antithrombus function. Secondly, on the surface of chitosan film more Alb and less Fib were adsorbed than on the surface of PS film, which demonstrated that chitosan has a better blood compatibility than polystyrene. Thirdly, the calculated layer number of the three proteins indicated that on both chitosan and PS substrates monolayer coatings form.

  1. Surface modification of diamond-like carbon films with protein via polydopamine inspired coatings

    Energy Technology Data Exchange (ETDEWEB)

    Tao Caihong [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Tianshui Middle Road 18th, Lanzhou 730000 (China); China and Graduate University of Chinese Academy of Sciences, Beijing 100080 (China); Yang Shengrong [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Tianshui Middle Road 18th, Lanzhou 730000 (China); Zhang Junyan, E-mail: zhangjunyan@lzb.ac.cn [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Tianshui Middle Road 18th, Lanzhou 730000 (China); Wang Jinqing [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Tianshui Middle Road 18th, Lanzhou 730000 (China)

    2009-10-15

    In this paper, we report a facile two-step approach to immobilize proteins onto DLC surfaces. The first step was a simple immersion of DLC in a solution of dopamine. Polydopamine was deposited on DLC as a stable anchor to present protein molecules. Then the protein ad-layer was deposited on it. The chemical components of the modified DLC surfaces were characterized by Fourier transform infrared spectra and X-ray photoelectron spectroscopy. The biocompatibility of it was evaluated in vitro by the tetrazolium salt method. And it was indicated that the BSA modified surface had good haemocompatibility properties, and was cytocompatible to PC-12 cells.

  2. Surface modification of diamond-like carbon films with protein via polydopamine inspired coatings

    International Nuclear Information System (INIS)

    In this paper, we report a facile two-step approach to immobilize proteins onto DLC surfaces. The first step was a simple immersion of DLC in a solution of dopamine. Polydopamine was deposited on DLC as a stable anchor to present protein molecules. Then the protein ad-layer was deposited on it. The chemical components of the modified DLC surfaces were characterized by Fourier transform infrared spectra and X-ray photoelectron spectroscopy. The biocompatibility of it was evaluated in vitro by the tetrazolium salt method. And it was indicated that the BSA modified surface had good haemocompatibility properties, and was cytocompatible to PC-12 cells.

  3. DARC: Mapping Surface Topography by Ray-Casting for Effective Virtual Screening at Protein Interaction Sites.

    Science.gov (United States)

    Gowthaman, Ragul; Miller, Sven A; Rogers, Steven; Khowsathit, Jittasak; Lan, Lan; Bai, Nan; Johnson, David K; Liu, Chunjing; Xu, Liang; Anbanandam, Asokan; Aubé, Jeffrey; Roy, Anuradha; Karanicolas, John

    2016-05-12

    Protein-protein interactions represent an exciting and challenging target class for therapeutic intervention using small molecules. Protein interaction sites are often devoid of the deep surface pockets presented by "traditional" drug targets, and crystal structures reveal that inhibitors typically engage these sites using very shallow binding modes. As a consequence, modern virtual screening tools developed to identify inhibitors of traditional drug targets do not perform as well when they are instead deployed at protein interaction sites. To address the need for novel inhibitors of important protein interactions, here we introduce an alternate docking strategy specifically designed for this regime. Our method, termed DARC (Docking Approach using Ray-Casting), matches the topography of a surface pocket "observed" from within the protein to the topography "observed" when viewing a potential ligand from the same vantage point. We applied DARC to carry out a virtual screen against the protein interaction site of human antiapoptotic protein Mcl-1 and found that four of the top-scoring 21 compounds showed clear inhibition in a biochemical assay. The Ki values for these compounds ranged from 1.2 to 21 μM, and each had ligand efficiency comparable to promising small-molecule inhibitors of other protein-protein interactions. These hit compounds do not resemble the natural (protein) binding partner of Mcl-1, nor do they resemble any known inhibitors of Mcl-1. Our results thus demonstrate the utility of DARC for identifying novel inhibitors of protein-protein interactions. PMID:26126123

  4. 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.

  5. Synthesis and evaluation of a conjugate vaccine composed of Staphylococcus aureus poly-N-acetyl-glucosamine and clumping factor A.

    Directory of Open Access Journals (Sweden)

    Tomás Maira-Litrán

    Full Text Available The increasing frequency, severity and antimicrobial resistance of Staphylococcus aureus infections has made the development of immunotherapies against this pathogen more urgent than ever. Previous immunization attempts using monovalent antigens resulted in at best partial levels of protection against S. aureus infection. We therefore reasoned that synthesizing a bivalent conjugate vaccine composed of two widely expressed antigens of S. aureus would result in additive/synergetic activities by antibodies to each vaccine component and/or in increased strain coverage. For this we used reductive amination, to covalently link the S. aureus antigens clumping factor A (ClfA and deacetylated poly-N-β-(1-6-acetyl-glucosamine (dPNAG. Mice immunized with 1, 5 or 10 µg of the dPNAG-ClfA conjugate responded in a dose-dependent manner with IgG to dPNAG and ClfA, whereas mice immunized with a mixture of ClfA and dPNAG developed significantly lower antibody titers to ClfA and no antibodies to PNAG. The dPNAG-ClfA vaccine was also highly immunogenic in rabbits, rhesus monkeys and a goat. Moreover, affinity-purified, antibodies to ClfA from dPNAG-ClfA immune serum blocked the binding of three S. aureus strains to immobilized fibrinogen. In an opsonophagocytic assay (OPKA goat antibodies to dPNAG-ClfA vaccine, in the presence of complement and polymorphonuclear cells, killed S. aureus Newman and, to a lower extent, S. aureus Newman ΔclfA. A PNAG-negative isogenic mutant was not killed. Moreover, PNAG antigen fully inhibited the killing of S. aureus Newman by antisera to dPNAG-ClfA vaccine. Finally, mice passively vaccinated with goat antisera to dPNAG-ClfA or dPNAG-diphtheria toxoid conjugate had comparable levels of reductions of bacteria in the blood 2 h after infection with three different S. aureus strains as compared to mice given normal goat serum. In conclusion, ClfA is an immunogenic carrier protein that elicited anti-adhesive antibodies that fail to

  6. Quantitative evaluation of interaction force between functional groups in protein and polymer brush surfaces.

    Science.gov (United States)

    Sakata, Sho; Inoue, Yuuki; Ishihara, Kazuhiko

    2014-03-18

    To understand interactions between polymer surfaces and different functional groups in proteins, interaction forces were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Various polymer brush surfaces were systematically prepared by surface-initiated atom transfer radical polymerization as well-defined model surfaces to understand protein adsorption behavior. The polymer brush layers consisted of phosphorylcholine groups (zwitterionic/hydrophilic), trimethylammonium groups (cationic/hydrophilic), sulfonate groups (anionic/hydrophilic), hydroxyl groups (nonionic/hydrophilic), and n-butyl groups (nonionic/hydrophobic) in their side chains. The interaction forces between these polymer brush surfaces and different functional groups (carboxyl groups, amino groups, and methyl groups, which are typical functional groups existing in proteins) were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Furthermore, the amount of adsorbed protein on the polymer brush surfaces was quantified by surface plasmon resonance using albumin with a negative net charge and lysozyme with a positive net charge under physiological conditions. The amount of proteins adsorbed on the polymer brush surfaces corresponded to the interaction forces generated between the functional groups on the cantilever and the polymer brush surfaces. The weakest interaction force and least amount of protein adsorbed were observed in the case of the polymer brush surface with phosphorylcholine groups in the side chain. On the other hand, positive and negative surfaces generated strong forces against the oppositely charged functional groups. In addition, they showed significant adsorption with albumin and lysozyme, respectively. These results indicated that the interaction force at the functional group level might be

  7. Staphylococcus aureus vaccines: Deviating from the carol.

    Science.gov (United States)

    Missiakas, Dominique; Schneewind, Olaf

    2016-08-22

    Staphylococcus aureus, a commensal of the human nasopharynx and skin, also causes invasive disease, most frequently skin and soft tissue infections. Invasive disease caused by drug-resistant strains, designated MRSA (methicillin-resistant S. aureus), is associated with failure of antibiotic therapy and elevated mortality. Here we review polysaccharide-conjugate and subunit vaccines that were designed to prevent S. aureus infection in patients at risk of bacteremia or surgical wound infection but failed to reach their clinical endpoints. We also discuss vaccines with ongoing trials for combinations of polysaccharide-conjugates and subunits. S. aureus colonization and invasive disease are not associated with the development of protective immune responses, which is attributable to a large spectrum of immune evasion factors. Two evasive strategies, assembly of protective fibrin shields via coagulases and protein A-mediated B cell superantigen activity, are discussed as possible vaccine targets. Although correlates for protective immunity are not yet known, opsonophagocytic killing of staphylococci by phagocytic cells offers opportunities to establish such criteria. PMID:27526714

  8. Shotgun proteomic analytical approach for studying proteins adsorbed onto liposome surface

    KAUST Repository

    Capriotti, Anna Laura

    2011-07-02

    The knowledge about the interaction between plasma proteins and nanocarriers employed for in vivo delivery is fundamental to understand their biodistribution. Protein adsorption onto nanoparticle surface (protein corona) is strongly affected by vector surface characteristics. In general, the primary interaction is thought to be electrostatic, thus surface charge of carrier is supposed to play a central role in protein adsorption. Because protein corona composition can be critical in modifying the interactive surface that is recognized by cells, characterizing its formation onto lipid particles may serve as a fundamental predictive model for the in vivo efficiency of a lipidic vector. In the present work, protein coronas adsorbed onto three differently charged cationic liposome formulations were compared by a shotgun proteomic approach based on nano-liquid chromatography-high-resolution mass spectrometry. About 130 proteins were identified in each corona, with only small differences between the different cationic liposome formulations. However, this study could be useful for the future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins into body fluids. © 2011 Springer-Verlag.

  9. Electrochemical Characterization of Protein Adsorption onto YNGRT-Au and VLGXE-Au Surfaces

    Directory of Open Access Journals (Sweden)

    Hanna Trzeciakiewicz

    2015-08-01

    Full Text Available The adsorption of the proteins CD13, mucin and bovine serum albumin on VLGXE-Au and YNGRT-Au interfaces was monitored by electrochemical impedance spectroscopy in the presence of [Fe(CN6]3−/4−. The hydrophobicity of the Au surface was tailored using specific peptides, blocking agents and diluents. The combination of blocking agents (ethanolamine or n-butylamine and diluents (hexanethiol or 2-mercaptoethanol was used to prepare various peptide-modified Au surfaces. Protein adsorption onto the peptide-Au surfaces modified with the combination of n-butylamine and hexanethiol produced a dramatic decrease in the charge transfer resistance, Rct, for all three proteins. In contrast, polar peptide-surfaces induced a minimal change in Rct for all three proteins. Furthermore, an increase in Rct was observed with CD13 (an aminopeptidase overexpressed in certain cancers in comparison to the other proteins when the VLGXE-Au surface was modified with n-butylamine as a blocking agent. The electrochemical data indicated that protein adsorption may be modulated by tailoring the peptide sequence on Au surfaces and that blocking agents and diluents play a key role in promoting or preventing protein adsorption. The peptide-Au platform may also be used for targeting cancer biomarkers with designer peptides.

  10. Electrochemical Characterization of Protein Adsorption onto YNGRT-Au and VLGXE-Au Surfaces.

    Science.gov (United States)

    Trzeciakiewicz, Hanna; Esteves-Villanueva, Jose; Soudy, Rania; Kaur, Kamaljit; Martic-Milne, Sanela

    2015-01-01

    The adsorption of the proteins CD13, mucin and bovine serum albumin on VLGXE-Au and YNGRT-Au interfaces was monitored by electrochemical impedance spectroscopy in the presence of [Fe(CN)6](3-/4-). The hydrophobicity of the Au surface was tailored using specific peptides, blocking agents and diluents. The combination of blocking agents (ethanolamine or n-butylamine) and diluents (hexanethiol or 2-mercaptoethanol) was used to prepare various peptide-modified Au surfaces. Protein adsorption onto the peptide-Au surfaces modified with the combination of n-butylamine and hexanethiol produced a dramatic decrease in the charge transfer resistance, Rct, for all three proteins. In contrast, polar peptide-surfaces induced a minimal change in Rct for all three proteins. Furthermore, an increase in Rct was observed with CD13 (an aminopeptidase overexpressed in certain cancers) in comparison to the other proteins when the VLGXE-Au surface was modified with n-butylamine as a blocking agent. The electrochemical data indicated that protein adsorption may be modulated by tailoring the peptide sequence on Au surfaces and that blocking agents and diluents play a key role in promoting or preventing protein adsorption. The peptide-Au platform may also be used for targeting cancer biomarkers with designer peptides. PMID:26262621

  11. Molecular interaction forces generated during protein adsorption to well-defined polymer brush surfaces.

    Science.gov (United States)

    Sakata, Sho; Inoue, Yuuki; Ishihara, Kazuhiko

    2015-03-17

    The molecular interaction forces generated during the adsorption of proteins to surfaces were examined by the force-versus-distance (f-d) curve measurements of atomic force microscopy using probes modified with appropriate molecules. Various substrates with polymer brush layers bearing zwitterionic, cationic, anionic, and hydrophobic groups were systematically prepared by surface-initiated atom transfer radical polymerization. Surface interaction forces on these substrates were analyzed by the f-d curve measurements using probes with the same polymer brush layer as the substrate. Repulsive forces, which decreased depending on the ionic strength, were generated between cationic or anionic polyelectrolyte brush layers; these were considered to be electrostatic interaction forces. A strong adhesive force was detected between hydrophobic polymer brush layers during retraction; this corresponded to the hydrophobic interaction between two hydrophobic polymer layers. In contrast, no significant interaction forces were detected between zwitterionic polymer brush layers. Direct interaction forces between proteins and polymer brush layers were then quantitatively evaluated by the f-d curve measurements using protein-immobilized probes consisting of negatively charged albumin and positively charged lysozyme under physiological conditions. In addition, the amount of protein adsorbed on the polymer brush layer was quantified by surface plasmon resonance measurements. Relatively large amounts of protein adsorbed to the polyelectrolyte brush layers with opposite charges. It was considered that the detachment of the protein after contact with the polymer brush layer hardly occurred due to salt formation at the interface. Both proteins adsorbed significantly on the hydrophobic polymer brush layer, which was due to hydrophobic interactions at the interface. In contrast, the zwitterionic polymer brush layer exhibited no significant interaction force with proteins and suppressed

  12. Engineering and Characterization of Peptides and Proteins at Surfaces and Interfaces: A Case Study in Surface-Sensitive Vibrational Spectroscopy.

    Science.gov (United States)

    Ding, Bei; Jasensky, Joshua; Li, Yaoxin; Chen, Zhan

    2016-06-21

    Understanding molecular structures of interfacial peptides and proteins impacts many research fields by guiding the advancement of biocompatible materials, new and improved marine antifouling coatings, ultrasensitive and highly specific biosensors and biochips, therapies for diseases related to protein amyloid formation, and knowledge on mechanisms for various membrane proteins and their interactions with ligands. Developing methods for measuring such unique systems, as well as elucidating the structure and function relationship of such biomolecules, has been the goal of our lab at the University of Michigan. We have made substantial progress to develop sum frequency generation (SFG) vibrational spectroscopy into a powerful technique to study interfacial peptides and proteins, which lays a foundation to obtain unique and valuable insights when using SFG to probe various biologically relevant systems at the solid/liquid interface in situ in real time. One highlighting feature of this Account is the demonstration of the power of combining SFG with other techniques and methods such as ATR-FTIR, surface engineering, MD simulation, liquid crystal sensing, and isotope labeling in order to study peptides and proteins at interfaces. It is necessary to emphasize that SFG plays a major role in these studies, while other techniques and methods are supplemental. The central role of SFG is to provide critical information on interfacial peptide and protein structure (e.g., conformation and orientation) in order to elucidate how surface engineering (e.g., to vary the structure) can ultimately affect surface function (e.g., to optimize the activity). This Account focuses on the most significant recent progress in research on interfacial peptides and proteins carried out by our group including (1) the development of SFG analysis methods to determine orientations of regular as well as disrupted secondary structures, and the successful demonstration and application of an isotope

  13. Protein overexport in a Saccharomyces cerevisiae mutant is not due to facilitated release of cell-surface proteins.

    Science.gov (United States)

    Alexieva, K I; Venkov, P V

    2000-01-01

    Saccharomyces cerevisiae strain MW11 is a temperature-sensitive mutant which exports twenty times more proteins at 37 degrees C than parental or wild-type strains do. To understand the mechanism underlying the protein overexport in the mutant the possibility of an altered cell-wall structure leading to facilitated release of cell-surface proteins was studied. Data on calcofluor white and zymolyase sensitivities, resistance to killer 1 toxin and determination of exported acid phosphatase and invertase did not provide evidence for alterations in the cell-wall structure that could explain the protein overexport phenotype. The results were obtained in experiments when transcription of mutated gene was discontinued which permits the full expression of the protein overexport phenotype.

  14. Modeling and simulation of protein-surface interactions: achievements and challenges.

    Science.gov (United States)

    Ozboyaci, Musa; Kokh, Daria B; Corni, Stefano; Wade, Rebecca C

    2016-01-01

    Understanding protein-inorganic surface interactions is central to the rational design of new tools in biomaterial sciences, nanobiotechnology and nanomedicine. Although a significant amount of experimental research on protein adsorption onto solid substrates has been reported, many aspects of the recognition and interaction mechanisms of biomolecules and inorganic surfaces are still unclear. Theoretical modeling and simulations provide complementary approaches for experimental studies, and they have been applied for exploring protein-surface binding mechanisms, the determinants of binding specificity towards different surfaces, as well as the thermodynamics and kinetics of adsorption. Although the general computational approaches employed to study the dynamics of proteins and materials are similar, the models and force-fields (FFs) used for describing the physical properties and interactions of material surfaces and biological molecules differ. In particular, FF and water models designed for use in biomolecular simulations are often not directly transferable to surface simulations and vice versa. The adsorption events span a wide range of time- and length-scales that vary from nanoseconds to days, and from nanometers to micrometers, respectively, rendering the use of multi-scale approaches unavoidable. Further, changes in the atomic structure of material surfaces that can lead to surface reconstruction, and in the structure of proteins that can result in complete denaturation of the adsorbed molecules, can create many intermediate structural and energetic states that complicate sampling. In this review, we address the challenges posed to theoretical and computational methods in achieving accurate descriptions of the physical, chemical and mechanical properties of protein-surface systems. In this context, we discuss the applicability of different modeling and simulation techniques ranging from quantum mechanics through all-atom molecular mechanics to coarse

  15. The S. aureus polysaccharide capsule and Efb-dependent fibrinogen shield act in concert to protect against phagocytosis

    Science.gov (United States)

    Kuipers, Annemarie; Stapels, Daphne A. C.; Weerwind, Lleroy T.; Ko, Ya-Ping; Ruyken, Maartje; Lee, Jean C.; van Kessel, Kok P.M.; Rooijakkers, Suzan H. M.

    2016-01-01

    Staphylococcus aureus has developed many mechanisms to escape from human immune responses. In order to resist phagocytic clearance, S. aureus expresses a polysaccharide capsule, which effectively masks the bacterial surface and surface-associated proteins, such as opsonins, from recognition by phagocytic cells. Additionally, secretion of the Extracellular fibrinogen binding protein (Efb) potently blocks phagocytic uptake of the pathogen. Efb creates a fibrinogen shield surrounding the bacteria by simultaneously binding complement C3b and fibrinogen at the bacterial surface. By means of neutrophil phagocytosis assays with fluorescently labeled encapsulated serotype 5 (CP5) and serotype 8 (CP8) strains we now compare the immune-modulating function of these shielding mechanisms. Our data indicate that, in highly encapsulated S. aureus strains, the polysaccharide capsule is able to prevent phagocytic uptake at plasma concentrations <10%, but loses its protective ability at higher concentrations of plasma. Interestingly, Efb shows a strong inhibitory effect on both capsule-negative as well as encapsulated strains at all tested plasma concentrations. Furthermore our results suggest that both shielding mechanisms can exist simultaneously and collaborate to provide optimal protection against phagocytosis at a broad range of plasma concentrations. Since opsonizing antibodies will be shielded from recognition by either mechanism, incorporating both capsular polysaccharides and Efb in future vaccines could be of great importance. PMID:27112346

  16. Surface-protein interactions on different stainless steel grades: effects of protein adsorption, surface changes and metal release

    OpenAIRE

    Hedberg, Y; Wang, X.; Hedberg, J; Lundin, M.; Blomberg, E.; Odnevall Wallinder, I.

    2013-01-01

    Implantation using stainless steels (SS) is an example where an understanding of protein-induced metal release from SS is important when assessing potential toxicological risks. Here, the protein-induced metal release was investigated for austenitic (AISI 304, 310, and 316L), ferritic (AISI 430), and duplex (AISI 2205) grades in a phosphate buffered saline (PBS, pH 7.4) solution containing either bovine serum albumin (BSA) or lysozyme (LSZ). The results show that both BSA and LSZ induce a sig...

  17. Surface Plasmon Resonance for Rapid Screening of Uranyl Affine Proteins

    International Nuclear Information System (INIS)

    A sensitive immunoassay based on SPR analysis was developed to measure uranyl cation (UO22+) affinity for any protein in a free state under physiological conditions. The technique involves immobilization of a specific monoclonal antibody (mAb) raised against UO22+ and 1, 10-phenanthroline-2, 9-dicarboxylic acid (DCP) used as a probe of UO22+ captured by the mAb. Calibration curves were established for accurate determination of UO22+ concentrations with a detection limit of 7 nM. The remaining free UO22+ could be accurately quantified from the different protein-metal equilibrium and a dose-response curve established for KD determination. This generic method was applied not only to proteins such as transferrin and albumin but also to small phosphonated ligands. Its robustness allows the fast UO22+ KD determination of any kind of macromolecules and small ligands using very few amount of compounds, thus opening new prospects in the field of uranium toxicity. (authors)

  18. Identification of polymer surface adsorbed proteins implicated in pluripotent human embryonic stem cell expansion.

    Science.gov (United States)

    Hammad, Moamen; Rao, Wei; Smith, James G W; Anderson, Daniel G; Langer, Robert; Young, Lorraine E; Barrett, David A; Davies, Martyn C; Denning, Chris; Alexander, Morgan R

    2016-08-16

    Improved biomaterials are required for application in regenerative medicine, biosensing, and as medical devices. The response of cells to the chemistry of polymers cultured in media is generally regarded as being dominated by proteins adsorbed to the surface. Here we use mass spectrometry to identify proteins adsorbed from a complex mouse embryonic fibroblast (MEF) conditioned medium found to support pluripotent human embryonic stem cell (hESC) expansion on a plasma etched tissue culture polystyrene surface. A total of 71 proteins were identified, of which 14 uniquely correlated with the surface on which pluripotent stem cell expansion was achieved. We have developed a microarray combinatorial protein spotting approach to test the potential of these 14 proteins to support expansion of a hESC cell line (HUES-7) and a human induced pluripotent stem cell line (ReBl-PAT) on a novel polymer (N-(4-Hydroxyphenyl) methacrylamide). These proteins were spotted to form a primary array yielding several protein mixture 'hits' that enhanced cell attachment to the polymer. A second array was generated to test the function of a refined set of protein mixtures. We found that a combination of heat shock protein 90 and heat shock protein-1 encourage elevated adherence of pluripotent stem cells at a level comparable to fibronectin pre-treatment. PMID:27466628

  19. Altering protein surface charge with chemical modification modulates protein–gold nanoparticle aggregation

    International Nuclear Information System (INIS)

    Gold nanoparticles (AuNP) can interact with a wide range of molecules including proteins. Whereas significant attention has focused on modifying the nanoparticle surface to regulate protein–AuNP assembly or influence the formation of the protein “corona,” modification of the protein surface as a mechanism to modulate protein–AuNP interaction has been less explored. Here, we examine this possibility utilizing three small globular proteins—lysozyme with high isoelectric point (pI) and established interactions with AuNP; α-lactalbumin with similar tertiary fold to lysozyme but low pI; and myoglobin with a different globular fold and an intermediate pI. We first chemically modified these proteins to alter their charged surface functionalities, and thereby shift protein pI, and then applied multiple methods to assess protein–AuNP assembly. At pH values lower than the anticipated pI of the modified protein, AuNP exposure elicits changes in the optical absorbance of the protein–NP solutions and other properties due to aggregate formation. Above the expected pI, however, protein–AuNP interaction is minimal, and both components remain isolated, presumably because both species are negatively charged. These data demonstrate that protein modification provides a powerful tool for modulating whether nanoparticle–protein interactions result in material aggregation. The results also underscore that naturally occurring protein modifications found in vivo may be critical in defining nanoparticle–protein corona compositions.

  20. Comparative Proteomic Analysis of Differential Proteins in Response to Aqueous Extract of Quercus infectoria Gall in Methicillin-Resistant Staphylococcus aureus

    Science.gov (United States)

    Khairon, Radhiah; Zin, Noraziah Mohamad; Abdul Rahman, Mariati

    2016-01-01

    The aim of this study is to analyze the differential proteins in MRSA ATCC 33591 treated with aqueous extract from Q. infectoria gall. Protein extracts were obtained from MRSA cells by sonication and were separated by 2D polyacrylamide gels. Protein spots of interest were extracted from the gels and identified using LC-ESI-QTOF MS. The concentration of Q. infectoria extract used for 2D-gel electrophoresis was subinhibitory concentration. Minimum inhibitory concentration (MIC) value of the extract against MRSA was 19.50 μg/mL with bacteriostatic action at 1x MIC from time-kill assay. However, the extract exhibited dose-dependent manner and was bactericidal at 4x MIC with more than 3 log10 CFU/mL reduction at 4 h. 2D-GE map showed that 18 protein spots were upregulated and another six were downregulated more than twofold (p < 0.05) after treatment with subinhibitory concentration. Out of six proteins being downregulated, four proteins were identified as ferritin and catalase, branched-chain alpha-keto acid dehydrogenase subunit E2, and succinyl-CoA ligase [ADP-forming] subunit beta. Seven upregulated proteins which have been successfully identified were 3-hydroxyacyl-CoA dehydrogenase, NAD binding domain protein, formate C-acetyltransferase, 3-hydroxyacyl-[acyl-carrier-protein] dehydratase FabZ, NAD dependent epimerase/dehydratase family protein, and phosphopantothenoyl cysteine decarboxylase. It is postulated that the main mechanism of aqueous extract from gall of Q. infectoria was most likely involved in energy metabolism and protein stress.

  1. Comparative Proteomic Analysis of Differential Proteins in Response to Aqueous Extract of Quercus infectoria Gall in Methicillin-Resistant Staphylococcus aureus

    Science.gov (United States)

    Khairon, Radhiah; Zin, Noraziah Mohamad; Abdul Rahman, Mariati

    2016-01-01

    The aim of this study is to analyze the differential proteins in MRSA ATCC 33591 treated with aqueous extract from Q. infectoria gall. Protein extracts were obtained from MRSA cells by sonication and were separated by 2D polyacrylamide gels. Protein spots of interest were extracted from the gels and identified using LC-ESI-QTOF MS. The concentration of Q. infectoria extract used for 2D-gel electrophoresis was subinhibitory concentration. Minimum inhibitory concentration (MIC) value of the extract against MRSA was 19.50 μg/mL with bacteriostatic action at 1x MIC from time-kill assay. However, the extract exhibited dose-dependent manner and was bactericidal at 4x MIC with more than 3 log10 CFU/mL reduction at 4 h. 2D-GE map showed that 18 protein spots were upregulated and another six were downregulated more than twofold (p < 0.05) after treatment with subinhibitory concentration. Out of six proteins being downregulated, four proteins were identified as ferritin and catalase, branched-chain alpha-keto acid dehydrogenase subunit E2, and succinyl-CoA ligase [ADP-forming] subunit beta. Seven upregulated proteins which have been successfully identified were 3-hydroxyacyl-CoA dehydrogenase, NAD binding domain protein, formate C-acetyltransferase, 3-hydroxyacyl-[acyl-carrier-protein] dehydratase FabZ, NAD dependent epimerase/dehydratase family protein, and phosphopantothenoyl cysteine decarboxylase. It is postulated that the main mechanism of aqueous extract from gall of Q. infectoria was most likely involved in energy metabolism and protein stress. PMID:27688912

  2. Regulation of ADAM12 cell-surface expression by protein kinase C epsilon

    DEFF Research Database (Denmark)

    Sundberg, Christina; Thodeti, Charles Kumar; Kveiborg, Marie;

    2004-01-01

    constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) epsilon induces ADAM12 translocation to the cell......The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembrane-anchored proteins are synthesized as proforms that have (from the N terminus): a prodomain; a metalloprotease-, disintegrin......-immunoprecipitated from membrane-enriched fractions of PMA-treated cells, 3) RD cells transfected with EGFP-tagged, myristoylated PKCepsilon expressed more ADAM12 at the cell surface than did non-transfected cells, and 4) RD cells transfected with a kinase-inactive PKCepsilon mutant did not exhibit ADAM12 cell...

  3. Investigating the BSA protein adsorption and bacterial adhesion of Al-alloy surfaces after creating a hierarchical (micro/nano) superhydrophobic structure.

    Science.gov (United States)

    Moazzam, Parisa; Razmjou, Amir; Golabi, Mohsen; Shokri, Dariush; Landarani-Isfahani, Amir

    2016-09-01

    Bacterial adhesion and subsequent biofilm formation on metals such as aluminum (Al) alloys lead to serious issues in biomedical and industrial fields from both an economical and health perspective. Here, we showed that a careful manipulation of Al surface characteristics via a facile two-steps superhydrophobic modification can provide not only biocompatibility and an ability to control protein adsorption and bacterial adhesion, but also address the issue of apparent long-term toxicity of Al-alloys. To find out the roles of surface characteristics, surface modification and protein adsorption on microbial adhesion and biofilm formation, the surfaces were systematically characterized by SEM, EDX, XPS, AFM, FTIR, water contact angle (WCA) goniometry, surface free energy (SFE) measurement, MTT, Bradford, Lowry and microtiter plate assays and also flow-cytometry and potentiostat analyses. Results showed that WCA and SFE changed from 70° to 163° and 36.3 to 0.13 mN m(-1) , respectively. The stable and durable modification led to a substantial reduction in static/dynamic BSA adsorption. The effect of such a treatment on the biofilm formation was analyzed by using three different bacteria of Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus. The microtiter plate assay and flow cytometry analysis showed that the modification not only could substantially reduce the bacterial adhesion but this biofouling resistance is independent of bacterium type. An excellent cell viability after exposure of HeLa cells to waters incubated with the modified samples was observed. Finally, the corrosion rate reduced sharply from 856.6 to 0.119 MPY after superhydrophobic modifications, which is an excellent stable corrosion inhibition property. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2220-2233, 2016. PMID:27104583

  4. Investigating the BSA protein adsorption and bacterial adhesion of Al-alloy surfaces after creating a hierarchical (micro/nano) superhydrophobic structure.

    Science.gov (United States)

    Moazzam, Parisa; Razmjou, Amir; Golabi, Mohsen; Shokri, Dariush; Landarani-Isfahani, Amir

    2016-09-01

    Bacterial adhesion and subsequent biofilm formation on metals such as aluminum (Al) alloys lead to serious issues in biomedical and industrial fields from both an economical and health perspective. Here, we showed that a careful manipulation of Al surface characteristics via a facile two-steps superhydrophobic modification can provide not only biocompatibility and an ability to control protein adsorption and bacterial adhesion, but also address the issue of apparent long-term toxicity of Al-alloys. To find out the roles of surface characteristics, surface modification and protein adsorption on microbial adhesion and biofilm formation, the surfaces were systematically characterized by SEM, EDX, XPS, AFM, FTIR, water contact angle (WCA) goniometry, surface free energy (SFE) measurement, MTT, Bradford, Lowry and microtiter plate assays and also flow-cytometry and potentiostat analyses. Results showed that WCA and SFE changed from 70° to 163° and 36.3 to 0.13 mN m(-1) , respectively. The stable and durable modification led to a substantial reduction in static/dynamic BSA adsorption. The effect of such a treatment on the biofilm formation was analyzed by using three different bacteria of Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus. The microtiter plate assay and flow cytometry analysis showed that the modification not only could substantially reduce the bacterial adhesion but this biofouling resistance is independent of bacterium type. An excellent cell viability after exposure of HeLa cells to waters incubated with the modified samples was observed. Finally, the corrosion rate reduced sharply from 856.6 to 0.119 MPY after superhydrophobic modifications, which is an excellent stable corrosion inhibition property. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2220-2233, 2016.

  5. PROTEIN ADSORPTION AND CELL ADHESION ON RGD-FUNCTIONALIZED SILICON SUBSTRATE SURFACES

    Institute of Scientific and Technical Information of China (English)

    Wei-fang Tong; Xiao-li Liu; Fei Pan; Zhao-qiang Wu; Wen-wen Jiang

    2013-01-01

    A method was developed to modify silicon surfaces with good protein resistance and specific cell attachment.A silicon surface was initially deposited using a block copolymer of N-vinylpyrrolidone (NVP) and 2-hydroxyethyl methacrylate (HEMA) (PVP-b-PHEMA) film through surface-initiated atom transfer radical polymerization and then further immobilized using a short arginine-glycine-aspartate (RGD) peptide.Our results demonstrate that the RGD-modified surfaces (Si-RGD) can suppress non-specific adsorption of proteins and induce the adhesion of L929 cells.The Si-RGD surface exhibited higher cell proliferation rates than the unmodified silicon surface.This research established a simple method for the fabrication of dual-functional silicon surface that combines antifouling and cell attachment promotion.

  6. Mapping lipid and detergent molecules at the surface of membrane proteins.

    Science.gov (United States)

    Cogdell, Richard J; Gardiner, Alastair T; Roszak, Aleksander W; Stončius, Sigitas; Kočovský, Pavel; Isaacs, Neil W

    2011-06-01

    Electron-density maps for the crystal structures of membrane proteins often show features suggesting binding of lipids and/or detergent molecules on the hydrophobic surface, but usually it is difficult to identify the bound molecules. In our studies, heavy-atom-labelled phospholipids and detergents have been used to unequivocally identify these binding sites at the surfaces of test membrane proteins, the reaction centres from Rhodobacter sphaeroides and Blastochloris viridis. The generality of this method is discussed in the present article.

  7. A surface derivatization strategy for combinatorial analysis of cell response to mixtures of protein domains

    OpenAIRE

    Chiang, Chunyi; Karuri, Stella W.; Kshatriya, Pradnya P.; Schwartz, Jeffrey; Schwarzbauer, Jean E.; Karuri, Nancy W.

    2011-01-01

    We report a robust strategy for conjugating mixtures of two or more protein domains to non-fouling polyurethane surfaces. In our strategy, the carbamate groups of polyurethane are reacted with zirconium alkoxide from the vapor phase to give a surface bound oxide that serves as a chemical layer that can be used to bond organics to the polymer substrate. An hydroxyalkylphosphonate monolayer was synthesized on this layer, which was then used to covalently bind primary amine groups in protein dom...

  8. Extraction of cell surface-associated proteins from living yeast cells.

    NARCIS (Netherlands)

    F.M. Klis; M. de Jong; S. Brul; P.W.J. de Groot

    2007-01-01

    To extract cell surface-associated proteins from living fungal cells, reducing agents such as beta-mercaptoethanol and dithiothreitol are often used. We show here that both compounds are moderately lipophilic and may perturb the plasma membrane, thus causing the release of cytosolic proteins, especi

  9. Surface-tethered polymers to influence protein adsorption and microbial adhesion

    NARCIS (Netherlands)

    Norde, Willem

    2007-01-01

    In various applications it is desired that biological cells or protein molecules are immobilized at surfaces. Examples are enzymes or cells in bioreactors and biosensors, immuno-proteins in solid-state diagnostics and proteinaceous farmacons in drug delivery systems. In order to retain biological ac

  10. Perspectives for in situ Scanning Tunnel Microscopic Imaging of Proteins at HOPG surfaces

    DEFF Research Database (Denmark)

    Andersen, Jens Enevold Thaulov; Thuesen, Marianne Hallberg; Møller, Per;

    1996-01-01

    We have investigated the behaviour of the four-copper fungal metalloenzyme laccase (MW~68kDa) at highly oriented pyrolytic graphite (HOPG) surfaces by ex situ and in situ STM. The four copper atoms ar suited to stimulate long-range inelastic tunnel modes through the protein. The proteins forms cr...

  11. Cell wall sorting signals in surface proteins of gram-positive bacteria.

    OpenAIRE

    Schneewind, O; Mihaylova-Petkov, D; Model, P

    1993-01-01

    Staphylococcal protein A is anchored to the cell wall, a unique cellular compartment of Gram-positive bacteria. The sorting signal sufficient for cell wall anchoring consists of an LPXTG motif, a C-terminal hydrophobic domain and a charged tail. Homologous sequences are found in many surface proteins of Gram-positive bacteria and we explored the universality of these sequences to serve as cell wall sorting signals. We show that several signals are able to anchor fusion proteins to the staphyl...

  12. Surface characterization of proteins using multi-fractal property of heat-denatured aggregates

    OpenAIRE

    Lahiri, Tapobrata; Mishra, Hrishikesh; Sarkar, Subrata; Misra, Krishna

    2008-01-01

    Multi-fractal property of heat-denatured protein aggregates (HDPA) is characteristic of its individual form. The visual similarity between digitally generated microscopic images of HDPA with that of surface-image of its individual X-ray structures in protein databank (PDB) displayed using Visual Molecular Dynamics (VMD) viewer is the basis of the study. We deigned experiments to view the fractal nature of proteins at different aggregate scales. Intensity based multi-fractal dimensions (ILMFD)...

  13. Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV.

    Science.gov (United States)

    Vieira, Monica L; de Morais, Zenaide M; Gonçales, Amane P; Romero, Eliete C; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2010-01-01

    Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease that affects populations worldwide. We have identified in proteomic studies a protein that is encoded by the gene LIC10314 and expressed in virulent strain of L. interrogans serovar Pomona. This protein was predicted to be surface exposed by PSORT program and contains a p83/100 domain identified by BLAST analysis that is conserved in protein antigens of several strains of Borrelia and Treponema spp. The proteins containing this domain have been claimed antigen candidates for serodiagnosis of Lyme borreliosis. Thus, we have cloned the LIC10314 and expressed the protein in Escherichia coli BL21-SI strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. This protein is conserved among several species of pathogenic Leptospira and absent in the saprophytic strain L. biflexa. We confirm by liquid-phase immunofluorescence assays with living organisms that this protein is most likely a new surface leptospiral protein. The ability of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC10314, named Lsa63 (Leptospiral surface adhesin of 63kDa), binds strongly to laminin and collagen IV in a dose-dependent and saturable fashion. In addition, Lsa63 is probably expressed during infection since it was recognized by antibodies of serum samples of confirmed-leptospirosis patients in convalescent phase of the disease. Altogether, the data suggests that this novel identified surface protein may be involved in leptospiral pathogenesis.

  14. Protein adsorption on the poly(L-lactic acid) surface modified by chitosan and its derivatives

    Institute of Scientific and Technical Information of China (English)

    JIAO YanPeng; ZHOU ChangRen; LI LiHua; DING Shan; LU Lu; LUO BingHong; LI Hong

    2009-01-01

    Surface modification of biomaterials has been adopted over the years to improve their biocompatibility.In this study,aiming to promote hydrophilicity and to introduce natural recognition sites onto poly(L-lactic acid) (PLLA) films,chitosan and its derivatives,carboxymethyl chitosan(CMC) and N-methylene phosphonic chitosan (NPC),were used to modify the surface of PLLA films by an entrapment method. Radiolabeled (12SI) proteins were used to measure the amount of protein adsorbed to PLLA surfaces. Fibronectin (Fn) was used to study the protein adsorption on the modified PLLA surfaces,including isotherm adsorption and adsorption kinetics of single protein,competitive adsorption of binary proteins system and serum multi-proteins and the desorption behavior in serum solution. The results showed that in the isotherm adsorption,Fn had a larger adsorption capacity on the CS-modified surface at lower concentrations,but had a high adsorption capacity at CMC-modified surface at higher concentrations. In the study of absorption kinetics,Fn had a fastest adsorption equilibrium and a highest equilibrium adsorption capacity at the CS-modified surface,while it was opposite at the PCS-modified surface. When BSA and serum were added,it had the greatest effect on the adsorption of Fn on the PCS-modified surface. After 6 hours soaking in the desorption study,Fn had reached desorption equilibrium on all the modified surfaces,which had different effects on the desorption rate and the remaining percentage of Fn.

  15. Protein immobilization on epoxy-activated thin polymer films: effect of surface wettability and enzyme loading.

    Science.gov (United States)

    Chen, Bo; Pernodet, Nadine; Rafailovich, Miriam H; Bakhtina, Asya; Gross, Richard A

    2008-12-01

    A series of epoxy-activated polymer films composed of poly(glycidyl methacrylate/butyl methacrylate/hydroxyethyl methacrylate) were prepared. Variation in comonomer composition allowed exploration of relationships between surface wettability and Candida antartica lipase B (CALB) binding to surfaces. By changing solvents and polymer concentrations, suitable conditions were developed for preparation by spin-coating of uniform thin films. Film roughness determined by AFM after incubation in PBS buffer for 2 days was less than 1 nm. The occurrence of single CALB molecules and CALB aggregates at surfaces was determined by AFM imaging and measurements of volume. Absolute numbers of protein monomers and multimers at surfaces were used to determine values of CALB specific activity. Increased film wettability, as the water contact angle of films increased from 420 to 550, resulted in a decreased total number of immobilized CALB molecules. With further increases in the water contact angle of films from 55 degrees to 63 degrees, there was an increased tendency of CALB molecules to form aggregates on surfaces. On all flat surfaces, two height populations, differing by more than 30%, were observed from height distribution curves. They are attributed to changes in protein conformation and/or orientation caused by protein-surface and protein-protein interactions. The fraction of molecules in these populations changed as a function of film water contact angle. The enzyme activity of immobilized films was determined by measuring CALB-catalyzed hydrolysis of p-nitrophenyl butyrate. Total enzyme specific activity decreased by decreasing film hydrophobicity.

  16. Novel Chromosomally Encoded Multidrug Efflux Transporter MdeA in Staphylococcus aureus

    OpenAIRE

    Huang, Jianzhong; O'Toole, Paul W.; Shen, Wei; Amrine-Madsen, Heather; Jiang, Xinhe; Lobo, Neethan; Palmer, Leslie M.; Voelker, LeRoy; Fan, Frank; Gwynn, Michael N.; McDevitt, Damien

    2004-01-01

    Antibiotic efflux is an important mechanism of resistance in pathogenic bacteria. Here we describe the identification and characterization of a novel chromosomally encoded multidrug resistance efflux protein in Staphylococcus aureus, MdeA (multidrug efflux A). MdeA was identified from screening an S. aureus open reading frame expression library for resistance to antibiotic compounds. When overexpressed, MdeA confers resistance on S. aureus to a range of quaternary ammonium compounds and antib...

  17. Single cell protein production from yacon extract using a highly thermosensitive and permeable mutant of the marine yeast Cryptococcus aureus G7a and its nutritive analysis.

    Science.gov (United States)

    Zhao, Chun-Hai; Zhang, Tong; Chi, Zhen-Ming; Chi, Zhe; Li, Jing; Wang, Xiang-Hong

    2010-06-01

    The intracellular protein in the highly thermosensitive and permeable mutant can be easily released when they are incubated both in the low-osmolarity water and at the non-permissive temperature (usually 37 degrees C). After the mutant was grown in the yacon extract for 45 h, the crude protein content in the highly thermosensitive and permeable mutant Z114 was 59.1% and over 61% of the total protein could be released from the cells treated at 37 degrees C. The mutant cells grown in the yacon extract still contained high level of essential amino acids and other nutrients. This means that the yacon extract could be used as the medium for growth of the highly thermosensitive and permeable mutant which contained high content of crude protein. PMID:19727833

  18. Development of a mimotope vaccine targeting the Staphylococcus aureus quorum sensing pathway.

    Directory of Open Access Journals (Sweden)

    John P O'Rourke

    Full Text Available A major hurdle in vaccine development is the difficulty in identifying relevant target epitopes and then presenting them to the immune system in a context that mimics their native conformation. We have engineered novel virus-like-particle (VLP technology that is able to display complex libraries of random peptide sequences on a surface-exposed loop in the coat protein without disruption of protein folding or VLP assembly. This technology allows us to use the same VLP particle for both affinity selection and immunization, integrating the power of epitope discovery and epitope mimicry of traditional phage display with the high immunogenicity of VLPs. Previously, we showed that using affinity selection with our VLP platform identifies linear epitopes of monoclonal antibodies and subsequent immunization generates the proper antibody response. To test if our technology could identify immunologic mimotopes, we used affinity selection on a monoclonal antibody (AP4-24H11 that recognizes the Staphylococcus aureus autoinducing peptide 4 (AIP4. AIP4 is a secreted eight amino acid, cyclized peptide produced from the S. aureus accessory gene regulator (agrIV quorum-sensing operon. The agr system coordinates density dependent changes in gene expression, leading to the upregulation of a host of virulence factors, and passive transfer of AP4-24H11 protects against S. aureus agrIV-dependent pathogenicity. In this report, we identified a set of peptides displayed on VLPs that bound with high specificity to AP4-24H11. Importantly, similar to passive transfer with AP4-24H11, immunization with a subset of these VLPs protected against pathogenicity in a mouse model of S. aureus dermonecrosis. These data are proof of principle that by performing affinity selection on neutralizing antibodies, our VLP technology can identify peptide mimics of non-linear epitopes and that these mimotope based VLP vaccines provide protection against pathogens in relevant animal models.

  19. Development of a mimotope vaccine targeting the Staphylococcus aureus quorum sensing pathway.

    Science.gov (United States)

    O'Rourke, John P; Daly, Seth M; Triplett, Kathleen D; Peabody, David; Chackerian, Bryce; Hall, Pamela R

    2014-01-01

    A major hurdle in vaccine development is the difficulty in identifying relevant target epitopes and then presenting them to the immune system in a context that mimics their native conformation. We have engineered novel virus-like-particle (VLP) technology that is able to display complex libraries of random peptide sequences on a surface-exposed loop in the coat protein without disruption of protein folding or VLP assembly. This technology allows us to use the same VLP particle for both affinity selection and immunization, integrating the power of epitope discovery and epitope mimicry of traditional phage display with the high immunogenicity of VLPs. Previously, we showed that using affinity selection with our VLP platform identifies linear epitopes of monoclonal antibodies and subsequent immunization generates the proper antibody response. To test if our technology could identify immunologic mimotopes, we used affinity selection on a monoclonal antibody (AP4-24H11) that recognizes the Staphylococcus aureus autoinducing peptide 4 (AIP4). AIP4 is a secreted eight amino acid, cyclized peptide produced from the S. aureus accessory gene regulator (agrIV) quorum-sensing operon. The agr system coordinates density dependent changes in gene expression, leading to the upregulation of a host of virulence factors, and passive transfer of AP4-24H11 protects against S. aureus agrIV-dependent pathogenicity. In this report, we identified a set of peptides displayed on VLPs that bound with high specificity to AP4-24H11. Importantly, similar to passive transfer with AP4-24H11, immunization with a subset of these VLPs protected against pathogenicity in a mouse model of S. aureus dermonecrosis. These data are proof of principle that by performing affinity selection on neutralizing antibodies, our VLP technology can identify peptide mimics of non-linear epitopes and that these mimotope based VLP vaccines provide protection against pathogens in relevant animal models. PMID:25379726

  20. Microscopic Investigation of Reversible Nanoscale Surface Size Dependent Protein Conjugation

    Directory of Open Access Journals (Sweden)

    Michael A. Carpenter

    2009-05-01

    Full Text Available Aβ1-40 coated 20 nm gold colloidal nanoparticles exhibit a reversible color change as pH is externally altered between pH 4 and 10. This reversible process may contain important information on the initial reversible step reported for the fibrillogenesis of Aβ (a hallmark of Alzheimer’s disease. We examined this reversible color change by microscopic investigations. AFM images on graphite surfaces revealed the morphology of Aβ aggregates with gold colloids. TEM images clearly demonstrate the correspondence between spectroscopic features and conformational changes of the gold colloid.

  1. Exploring the Plant–Microbe Interface by Profiling the Surface-Associated Proteins of Barley Grains

    DEFF Research Database (Denmark)

    Sultan, Abida; Andersen, Birgit; Svensson, Birte;

    2016-01-01

    xylanase inhibitors. To gain insight into the importance of the microbial consortia and their interaction with barley grains, we used a combined gel-based (2-DE coupled to MALDI-TOF-TOF MS) and gel-free (LC–MS/MS) proteomics approach complemented with enzyme activity assays to profile the surface......-associated proteins and xylanolytic activities of two barley cultivars. The surface-associated proteome was dominated by plant proteins with roles in defense and stress-responses, while the relatively less abundant microbial (bacterial and fungal) proteins were involved in cell-wall and polysaccharide degradation...

  2. Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein

    Directory of Open Access Journals (Sweden)

    Han Lanlan

    2011-10-01

    Full Text Available Abstract Background Previous studies have revealed that the C-terminal region of the S-layer protein from Lactobacillus is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB. In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of Lactobacillus crispatus K2-4-3 isolated from chicken intestine. Results Multiple sequence alignment revealed that the C-terminal region (LcsB of Lb. crispatus K2-4-3 SlpB had a high similarity with the cell wall binding domains SA and CbsA of Lactobacillus acidophilus and Lb. crispatus. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP or beta-galactosidase (Gal was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in Escherichia coli, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of gfp:lcsB was inserted into the Lactococcus lactis expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the nisA promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of L. lactis with the aid of the LcsB anchor. Conclusion The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria in vitro and in vivo, and has also the potential for biotechnological

  3. The effect of polymer surface modification on polymer-protein interaction via interfacial polymerization and hydrophilic polymer grafting

    Science.gov (United States)

    Protein membrane separation is prone to fouling on the membrane surface resulting from protein adsorption onto the surface. Surface modification of synthetic membranes is one way to reduce fouling. We investigated surface modification of polyethersulfone (PES) as a way of improving hydrophilicity ...

  4. Analyses of Interactions Between Heparin and the Apical Surface Proteins of Plasmodium falciparum

    Science.gov (United States)

    Kobayashi, Kyousuke; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Ishiwa, Akiko; Gong, Haiyan; Recuenco, Frances C.; Iwanaga, Tatsuya; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

    2013-11-01

    Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

  5. Ultrasensitive probing of the protein resistance of PEG surfaces by secondary ion mass spectrometry

    DEFF Research Database (Denmark)

    Kingshott, P.; McArthur, S.; Thissen, H.;

    2002-01-01

    The highly sensitive surface analytical techniques X-ray photoelectron spectroscopy (XPS) and time-of-flight static secondary ion mass spectrometry (ToF-SIMS) were used to test the resistance of poly(ethylene glycol) (PEG) coatings towards adsorption of lysozyme (LYS) and fibronectin (FN). PEG...... temperature to maximise the graft density of the PEG chains. XPS showed that the grafted density of PEG chains was slightly higher on the allylamine surface. XPS detected no adsorption of either protein on either PEG coating. ToF-SIMS analysis, on the other hand, found, in the positive ion spectra, minute...... but no changes assignable to adsorbed proteins on the higher density PEG coating. These results demonstrate the importance of utilising highly sensitive techniques to study protein adsorption on surfaces intended to be protein resistant, and that both positive and negative ion ToF-SIMS spectra should be acquired...

  6. Comprehensive proteomic analysis of Trypanosoma cruzi epimastigote cell surface proteins by two complementary methods

    DEFF Research Database (Denmark)

    Queiroz, Rayner M L; Charneau, Sébastien; Motta, Flávia N;

    2013-01-01

    Trypanosoma cruzi is a protozoan that causes Chagas' disease, a neglected infectious illness that affects millions of people, mostly in Latin America. Here, the cell surface subproteome of the T. cruzi epimastigote life form was characterized. In order to prepare samples enriched in epimastigote...... plasma membrane protein, two distinct methodologies were optimized and evaluated. The first methodology was based on cell surface trypsinization (Shave) of intact living cells while the second approach used biotinylation of cell surface proteins followed by streptavidin affinity chromatography isolation...

  7. Deposition at glancing angle, surface roughness, and protein adsorption: Monte Carlo simulations.

    Science.gov (United States)

    Zhdanov, Vladimir P; Rechendorff, Kristian; Hovgaard, Mads B; Besenbacher, Flemming

    2008-06-19

    To generate rough surfaces in Monte Carlo simulations, we use the 2 + 1 solid-on-solid model of deposition with rapid transient diffusion of newly arrived atoms supplied at glancing angle. The surfaces generated are employed to scrutinize the effect of surface roughness on adsorption of globular and anisotropic rodlike proteins. The obtained results are compared with the available experimental data for Ta deposition at glancing angle and for the bovine serum albumin and fibrinogen uptake on the corresponding Ta films.

  8. Isolation of two biologically active cell surface proteins from Brucella abortus by chromatofocusing

    Energy Technology Data Exchange (ETDEWEB)

    Tabatabai, L.B.; Deyoe, B.L.

    1983-01-01

    Brucella abortus contains a group of immunogenic cell surface proteins which have potential value as a vaccine or as a diagnostic reagent for the prevention and diagnosis of bovine brucellosis. Under nondenaturing conditions, these proteins range in molecular weight from 10,000-124,000, as determined by high performance liquid chromatography (HPLC) on TSK 3000sw. By analytical isoelectrofocusing, 6 major protein bands could be distinguished with pI's ranging from 4.0 to 6.0 and 3 additional major proteins with pI's of 7.5, 9.5, and 10. By chromatofocusing on Polybuffer Exchanger 94 with a pH gradient from 6-4, two of the six proteins from pI 4-6 were separated, a pI 4.9 and a pI 4.7 protein; a third fraction contained the high pI proteins. The former two proteins were homogeneous by analytical isoelectrofocusing, and a molecular weight of 54,000 daltons was found for both protein species by HPLC on TSK 3000sw. The pI 4-6 and not the pI 9.5 and 10 proteins, could be radiolabeled when intact cells were radioiodinated with diazotized (/sup 125/I)-iodosulfanilic acid. Biological activity of the proteins as assessed in lemmings indicated that immunization with the pI 4.7 and 4.9 proteins afforded better protection against experimental brucellosis than immunization with the high pI proteins. These results support our view that a single surface protein may be sufficient for the prevention of experimental brucellosis.

  9. Utilizing Yeast Surface Human Proteome Display Libraries to Identify Small Molecule-Protein Interactions.

    Science.gov (United States)

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    The identification of proteins that interact with small bioactive molecules is a critical but often difficult and time-consuming step in understanding cellular signaling pathways or molecular mechanisms of drug action. Numerous methods for identifying small molecule-interacting proteins have been developed and utilized, including affinity-based purification followed by mass spectrometry analysis, protein microarrays, phage display, and three-hybrid approaches. Although all these methods have been used successfully, there remains a need for additional techniques for analyzing small molecule-protein interactions. A promising method for identifying small molecule-protein interactions is affinity-based selection of yeast surface-displayed human proteome libraries. Large and diverse libraries displaying human protein fragments on the surface of yeast cells have been constructed and subjected to FACS-based enrichment followed by comprehensive exon microarray-based output analysis to identify protein fragments with affinity for small molecule ligands. In a recent example, a proteome-wide search has been successfully carried out to identify cellular proteins binding to the signaling lipids PtdIns(4,5)P2 and PtdIns(3,4,5)P3. Known phosphatidylinositide-binding proteins such as pleckstrin homology domains were identified, as well as many novel interactions. Intriguingly, many novel nuclear phosphatidylinositide-binding proteins were discovered. Although the existence of an independent pool of nuclear phosphatidylinositides has been known about for some time, their functions and mechanism of action remain obscure. Thus, the identification and subsequent study of nuclear phosphatidylinositide-binding proteins is expected to bring new insights to this important biological question. Based on the success with phosphatidylinositides, it is expected that the screening of yeast surface-displayed human proteome libraries will be of general use for the discovery of novel small

  10. SarA positively controls bap-dependent biofilm formation in Staphylococcus aureus.

    Science.gov (United States)

    Trotonda, María Pilar; Manna, Adhar C; Cheung, Ambrose L; Lasa, Iñigo; Penadés, José R

    2005-08-01

    The biofilm-associated protein Bap is a staphylococcal surface protein involved in biofilm formation. We investigated the influence of the global regulatory locus sarA on bap expression and Bap-dependent biofilm formation in three unrelated Staphylococcus aureus strains. The results showed that Bap-dependent biofilm formation was diminished in the sarA mutants by an agr-independent mechanism. Complementation studies using a sarA clone confirmed that the defect in biofilm formation was due to the sarA mutation. As expected, the diminished capacity to form biofilms in the sarA mutants correlated with the decreased presence of Bap in the bacterial surface. Using transcriptional fusion and Northern analysis data, we demonstrated that the sarA gene product acts as an activator of bap expression. Finally, the bap promoter was characterized and the transcriptional start point was mapped by the rapid amplification of cDNA ends technique. As expected, we showed that purified SarA protein binds specifically to the bap promoter, as determined by gel shift and DNase I footprinting assays. Based on the previous studies of others as well as our work demonstrating the role for SarA in icaADBC and bap expression, we propose that SarA is an essential regulator controlling biofilm formation in S. aureus.

  11. Immobilization of multivalent glycoprobes on gold surfaces for sensing proteins and macrophages.

    Science.gov (United States)

    Gade, Madhuri; Khandelwal, Puneet; Sangabathuni, Sivakoti; Bavireddi, Harikrishna; Murthy, Raghavendra Vasudeva; Poddar, Pankaj; Kikkeri, Raghavendra

    2016-04-01

    The multivalent display of carbohydrates on the cell surface provides cooperative binding to improve the specific biological events. In addition to multivalency, the spatial arrangement and orientation of sugars with respect to external stimuli also trigger carbohydrate-protein interactions. Herein, we report a non-covalent host-guest strategy to immobilize heptavalent glyco-β-cyclodextrin on gold-coated glass slides to study multivalent carbohydrate-protein interactions. We have found that the localization of sugar entities on surfaces using β-cyclodextrin (β-CD) chemistry increased the avidity of carbohydrate-protein and carbohydrate-macrophage interactions compared to monovalent-β-CD sugar coated surfaces. This platform is expected to be a promising tool to amplify the avidity of sugar-mediated interactions on surfaces and contribute to the development of next generation bio-medical products. PMID:26934683

  12. An Investigation of Freezing of Supercooled Water on Anti-Freeze Protein Modified Surfaces

    Institute of Scientific and Technical Information of China (English)

    Thibaut V J Charpentier; Anne Neville; Paul Millner; Rob Hewson; Ardian Morina

    2013-01-01

    This work investigates how functionalization ofaluminium surfaces with natural type Ⅲ Anti-Freeze Protein (AFP) affects the mechanism of heterogeneous ice nucleation.First the bulk ice nucleation properties of distilled water and aqueous solution of AFP were evaluated by differential scanning calorimetry.Then the modified surface was characterized by Secondary Ions Mass Spectroscopy (SIMS),Fourier Transform InfraRed (FTIR) spectroscopy and contact angle measurement.Freezing experiments were then conducted in which water droplets underwent a slow controlled cooling.This study shows that compared to uncoated aluminium,the anti-freeze proteins functionalized surfaces exhibit a higher and narrower range of freezing temperature.It was found that these proteins that keep living organisms from freezing in cold environment act in the opposite way once immobilized on surfaces by promoting ice nucleation.Some suggestions regarding the mechanism of action of the observed phenomena were proposed based on the Classical Nucleation Theory (CNT).

  13. Multiple Plasmodium falciparum Merozoite Surface Protein 1 Complexes Mediate Merozoite Binding to Human Erythrocytes.

    Science.gov (United States)

    Lin, Clara S; Uboldi, Alessandro D; Epp, Christian; Bujard, Hermann; Tsuboi, Takafumi; Czabotar, Peter E; Cowman, Alan F

    2016-04-01

    Successful invasion of human erythrocytes byPlasmodium falciparummerozoites is required for infection of the host and parasite survival. The early stages of invasion are mediated via merozoite surface proteins that interact with human erythrocytes. The nature of these interactions are currently not well understood, but it is known that merozoite surface protein 1 (MSP1) is critical for successful erythrocyte invasion. Here we show that the peripheral merozoite surface proteins MSP3, MSP6, MSPDBL1, MSPDBL2, and MSP7 bind directly to MSP1, but independently of each other, to form multiple forms of the MSP1 complex on the parasite surface. These complexes have overlapping functions that interact directly with human erythrocytes. We also show that targeting the p83 fragment of MSP1 using inhibitory antibodies inhibits all forms of MSP1 complexes and disrupts parasite growthin vitro.

  14. Cytolytic pore-forming protein associated with the surface membrane of Naegleria fowleri

    Energy Technology Data Exchange (ETDEWEB)

    Lowrey, D.M.

    1985-01-01

    Whole cell homogenates of Naegleria fowleri were examined by hemolytic and /sup 51/Cr-release assays for the presence of cytolytic molecules which may participate in the cytopathogenic action of this amoeba. Two distinct cytolytic activities were found. A surface membrane cytolysin was identified which was found to be avidly associated with membranes possessing an equilibrium density of 1.135 g/cm/sup 3/ in isopycnic sucrose gradients. The activity of the surface membrane cytolysin was not affected by heating at 75/sup 0/C for 30 min. The second cytolytic activity was found in putative lysosomes possessing an equilibrium density of 1.162 g/cm/sup 3/ and was completely inactivated by heating at 75/sup 0/C for 30 min. Cytolysis produced in the presence of both cytolysins was consistently synergistic with respect to the activity of either cytolysin alone. The lesions produced on erythrocytes by this cooperative process were characterized by electron microscopy as transmembrane pores resembling a number of other cytolytic effector molecules including the ninth component of complement, perforins of cytolytic T lymphocytes, and the alphatoxin of Staphylococcus aureus.

  15. Comparative Opsonic and Protective Activities of Staphylococcus aureus Conjugate Vaccines Containing Native or Deacetylated Staphylococcal Poly-N-Acetyl-β-(1-6)-Glucosamine

    OpenAIRE

    Maira-Litrán, Tomás; Kropec, Andrea; Goldmann, Donald A.; Pier, Gerald B.

    2005-01-01

    Staphylococcus aureus and Staphylococcus epidermidis both synthesize the surface polysaccharide poly-N-acetyl-β-(1-6)-glucosamine (PNAG), which is produced in vitro with a high level (>90%) of the amino groups substituted by acetate. Here, we examined the role of the acetate substituents of PNAG in generating opsonic and protective antibodies. PNAG and a deacetylated form of the antigen (dPNAG; 15% acetylation) were conjugated to the carrier protein diphtheria toxoid (DT) and used to immunize...

  16. Investigation of cellular and protein interactions with model self-assembled monolayer surfaces

    Science.gov (United States)

    Tegoulia, Vassiliki Apostolou

    Self-assembled monolayers (SAMs) of alkanethiolates on gold have been used to investigate the effect of substrate surface properties on bacterial and blood cell adhesion in the presence and absence of blood proteins. Protein adsorption and binding strength on SAMs as well as complement activation by these model surfaces were also studied. It is hoped that information gained, regarding factors that influence biological processes, will lead to strategies for designing materials and surfaces that specifically inhibit cell adhesion and protein adsorption. Single component SAMs of the general formula HS(CH2) 10X, where X = CH3, CH2OH. COOH and CH2(OCH 2CH2)3OH, and two component mixed SAMs created from binary solutions of HS(CH2), OCH3 and HS(CH 2)10CH2OH, were used. Adhesion was investigated under well-defined flow conditions. Adhesion was found to be higher for the hydrophobic methyl and minimal for the tri(ethyleneoxide) terminated SAM. Preincubation of the SAMs with fibrinogen led to an increase in cell adhesion for bacteria and a decrease for leukocyte adhesion. The effect of surface chemistry on protein adsorption was studied for three blood proteins, fibrinogen, fibronectin and albumin. Adsorption was found to be higher on the hydrophobic CH3 surface and lower but comparable for the other surfaces while proteins adsorbed strongly on all surfaces. SAMs were also used to evaluate complement activation by foreign surfaces. The hydroxyl rich SAMs were found to activate complement more significantly than the anionic carboxyl and the hydrophobic methyl terminated SAMs. A surface modification was introduced to incorporate a zwitterionic phosphorylcholine (PC) group on a hydroxyl monolayer in an effort to create a biomimetic surface that could minimize cell adhesion and protein adsorption. The good antifouling properties of the phosphorylcholine modified surface led to the synthesis of a novel phosphorylcholine functionalized thiol. Single component and two component

  17. Identification of Staphylococcus aureus Colony-Spreading Stimulatory Factors from Mammalian Serum

    OpenAIRE

    Yosuke Omae; Kazuhisa Sekimizu; Chikara Kaito

    2014-01-01

    Staphylococcus aureus forms giant colonies on soft-agar surfaces, which is called colony-spreading. In the present study, we searched for host factors that influence S. aureus colony-spreading activity. The addition of calf serum, porcine serum, or silkworm hemolymph to soft-agar medium stimulated S. aureus colony-spreading activity. Gel filtration column chromatography of calf serum produced a high molecular weight fraction and a low molecular weight fraction, both of which exhibited colony-...

  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. In various protein complexes, disordered protomers have large per-residue surface areas and area of protein-, DNA- and RNA-binding interfaces.

    Science.gov (United States)

    Wu, Zhonghua; Hu, Gang; Yang, Jianyi; Peng, Zhenling; Uversky, Vladimir N; Kurgan, Lukasz

    2015-09-14

    We provide first large scale analysis of the peculiarities of surface areas of 5658 dissimilar (below 50% sequence similarity) proteins with known 3D-structures that bind to proteins, DNA or RNAs. We show here that area of the protein surface is highly correlated with the protein length. The size of the interface surface is only modestly correlated with the protein size, except for RNA-binding proteins where larger proteins are characterized by larger interfaces. Disordered proteins with disordered interfaces are characterized by significantly larger per-residue areas of their surfaces and interfaces when compared to the structured proteins. These result are applicable for proteins involved in interaction with DNA, RNA, and proteins and suggest that disordered proteins and binding regions are less compact and more likely to assume extended shape. We demonstrate that disordered protein binding residues in the interfaces of disordered proteins drive the increase in the per residue area of these interfaces. Our results can be used to predict in silico whether a given protomer from the DNA, RNA or protein complex is likely to be disordered in its unbound form.

  20. Mitogen-activated protein kinases mediate Mycobacterium tuberculosis–induced CD44 surface expression in monocytes

    Indian Academy of Sciences (India)

    Natarajan Palaniappan; S Anbalagan; Sujatha Narayanan

    2012-03-01

    CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are involved in M. tuberculosis–induced CD44 surface expression in monocytic cells are currently unknown. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv and H37Ra induced distinct, time-dependent, phosphorylation of mitogen-activated protein kinase kinase-1, extracellular signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, p38 mitogen-activated protein kinase and c-jun N-terminal kinases. The strains also differed in their usage of CD14 and human leukocyte antigen-DR (HLA-DR) receptors in mediating mitogen-activated protein kinase activation. M. tuberculosis H37Rv strain induced lower CD44 surface expression and tumour necrosis factor-alpha levels, whereas H37Ra the reverse. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and c-jun N-terminal kinase, we report that inhibition of extracellular signal regulated kinase 1/2 and c-jun N-terminal kinases increases, but that inhibition of p38 mitogen-activated protein kinase decreases M. tuberculosis–induced CD44 surface expression in THP-1 human monocytes.

  1. Interaction of Moringa oleifera seed protein with a mineral surface and the influence of surfactants.

    Science.gov (United States)

    Kwaambwa, Habauka M; Hellsing, Maja S; Rennie, Adrian R; Barker, Robert

    2015-06-15

    The paper describes the adsorption of purified protein from seeds of Moringa oleifera to a sapphire interface and the effects of addition of the anionic surfactant sodium dodecylsulfate (SDS) and the cationic surfactant hexadecyltrimethylammonium bromide (CTAB). Neutron reflection was used to determine the structure and composition of interfacial layers adsorbed at the solid/solution interface. The maximum surface excess of protein was found to be about 5.3 mg m(-2). The protein does not desorb from the solid/liquid interface when rinsed with water. Addition of SDS increases the reflectivity indicating co-adsorption. It was observed that CTAB is able to remove the protein from the interface. The distinct differences to the behavior observed previously for the protein at the silica/water interface are identified. The adsorption of the protein to alumina in addition to other surfaces has shown why it is an effective flocculating agent for the range of impurities found in water supplies. The ability to tailor different surface layers in combination with various surfactants also offers the potential for adsorbed protein to be used in separation technologies. PMID:25746187

  2. Interaction of Moringa oleifera seed protein with a mineral surface and the influence of surfactants.

    Science.gov (United States)

    Kwaambwa, Habauka M; Hellsing, Maja S; Rennie, Adrian R; Barker, Robert

    2015-06-15

    The paper describes the adsorption of purified protein from seeds of Moringa oleifera to a sapphire interface and the effects of addition of the anionic surfactant sodium dodecylsulfate (SDS) and the cationic surfactant hexadecyltrimethylammonium bromide (CTAB). Neutron reflection was used to determine the structure and composition of interfacial layers adsorbed at the solid/solution interface. The maximum surface excess of protein was found to be about 5.3 mg m(-2). The protein does not desorb from the solid/liquid interface when rinsed with water. Addition of SDS increases the reflectivity indicating co-adsorption. It was observed that CTAB is able to remove the protein from the interface. The distinct differences to the behavior observed previously for the protein at the silica/water interface are identified. The adsorption of the protein to alumina in addition to other surfaces has shown why it is an effective flocculating agent for the range of impurities found in water supplies. The ability to tailor different surface layers in combination with various surfactants also offers the potential for adsorbed protein to be used in separation technologies.

  3. Selective radiolabeling of cell surface proteins to a high specific activity

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, J.A.; Lau, A.L.; Cunningham, D.D.

    1987-02-10

    A procedure was developed for selective radiolabeling of membrane proteins on cells to higher specific activities than possible with available techniques. Cell surface amino groups were derivatized with /sup 125/I-(hydroxyphenyl)propionyl groups via /sup 125/I-sulfosuccinimidyl (hydroxyphenyl)propionate (/sup 125/II-sulfo-SHPP). This reagent preferentially labeled membrane proteins exposed at the cell surface of erythrocytes as assessed by the degree of radiolabel incorporation into erythrocyte ghost proteins and hemoglobin. Comparison with the lactoperoxidase-(/sup 125/I)iodide labeling technique revealed that /sup 125/I-sulfo-SHPP labeled cell surface proteins to a much higher specific activity and hemoglobin to a much lower specific activity. Additionally, this reagent was used for selective radiolabeling of membrane proteins on the cytoplasmic face of the plasma membrane by blocking exofacial amino groups with uniodinated sulfo-SHPP, lysing the cells, and then incubating them with /sup 125/I-sulfo-SHPP. Exclusive labeling of either side of the plasma membrane was demonstrated by the labeling of some marker proteins with well-defined spacial orientations on erythroctyes. Transmembrane proteins such as the epidermal growth factor receptor on cultured cells could also be labeled differentially from either side of the plasma membrane.

  4. Synthesis of an endothelial cell mimicking surface containing thrombomodulin and endothelial protein C receptor

    Science.gov (United States)

    Kador, Karl Erich

    Synthetic materials for use in blood contacting applications have been studied for many years with limited success. One of the main areas of need for these materials is the design of synthetic vascular grafts for use in the hundreds of thousands of patients who have coronary artery bypass grafting, many without suitable veins for autologous grafts. The design of these grafts is constrained by two common modes of failure, the formation of intimal hyperplasia (IH) and thrombosis. IH formation has been previously linked to a mismatching of the mechanical properties of the graft and has been overcome by creating grafts using materials whose compliance mimics that of the native artery. Several techniques and surface modification have been designed to limit thrombosis on the surface of synthetic materials. One which has shown the greatest promise is the immobilization of Thrombomodulin (TM), a protein found on the endothelial cell membrane lining native blood vessels involved in the activation of the anticoagulant Protein C (PC). While TM immobilization has been shown to arrest thrombin formation and limit fibrous formations in in-vitro and in-vivo experiments, it has shown to be transport limiting under arterial flow. On the endothelial cell surface, TM is co-localized with Endothelial Protein C Receptor (EPCR), which increases PC transport onto the cell surface and increases PC activation via TM between 20-100 fold. This dissertation will describe the chemical modification of medical grade polyurethane (PU), whose compliance has been shown to match that of native arteries. This modification will enable the immobilization of two proteins on an enzymatically relevant scale estimated at less than 10 nm. This dissertation will further describe the immobilization of the proteins TM and EPCR, and analyze the ability of a surface co-immobilized with these proteins to activate the anticoagulant PC. Finally, it will compare the ability of this co-immobilized surface to delay

  5. The surface-associated protein of Staphylococcus saprophyticus is a lipase.

    Science.gov (United States)

    Sakinc, Türkan; Woznowski, Magdalena; Ebsen, Michael; Gatermann, Sören G

    2005-10-01

    Staphylococcus saprophyticus surface-associated protein (Ssp) was the first surface protein described for this organism. Ssp-positive strains display a fuzzy layer of surface-associated material in electron micrographs, whereas Ssp-negative strains appear to be smooth. The physiologic function of Ssp, however, has remained elusive. To clone the associated gene, we determined the N-terminal sequence, as well as an internal amino acid sequence, of the purified protein. We derived two degenerate primers from these peptide sequences, which we used to identify the ssp gene from genomic DNA of S. saprophyticus 7108. The gene was cloned by PCR techniques and was found to be homologous to genes encoding staphylococcal lipases. In keeping with this finding, strains 7108 and 9325, which are Ssp positive, showed lipase activity on tributyrylglycerol agar plates, whereas the Ssp-negative strain CCM883 did not. Association of enzyme activity with the cloned DNA was proven by introducing the gene into Staphylococcus carnosus TM300. When wild-type strain 7108 and an isogenic mutant were analyzed by transmission electron microscopy, strain 7108 exhibited the fuzzy surface layer, whereas the mutant appeared to be smooth. Lipase activity and the surface appendages could be restored by reintroduction of the cloned gene into the mutant. Experiments using immobilized collagen type I did not provide evidence for the involvement of Ssp in adherence to this matrix protein. Our experiments thus provided evidence that Ssp is a surface-associated lipase of S. saprophyticus.

  6. Key role for clumping factor B in Staphylococcus aureus nasal colonization of humans

    NARCIS (Netherlands)

    H.F.L. Wertheim (Heiman); E.J. Walsh (Evelyn); R.S.R. Choudhurry (Roos); D.C. Melles (Damian); H.A.M. Boelens (Hélène); H. Miajlovic (Helen); H.A. Verbrugh (Henri); T.J. Foster (Timothy); A.F. van Belkum (Alex)

    2008-01-01

    textabstractBACKGROUND: Staphylococcus aureus permanently colonizes the vestibulum nasi of one-fifth of the human population, which is a risk factor for autoinfection. The precise mechanisms whereby S. aureus colonizes the nose are still unknown. The staphylococcal cell-wall protein clumping factor

  7. 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...

  8. 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...% of patients with S. aureus IE (p =0.05). In conclusion, CoNS IE was associated with a long diagnostic delay and high in-hospital mortality, whereas post-discharge prognosis was better in this group of patients compared to patients with IE due to S. aureus....

  9. The role of staphylothrombin-mediated fibrin deposition in catheter-related Staphylococcus aureus infections.

    Science.gov (United States)

    Vanassche, Thomas; Peetermans, Marijke; Van Aelst, Lucas N L; Peetermans, Willy E; Verhaegen, Jan; Missiakas, Dominique M; Schneewind, Olaf; Hoylaerts, Marc F; Verhamme, Peter

    2013-07-01

    Staphylococcus aureus (S. aureus) is a frequent cause of catheter-related infections. S. aureus secretes the coagulases staphylocoagulase and von Willebrand factor-binding protein, both of which form a staphylothrombin complex upon binding to prothrombin. Although fibrinogen and fibrin facilitate the adhesion of S. aureus to catheters, the contribution of staphylothrombin-mediated fibrin has not been examined. In this study, we use a S. aureus mutant lacking both coagulases (Δcoa/vwb) and dabigatran, a pharmacological inhibitor of both staphylothrombin and thrombin, to address this question. Genetic absence or chemical inhibition of pathogen-driven coagulation reduced both fibrin deposition and the retention of S. aureus on catheters in vitro. In a mouse model of jugular vein catheter infection, dabigatran reduced bacterial load on jugular vein catheters, as well as metastatic kidney infection. Importantly, inhibition of staphylothrombin improved the efficacy of vancomycin treatment both in vitro and in the mouse model. PMID:23532100

  10. The influence of the surface properties of silicon-fluorine hydrogel on protein adsorption.

    Science.gov (United States)

    Xie, Haijiao; Zhao, Zhengbai; An, Shuangshuang; Jiang, Yong

    2015-12-01

    A range of fluorinated hydrogels were synthesized using the copolymerization of 1, 1, 1, 3, 3, 3-hexafluoroisopropyl methacrylate (HFMA) or 1H, 1H, 7H-dodecafluoroheptyl methacrylate (DFMA) with hydrophilic monomers. Bovine serum albumin (BSA) and Lysozyme (LZM) were chosen as model proteins to investigate the performance of protein adsorption on the surface of these fluorinated hydrogels. It was found that the performance of the fluorinated hydrogels toward protein adsorption was different for different proteins; simultaneously, the amount of protein adsorption was related to but not linear with the fluorine content on the hydrogel surface. With increasing HFMA content, the mass of BSA adsorption increased in the first stage and then decreased, meanwhile the mass of LZM adsorption exhibited an upward trend in general. In addition, the amount of protein adsorption was also related to the type and length of the fluorinated groups. The hydrogels made from DFMA behaved better than HFMA hydrogels in terms of reducing protein adsorption. This study might provide further reference in choosing fluorine monomer to prepare protein-repelling hydrogels.

  11. Comparative proteomic analysis of surface proteins of Trichinella spiralis muscle larvae and intestinal infective larvae.

    Science.gov (United States)

    Liu, Ruo Dan; Cui, Jing; Liu, Xiao Lin; Jiang, Peng; Sun, Ge Ge; Zhang, Xi; Long, Shao Rong; Wang, Li; Wang, Zhong Quan

    2015-10-01

    The critical step for Trichinella spiralis infection is that muscle larvae (ML) are activated to intestinal infective larvae (IIL) and invade intestinal epithelium to further develop. The IIL is its first invasive stage, surface proteins are directly exposed to host environment and are crucial for larval invasion and development. In this study, shotgun LC-MS/MS was used to analyze surface protein profiles of ML and IIL. Totally, 41 proteins common to both larvae, and 85 ML biased and 113 IIL biased proteins. Some proteins (e.g., putative scavenger receptor cysteine-rich domain protein and putative onchocystatin) were involved in host-parasite interactions. Gene ontology analysis revealed that proteins involved in generation of precursor metabolites and energy; and nucleobase, nucleoside, nucleotide and nucleic acid metabolic process were enriched in IIL at level 4. Some IIL biased proteins might play important role in larval invasion and development. qPCR results confirmed the high expression of some genes in IIL. Our study provides new insights into larval invasion, host-Trichinella interaction and for screening vaccine candidate antigens. PMID:26184560

  12. Exploring the transcriptome of Staphylococcus aureus in its natural niche.

    Science.gov (United States)

    Chaves-Moreno, Diego; Wos-Oxley, Melissa L; Jáuregui, Ruy; Medina, Eva; Oxley, Andrew Pa; Pieper, Dietmar H

    2016-01-01

    Staphylococcus aureus is an important human pathogen and commensal, where the human nose is the predominant reservoir. To better understand its behavior in this environmental niche, RNA was extracted from the anterior nares of three documented S. aureus carriers and the metatranscriptome analyzed by RNAseq. In addition, the in vivo transcriptomes were compared to previously published transcriptomes of two in vitro grown S. aureus strains. None of the in vitro conditions, even growth in medium resembling the anterior nares environment, mimicked in vivo conditions. Survival in the nose was strongly controlled by the limitation of iron and evident by the expression of iron acquisition systems. S. aureus populations in different individuals clearly experience different environmental stresses, which they attempt to overcome by the expression of compatible solute biosynthetic pathways, changes in their cell wall composition and synthesis of general stress proteins. Moreover, the expression of adhesins was also important for colonization of the anterior nares. However, different S. aureus strains also showed different in vivo behavior. The assessment of general in vivo expression patterns and commonalities between different S. aureus strains will in the future result in new knowledge based strategies for controlling colonization. PMID:27641137

  13. The Possible Role of Staphylococcus epidermidis LPxTG Surface Protein SesC in Biofilm Formation.

    Science.gov (United States)

    Khodaparast, Laleh; Khodaparast, Ladan; Shahrooei, Mohammad; Stijlemans, Benoit; Merckx, Rita; Baatsen, Pieter; O'Gara, James P; Waters, Elaine; Van Mellaert, Lieve; Van Eldere, Johan

    2016-01-01

    Staphylococcus epidermidis is the most common cause of device-associated infections. It has been shown that active and passive immunization in an animal model against protein SesC significantly reduces S. epidermidis biofilm-associated infections. In order to elucidate its role, knock-out of sesC or isolation of S. epidermidis sesC-negative mutants were attempted, however, without success. As an alternative strategy, sesC was introduced into Staphylococcus aureus 8325-4 and its isogenic icaADBC and srtA mutants, into the clinical methicillin-sensitive S. aureus isolate MSSA4 and the MRSA S. aureus isolate BH1CC, which all lack sesC. Transformation of these strains with sesC i) changed the biofilm phenotype of strains 8325-4 and MSSA4 from PIA-dependent to proteinaceous even though PIA synthesis was not affected, ii) converted the non-biofilm-forming strain 8325-4 ica::tet to a proteinaceous biofilm-forming strain, iii) impaired PIA-dependent biofilm formation by 8325-4 srtA::tet, iv) had no impact on protein-mediated biofilm formation of BH1CC and v) increased in vivo catheter and organ colonization by strain 8325-4. Furthermore, treatment with anti-SesC antibodies significantly reduced in vitro biofilm formation and in vivo colonization by these transformants expressing sesC. These findings strongly suggest that SesC is involved in S. epidermidis attachment to and subsequent biofilm formation on a substrate.

  14. Secretome Analysis Defines the Major Role of SecDF in Staphylococcus aureus Virulence

    OpenAIRE

    Chantal Quiblier; Kati Seidl; Bernd Roschitzki; Zinkernagel, Annelies S.; Brigitte Berger-Bächi; Senn, Maria M.

    2013-01-01

    The Sec pathway plays a prominent role in protein export and membrane insertion, including the secretion of major bacterial virulence determinants. The accessory Sec constituent SecDF has been proposed to contribute to protein export. Deletion of Staphylococcus aureus secDF has previously been shown to reduce resistance, to alter cell separation, and to change the expression of certain virulence factors. To analyse the impact of the secDF deletion in S. aureus on protein secretion, a quantita...

  15. Two cell surface proteins bind the sponge Microciona prolifera aggregation factor.

    Science.gov (United States)

    Varner, J A; Burger, M M; Kaufman, J F

    1988-06-15

    Two extracellular matrix cell surface proteins which bind the proteoglycan-like aggregation factor from the marine sponge Microciona prolifera (MAF) and which may function as physiological receptors for MAF were identified and characterized for the first time. By probing nitrocellulose blots of nonreducing sodium dodecyl sulfate gels containing whole sponge cell protein with iodinated MAF, a 210- and a 68-kDa protein, which have native molecular masses of approximately 200-400 and 70 kDa, were identified. MAF binding to blots is species-specific. It is also sensitive to reduction and is completely abolished by pretreatment of live cells with proteases, as was cellular aggregation, indicating that the 210- and 68-kDa proteins may be located on the cell surface. The additional observations that the 68 kDa is an endoglycosidase F-sensitive glycoprotein and that antisera against whole sponge cells or membranes can immunoprecipitate the 210 kDa when prebound to intact cells are consistent with a cell surface location. Both proteins can be isolated from sponge cell membranes and from the sponge skeleton (insoluble extracellular matrix), but the 210-kDa MAF-binding protein can also be found in the soluble extracellular matrix (buffer washes of cells and skeleton) as well. A third MAF-binding protein of molecular mass 95 kDa was also found in the sponge extracellular matrix but rarely on cells. Both of the cell-associated 210- and 68-kDa proteins are nonintegral membrane proteins, based on Triton X-114 phase separation, flotation of liposomes containing sponge membrane lysates, and their extraction from membranes by buffer washes. Both proteins bind MAF affinity resins, indicating that they each exhibit a moderate affinity for MAF under native conditions. They can also be separated from each other and from the bulk of the protein in an octylpolyoxyethylene extract of membranes by fast protein liquid chromatography Mono Q anion exchange chromatography, as assessed by native

  16. Expression, purification, crystallization and preliminary crystallographic analysis of a putative Clostridium difficile surface protein Cwp19

    International Nuclear Information System (INIS)

    Cwp19 is a putatively surface-located protein from Clostridium difficile. A recombinant N-terminal protein (residues 27–401) lacking the signal peptide and the C-terminal cell-wall-binding repeats (PFam04122) was crystallized using the sitting-drop vapour-diffusion method and diffracted to 2 Å resolution. Cwp19 is a putatively surface-located protein from Clostridium difficile. A recombinant N-terminal protein (residues 27–401) lacking the signal peptide and the C-terminal cell-wall-binding repeats (PFam04122) was crystallized using the sitting-drop vapour-diffusion method and diffracted to 2 Å resolution. The crystal appeared to belong to the primitive monoclinic space group P21, with unit-cell parameters a = 109.1, b = 61.2, c = 109.2 Å, β = 111.85°, and is estimated to contain two molecules of Cwp19 per asymmetric unit

  17. Molecular kinetics of proteins at the surface of porcine sperm before and during fertilization.

    Science.gov (United States)

    Tsai, P S; Gadella, B M

    2009-01-01

    Fertilization is a decisive moment in life and enables the combination of the DNA from two gametes to ultimately form a new organism. The sperm surface, especially the head area, has distinguishable subdomains that are involved in distinct fertilization processes. It is known that the sperm head surface undergoes constant remodelling during epididymal maturation and migration in the male and female genital tract. But intriguingly, the identity, origin and spatial ordering of proteins at the sperm surface that are involved in mammalian fertilization are essentially unknown. This review deals with sperm surface protein modifications that are under somatic cell control. As soon as the sperm is released from the seminiferous tubules it is subjected to these modifications. These surface reorganisations continue until the sperm reside in the fallopian tube where they meet the oocyte and may fertilize it. Most likely, a selective process allows only functionally mature and intact sperm to optimally interact and fertilize the oocyte. Recent data suggest that even the perivitelline fluid is involved in sperm surface remodelling as it contains factors which could facilitate the first penetrating sperm to fertilize the oocyte. In this contribution, the kinetics of proteins at the sperm surface will be overviewed. Better understanding of this would help to design strategies to improve male fertility or to devise novel contraceptives.

  18. Gradation of proteins and cells attached to the surface of bio-inert zwitterionic polymer brush.

    Science.gov (United States)

    Li, Lifu; Nakaji-Hirabayashi, Tadashi; Kitano, Hiromi; Ohno, Kohji; Kishioka, Takahiro; Usui, Yuki

    2016-08-01

    A self-assembled monolayer (SAM) of a 2-bromoisobutyryl end group-carrying initiator for atom transfer radical polymerization (ATRP) was constructed on the surface of silicon wafer or glass substrates via a silane-coupling reaction. When the initiator SAM was irradiated with UV light at 254nm, the surface density of bromine atoms was reduced by the scission of CBr bonds as observed by XPS. With the surface-initiated ATRP of the zwitterionic vinyl monomer, carboxymethyl betaine (CMB), the surface density of PCMB brushes could be easily varied by changing the irradiation period of UV light prior to the polymerization. Furthermore, by using a UV-cut shutter sliding above the initiator SAM-modified substrate at a constant speed, the degree of bromine atom removal could be linearly varied along the direction of movement of the shutter. Consequently, the amount of both proteins adsorbed and cells adhered to the PCMB brush-covered substrate could easily be controlled by the gradation of the surface density of PCMB brushes, which suppressed protein adsorption and cell adhesion. Such a technique is very simple and useful for the regulation of the surface density of adsorbed proteins and adhered cells on an originally bio-inert surface. PMID:27085477

  19. Activity of Tri-N-Butyl Tin maleate in carpets against Staphylococcus aureus and Aspergillus niger, verified through two methodologies: Inhibition Halo (HZ and Inhibition Surface (Print Atividade de tri-n-butyl tin maleate em carpetes contra Staphylococcus aureus e Aspergillus niger, verificada através de duas metodologias: Zona de Inibição (ZI e Superfície de Inibição (Impressão

    Directory of Open Access Journals (Sweden)

    Satiko Uehara

    2008-06-01

    Full Text Available The aim of the present study was to verify the activity of the Tri-N-Butyl Tin maleate compound against Staphylococcus aureus and Aspergillus niger, after its industrial application in 40 samples of carpets of different materials (polypropylene, polyester, polyamide and wool. The qualitative assays were performed through two methodologies: Inhibition Halo (HZ and Inhibition of Surface (Print. The carpet with the product inhibited 100% of bacterial (Staphylococcus aureus and fungi (Aspergillus niger growth, under the conditions of this study. The microbial inhibition was higher in upper portion of carpets. The methodologies employed appear to be adequate to test the bactericide and fungicide activities of the Tri-N-Butyl Tin maleate. The print methodology confirmed the results obtained by the inhibition zone assay. Further studies using the same methodologies are needed to confirm our results.O objetivo do presente estudo foi verificar a atividade do composto maleato de estanho tri-n-butílico contra Staphylococcus aureus e Aspergillus niger, após sua aplicação industrial em 40 amostras de carpetes de diferentes materiais (polipropileno, poliéster, poliamida e lã. Os ensaios qualitativos foram realizados através de duas metodologias: Zona de Inibição (ZI e Superfície de Inibição (Impressão. Os carpetes tratados com o produto apresentaram 100% de inibição de crescimento bacteriano (Staphylococcus aureus e fúngico (Aspergillus niger, sob as condições desse estudo. A inibição de crescimento microbiano foi mais elevada na porção superior dos carpetes. As metodologias empregadas parecem ser adequadas para testar a atividade bactericida e fungicida do maleato de estanho tri-n-butílico. A metodologia de impressão confirmou os resultados obtidos no ensaio de zona de inibição. Estudos futuros utilizando as mesmas metodologias são necessários para confirmação destes dados.

  20. The impact of a carbon nanotube on the cholesterol domain localized on a protein surface

    CERN Document Server

    Gburski, Zygmunt; Raczynski, Przemyslaw; 10.1016/j.ssc.2009.12.005

    2011-01-01

    The influence of a single walled carbon nanotube on the structure of a cholesterol cluster (domain) developed over the surface of the endothelial protein 1LQV has been investigated using the classical molecular dynamics (MD) simulation technique. We have observed a substantial impact of carbon nanotube on the arrangement of the cholesterol domain. The carbon nanotube can drag out cholesterol molecules, remarkable reducing the volume of the domain settled down on the protein.

  1. Cell surface molecules and fibronectin-mediated cell adhesion: effect of proteolytic digestion of membrane proteins

    OpenAIRE

    1982-01-01

    Proteases have been used as a tool to investigate the role of surface molecules in fibronectin-mediated cell adhesion. Proteolytic digestion of membrane-proteins by pronase (1 mg/ml for 20 min at 37 degrees C) completely inhibited adhesion of baby hamster kidney (BHK) fibroblasts on fibronectin-coated plastic dishes. Various degrees of inhibition were also obtained after treatment with proteinase K, chymotrypsin, papain, subtilopeptidase A, and thermolysin. Protein synthesis was required to r...

  2. Exhaustive comparison and classification of ligand-binding surfaces in proteins

    OpenAIRE

    Murakami, Yoichi; Kinoshita, Kengo; Kinjo, Akira R.; Nakamura, Haruki

    2013-01-01

    Many proteins function by interacting with other small molecules (ligands). Identification of ligand-binding sites (LBS) in proteins can therefore help to infer their molecular functions. A comprehensive comparison among local structures of LBSs was previously performed, in order to understand their relationships and to classify their structural motifs. However, similar exhaustive comparison among local surfaces of LBSs (patches) has never been performed, due to computational complexity. To e...

  3. Study of antioxidant activity of sheep visceral protein hydrolysate: Optimization using response surface methodology

    OpenAIRE

    Meshginfar, Nasim; Sadeghi-Mahoonak, Alireza; Ziaiifar, Aman Mohammad; Ghorbani, Mohammad; Kashaninejad, Mahdi

    2014-01-01

    BACKGROUND The main objective of this experiment was optimal use of none edible protein source to increase nutritional value of production with high biological function, including antioxidant activity. METHODS Sheep visceral (stomach and intestine) was used as substrate. Response surface methodology (RSM) was used to optimize hydrolysis conditions for preparing protein hydrolysate from the sheep visceral, using alcalase 2.4 l enzyme. The investigated factors were temperature (43-52 °C), time ...

  4. Mycoplasma pneumoniae Infection: Role of a Surface Protein in the Attachment Organelle

    Science.gov (United States)

    Hu, P. C.; Cole, R. M.; Huang, Y. S.; Graham, J. A.; Gardner, D. E.; Collier, A. M.; Clyde, W. A.

    1982-04-01

    Attachment of Mycoplasma pneumoniae to host cells by means of a specialized terminus initiates infection. Monoclonal antibodies to a surface protein (P1) inhibit this process, and react with a region of the tip covered with peplomer-like particles. Since antibodies against the P1 protein are generated by natural and experimental infection and by immunization, the substance may be an important determinant of protective immunity.

  5. Identification of Pneumococcal Surface Protein A as a Lactoferrin-Binding Protein of Streptococcus pneumoniae

    OpenAIRE

    Hammerschmidt, Sven; Bethe, Gesina; H. Remane, Petra; Chhatwal, Gursharan S.

    1999-01-01

    Lactoferrin (Lf), an iron-sequestering glycoprotein, predominates in mucosal secretions, where the level of free extracellular iron (10−18 M) is not sufficient for bacterial growth. This represents a mechanism of resistance to bacterial infections by prevention of colonization of the host by pathogens. In this study we were able to show that Streptococcus pneumoniae specifically recognizes and binds the iron carrier protein human Lf (hLf). Pretreatment of pneumococci with proteases reduced hL...

  6. Human MHC class I antigens are associated with a 90-kDa cell surface protein.

    Science.gov (United States)

    Ferm, M T; Grönberg, A

    1991-08-01

    Human MHC class I proteins are expressed on almost all nucleated cells as a heavy chain (about 45 kDa) non-covalently associated with beta 2-microglobulin (12 kDa). In this report we show that MHC class I (MHC-I) proteins can also be associated with a 90-kDa protein in the cell membrane. Surface-radiolabelled cells were treated with dithiobis succinimidyl propionate (DSP) in order to preserve multimer protein complexes during cell lysis. The lysates were immunoprecipitated and analysed by SDS-PAGE and autoradiography. Immunoprecipitation of human MHC-I proteins co-precipitated another protein of about 90 kDa in molecular weight-p90. p90 was coprecipitated from all the MHC-I expressing cells tested: U937, Raji, Molt-4 and IFN-gamma treated K562, but not from untreated, MHC-I negative K562. A 90-kDa protein was also co-precipitated with MHC-I from fresh peripheral blood mononuclear cells (PBMC). Furthermore, p90 was coprecipitated by different MoAbs to the MHC-I heavy chain or beta 2-microglobulin, but not by control antibodies. Two additional co-precipitating proteins at 34 kDa and 28 kDa were seen in MHC-I precipitates from Raji cells. Our results suggest that MHC-I proteins and the 90-kDa protein are associated in the cell membrane, probably by a close but weak, non-covalent interaction. Two additional cell surface proteins at 34 kDa and 28 kDa seem to be MHC-I associated on Raji Burkitt's lymphoma cells.

  7. Evaluation of the Staphylococcus aureus Class C Nonspecific Acid Phosphatase (SapS) as a Reporter for Gene Expression and Protein Secretion in Gram-Negative and Gram-Positive Bacteria▿

    OpenAIRE

    Du Plessis, Erika; Theron, Jacques; Berger, Eldie; Louw, Maureen

    2007-01-01

    A phosphatase secreted by Staphylococcus aureus strain 154 has previously been characterized and classified as a new member of the bacterial class C family of nonspecific acid phosphatases. As the acid phosphatase activity can be easily detected with a cost-effective plate screen assay, quantitatively measured by a simple enzyme assay, and detected by zymography, its potential use as a reporter system was investigated. The S. aureus acid phosphatase (sapS) gene has been cloned and expressed f...

  8. Antigenicity and Immunogenicity of Rotavirus VP6 Protein Expressed on the Surface of Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    L. E. Esteban

    2013-01-01

    Full Text Available Group A rotaviruses are the major etiologic agents of acute gastroenteritis worldwide in children and young animals. Among its structural proteins, VP6 is the most immunogenic and is highly conserved within this group. Lactococcus lactis is a food-grade, Gram-positive, and nonpathogenic lactic acid bacteria that has already been explored as a mucosal delivery system of heterologous antigens. In this work, the nisin-controlled expression system was used to display the VP6 protein at the cell surface of L. lactis. Conditions for optimal gene expression were established by testing different nisin concentrations, cell density at induction, and incubation times after induction. Cytoplasmic and cell wall protein extracts were analyzed by Western blot and surface expression was confirmed by flow cytometry. Both analysis provided evidence that VP6 was efficiently expressed and displayed on the cell surface of L. lactis. Furthermore, the humoral response of mice immunized with recombinant L. lactis was evaluated and the displayed recombinant VP6 protein proved to be immunogenic. In conclusion, this is the first report of displaying VP6 protein on the surface of L. lactis to induce a specific immune response against rotavirus. These results provide the basis for further evaluation of this VP6-displaying L. lactis as a mucosal delivery vector in a mouse model of rotavirus infection.

  9. Electrochemical Characterization of Protein Adsorption onto YNGRT-Au and VLGXE-Au Surfaces

    OpenAIRE

    Hanna Trzeciakiewicz; Jose Esteves-Villanueva; Rania Soudy; Kamaljit Kaur; Sanela Martic-Milne

    2015-01-01

    The adsorption of the proteins CD13, mucin and bovine serum albumin on VLGXE-Au and YNGRT-Au interfaces was monitored by electrochemical impedance spectroscopy in the presence of [Fe(CN)6]3−/4−. The hydrophobicity of the Au surface was tailored using specific peptides, blocking agents and diluents. The combination of blocking agents (ethanolamine or n-butylamine) and diluents (hexanethiol or 2-mercaptoethanol) was used to prepare various peptide-modified Au surfaces. Protein adsorption onto t...

  10. Aquifex aeolicus PilT, Homologue of a Surface Motility Protein, Is a Thermostable Oligomeric NTPase

    OpenAIRE

    Herdendorf, Timothy J.; McCaslin, Darrell R.; Forest, Katrina T.

    2002-01-01

    Bacterial surface motility works by retraction of surface-attached type IV pili. This retraction requires the PilT protein, a member of a large family of putative NTPases from type II and IV secretion systems. In this study, the PilT homologue from the thermophilic eubacterium Aquifex aeolicus was cloned, overexpressed, and purified. A. aeolicus PilT was shown to be a thermostable ATPase with a specific activity of 15.7 nmol of ATP hydrolyzed/min/mg of protein. This activity was abolished whe...

  11. 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.

  12. 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. PMID:25687923

  13. Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily.

    Science.gov (United States)

    Matsunaga, James; Barocchi, Michele A; Croda, Julio; Young, Tracy A; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A; Reis, Mitermayer G; Riley, Lee W; Haake, David A; Ko, Albert I

    2003-08-01

    Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudogene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis. PMID:12890019

  14. Identification of novel surface-exposed proteins of Rickettsia rickettsii by affinity purification and proteomics.

    Directory of Open Access Journals (Sweden)

    Wenping Gong

    Full Text Available Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs, which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens.

  15. Hydrophobic surface protein masking by the opportunistic fungal pathogen Candida albicans.

    OpenAIRE

    Hazen, K C; Hazen, B W

    1992-01-01

    Ultrastructural and biochemical analyses of hydrophobic and hydrophilic yeast cell surface proteins of Candida albicans were performed. Hydrophobic and hydrophilic yeast cells were obtained by growth at 23 and 37 degrees C, respectively. In addition, hydrophilic yeast cells were converted to surface hydrophobicity by treatment with tunicamycin and dithiothreitol. When freeze-etched cells were examined, the temperature-induced hydrophilic cells had long (0.198 micron), compact, evenly distribu...

  16. Hydrophilic crosslinked-polymeric surface capable of effective suppression of protein adsorption

    Science.gov (United States)

    Kamon, Yuri; Inoue, Naoko; Mihara, Erika; Kitayama, Yukiya; Ooya, Tooru; Takeuchi, Toshifumi

    2016-08-01

    We investigated the nonspecific adsorption of proteins towards three hydrophilic crosslinked-polymeric thin layers prepared by surface-initiated atom transfer radical polymerization using N,N‧-methylenebisacrylamide, 2-(methacryloyloxy)ethyl-[N-(2-methacryloyloxy)ethyl]phosphorylcholine (MMPC), or 6,6‧-diacryloyl-trehalose crosslinkers. Protein binding experiments were performed by surface plasmon resonance with six proteins of different pI values including α-lactalbumin, bovine serum albumin (BSA), myoglobin, ribonuclease A, cytochrome C, and lysozyme in buffer solution at pH 7.4. All of the obtained crosslinked-polymeric thin layers showed low nonspecific adsorption of negatively charged proteins at pH 7.4 such as α-lactalbumin, BSA, and myoglobin. Nonspecific adsorption of positively charged proteins including ribonuclease A, cytochrome C, and lysozyme was the lowest for poly(MMPC). These results suggest poly(MMPC) can effectively reduce nonspecific adsorption of a wide range of proteins that are negatively or positively charged at pH 7.4. MMPC is a promising crosslinker for a wide range of polymeric materials requiring low nonspecific protein binding.

  17. Using extremely halophilic bacteria to understand the role of surface charge and surface hydration in protein evolution, folding, and function

    Science.gov (United States)

    Hoff, Wouter; Deole, Ratnakar; Osu Collaboration

    2013-03-01

    Halophilic Archaea accumulate molar concentrations of KCl in their cytoplasm as an osmoprotectant, and have evolved highly acidic proteomes that only function at high salinity. We examine osmoprotection in the photosynthetic Proteobacteria Halorhodospira halophila. We find that H. halophila has an acidic proteome and accumulates molar concentrations of KCl when grown in high salt media. Upon growth of H. halophila in low salt media, its cytoplasmic K + content matches that of Escherichia coli, revealing an acidic proteome that can function in the absence of high cytoplasmic salt concentrations. These findings necessitate a reassessment of two central aspects of theories for understanding extreme halophiles. We conclude that proteome acidity is not driven by stabilizing interactions between K + ions and acidic side chains, but by the need for maintaining sufficient solvation and hydration of the protein surface at high salinity through strongly hydrated carboxylates. We propose that obligate protein halophilicity is a non-adaptive property resulting from genetic drift in which constructive neutral evolution progressively incorporates weakly stabilizing K + binding sites on an increasingly acidic protein surface.

  18. Crystal structures of Bbp from Staphylococcus aureus reveal the ligand binding mechanism with Fibrinogen α.

    Science.gov (United States)

    Zhang, Xinyue; Wu, Meng; Zhuo, Wei; Gu, Jinke; Zhang, Sensen; Ge, Jingpeng; Yang, Maojun

    2015-10-01

    Bone sialoprotein-binding protein (Bbp), a MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) family protein expressed on the surface of Staphylococcus aureus (S. aureus), mediates adherence to fibrinogen α (Fg α), a component in the extracellular matrix of the host cell and is important for infection and pathogenesis. In this study, we solved the crystal structures of apo-Bbp(273-598) and Bbp(273-598)-Fg α(561-575) complex at a resolution of 2.03 Å and 1.45 Å, respectively. Apo-Bbp(273-598) contained the ligand binding region N2 and N3 domains, both of which followed a DE variant IgG fold characterized by an additional D1 strand in N2 domain and D1' and D2' strands in N3 domain. The peptide mapped to the Fg α(561-575) bond to Bbp(273-598) on the open groove between the N2 and N3 domains. Strikingly, the disordered C-terminus in the apo-form reorganized into a highly-ordered loop and a β-strand G'' covering the ligand upon ligand binding. Bbp(Ala298-Gly301) in the N2 domain of the Bbp(273-598)-Fg α(561-575) complex, which is a loop in the apo-form, formed a short α-helix to interact tightly with the peptide. In addition, Bbp(Ser547-Gln561) in the N3 domain moved toward the binding groove to make contact directly with the peptide, while Bbp(Asp338-Gly355) and Bbp(Thr365-Tyr387) in N2 domain shifted their configurations to stabilize the reorganized C-terminus mainly through strong hydrogen bonds. Altogether, our results revealed the molecular basis for Bbp-ligand interaction and advanced our understanding of S. aureus infection process. PMID:26349459

  19. Immune blot analysis of viral surface proteins in serum and liver of patients with chronic hepatitis B virus infection.

    Science.gov (United States)

    Gerken, G; Manns, M; Gerlich, W H; Hess, G; Meyer zum Büschenfelde, K H

    1989-12-01

    The small and the middle surface proteins of hepatitis virus form either the virion or the 22 nm particle both of which are secreted. The large surface protein by itself remains cell bound in artificially transfected cell culture unless it is accompanied by an excess of the smaller protens. Its behavior in vivo is not yet well studied. Using specific monoclonal antibodies for immunoblotting, we found an abundance of small surface protein in the serum of chronic virus carriers and moderate amounts in the liver irrespective of viremia. The large surface protein was present in the serum and the liver of viremic carriers. In nonviremic carriers, the large protein was absent from serum, but in the liver a shorter form of the large protein was readily detectable. These findings suggest a complex regulatory mechanism of the viral surface protein depending on the expression of other viral gene products. PMID:2621452

  20. Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Sasnauskas Kęstutis

    2011-05-01

    Full Text Available Abstract Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN and measles hemagglutinin (MeH in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A and is closely associated with small heat shock proteins (sHsps that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of

  1. Mapping hydrophobicity on the protein molecular surface at atom-level resolution.

    Directory of Open Access Journals (Sweden)

    Dan V Nicolau

    Full Text Available A precise representation of the spatial distribution of hydrophobicity, hydrophilicity and charges on the molecular surface of proteins is critical for the understanding of the interaction with small molecules and larger systems. The representation of hydrophobicity is rarely done at atom-level, as this property is generally assigned to residues. A new methodology for the derivation of atomic hydrophobicity from any amino acid-based hydrophobicity scale was used to derive 8 sets of atomic hydrophobicities, one of which was used to generate the molecular surfaces for 35 proteins with convex structures, 5 of which, i.e., lysozyme, ribonuclease, hemoglobin, albumin and IgG, have been analyzed in more detail. Sets of the molecular surfaces of the model proteins have been constructed using spherical probes with increasingly large radii, from 1.4 to 20 Å, followed by the quantification of (i the surface hydrophobicity; (ii their respective molecular surface areas, i.e., total, hydrophilic and hydrophobic area; and (iii their relative densities, i.e., divided by the total molecular area; or specific densities, i.e., divided by property-specific area. Compared with the amino acid-based formalism, the atom-level description reveals molecular surfaces which (i present an approximately two times more hydrophilic areas; with (ii less extended, but between 2 to 5 times more intense hydrophilic patches; and (iii 3 to 20 times more extended hydrophobic areas. The hydrophobic areas are also approximately 2 times more hydrophobicity-intense. This, more pronounced "leopard skin"-like, design of the protein molecular surface has been confirmed by comparing the results for a restricted set of homologous proteins, i.e., hemoglobins diverging by only one residue (Trp37. These results suggest that the representation of hydrophobicity on the protein molecular surfaces at atom-level resolution, coupled with the probing of the molecular surface at different geometric

  2. 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

  3. Role of a cell surface-associated protein in adherence and dental caries.

    OpenAIRE

    Bowen, W. H.; Schilling, K.; Giertsen, E; Pearson, S.; Lee, S. F.; Bleiweis, A; Beeman, D

    1991-01-01

    Insertional inactivation of the Streptococcus mutans spaP gene was used to construct an isogenic mutant (834) of strain NG8 (serotype c) which lacked the major cell surface-associated protein referred to as P1 (15). Results of several studies suggest that P1 is involved in the adherence of S. mutans to saliva-coated apatite surfaces. With an in vitro model system of hydroxyapatite (HA) beads coated with parotid saliva (PS) and additional HA surfaces coated with PS and in situ-formed glucan, i...

  4. Competitive Protein Adsorption of Albumin and Immunoglobulin G from Human Serum onto Polymer Surfaces

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hou, Xiaolin

    2010-01-01

    Competitive protein adsorption from human serum onto unmodified polyethylene terephthalate (PET) surfaces and plasma-polymerized PET surfaces, using the monomer diethylene glycol vinyl ether (DEGVE), has been investigated using radioactive labeling. Albumin and immunoglobulin G (IgG) labeled...... with two different iodine isotopes have been added to human serum solutions of different concentrations, and adsorption has been performed using adsorption times from approximately 5 s to 24 h. DEGVE surfaces showed indications of being nonfouling regarding albumin and IgG adsorption during competitive...

  5. Label-free Raman mapping of surface distribution of protein a and IgG biomolecules.

    Science.gov (United States)

    Combs, Zachary A; Chang, Sehoon; Clark, Tolecia; Singamaneni, Srikanth; Anderson, Kyle D; Tsukruk, Vladimir V

    2011-03-15

    We have demonstrated a nanoengineered substrate composed of micropatterned silver nanoparticles to be used for the label-free mapping of adsorbed biomolecules. We utilized surface-enhanced Raman scattering (SERS) phenomenon to monitor the known bioanalytes, protein A and human immunoglobulin G (IgG). The SERS substrate was composed of a poly(alylamine hydrochloride) (PAH)/poly(styrenesulfonate) (PSS) layer-by-layer (LbL) nanocoating micropatterned with silver nanoparticles confined to microscopic stripes. Selective adsorption of biomacromolecules is facilitated by the amine-terminated LbL nanocoating, which prevents the surface adsorption of positively charged protein A across the surface except on the patterned regions containing negatively charged silver nanoparticles. Furthermore, adsorption of IgG on predetermined regions is facilitated by the selective binding of the Fc region of IgG to protein A. This label-free SERS approach provides accurate, selective, and fast detection of protein A and IgG solutions with a nanomolar concentration, down to below 1 nM for IgG in solution. This method could also be utilized for the facile detection of proteins in field conditions as well as in clinical, forensic, industrial, and environmental laboratories. PMID:21294559

  6. Erratum: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing.

    Science.gov (United States)

    2015-01-01

    The author's email has been corrected in the publication of Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing. There was an error with the author, Jerry Zhou's, email. The author's email has been updated to: j.zhou@uws.edu.au from: jzho7551@mail.usyd.edu.au. PMID:26167960

  7. Dynamics of Agglutinin-Like Sequence (ALS) Protein Localization on the Surface of Candida Albicans

    Science.gov (United States)

    Coleman, David Andrew

    2009-01-01

    The ALS gene family encodes large cell-surface glycoproteins associated with "C. albicans" pathogenesis. Als proteins are thought to act as adhesin molecules binding to host tissues. Wide variation in expression levels among the ALS genes exists and is related to cell morphology and environmental conditions. "ALS1," "ALS3," and "ALS4" are three of…

  8. Preparation and recognition of surface molecularly imprinted core-shell microbeads for protein in aqueous solutions

    International Nuclear Information System (INIS)

    In this paper, a surface molecular imprinting technique was reported for preparing core-shell microbeads of protein imprinting, and bovine hemoglobin or bovine serum albumin were used as model proteins for studying the imprinted core-shell microbeads. 3-Aminophenylboronic acid (APBA) was polymerized onto the surface of polystyrene microbead in the presence of the protein templates to create protein-imprinted core-shell microbeads. The various samples were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and Brunauer-Emmett-Teller (BET) methods. The effect of pH on rebinding of the template hemoglobin, the specific binding and selective recognition were studied for the imprinted microbeads. The results show that the bovine hemoglobin-imprinted core-shell microbeads were successfully created. The shell was a sort of imprinted thin films with porous structure and larger surface areas. The imprinted microbeads have good selectivity for templates and high stability. Due to the recognition sites locating at or closing to the surface, these imprinted microbeads have good property of mass-transport. Unfortunately, the imprint technology was not successfully applied to imprinting bovine serum albumin (BSA).

  9. 'MYCOPLASMA PNEUMONIAE' INFECTION: ROLE OF A SURFACE PROTEIN IN THE ATTACHMENT ORGANELLE

    Science.gov (United States)

    Attachment of Mycoplasma pneumoniae to host cells by means of a specialized terminus initiates infection. Monoclonal antibodies to a surface protein (Pl) inhibit this process, and react with a region of the tip covered with peplomer-like particles. Since antibodies against the Pl...

  10. A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jarmander Johan

    2012-09-01

    Full Text Available Abstract Background The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H:gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. Results The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His6-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H:gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His6-tag facing the medium. Conclusions Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His6-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent

  11. Comparison of five methods for direct extraction of surface proteins from Listeria monocytogenes for proteomic analysis by orbitrap mass spectrometry.

    Science.gov (United States)

    Tiong, Hung King; Hartson, Steven; Muriana, Peter M

    2015-03-01

    Extracts of surface proteins, with minimal artifacts from contaminating cytosolic components, are highly desirable for investigating surface factors involved in the attachment and formation of biofilms by bacteria that are problematic in commercial food processing facilities. In this study, we compared the protein profiles of the food pathogen, Listeria monocytogenes, recovered after applying different surface protein extraction methods compiled from the literature: trypsin-enzymatic shaving with BICAM/sucrose or Tris/sucrose buffers (Tryp B+S, Tryp T+S), Tris-buffered urea (UB), lithium chloride (LiCl) and Tris-buffered urea applied with hypotonic-stressed cells (UB-Ghost), and subjected them to liquid chromatography tandem mass spectrometry and protein identification. The data indicate that the UB-Ghost extraction method provides a cleaner extract of surface proteins including the predicted (this study and the literature) or validated members (literature) from L. monocytogenes. This was determined by an accumulative lower unique peptide number exhibited by mass spectrometry for total cytoplasmic proteins among different surface extracts, with a majority of proteins demonstrating hydrophilic properties. The extracted proteins were from different functional categories and have associations with the cell surface, intermediary metabolism, information pathways, or functionally unknown proteins as suggested by in silico analyses performed by other groups (Leger and ListiList). The utilization of an optimized method for surface protein extraction should greatly facilitate identification by LC-MS/MS that could be useful to anyone working on molecular proteomics of bacterial surfaces.

  12. Evaluation of the Effectiveness of Surfactants and Denaturants to Elute and Denature Adsorbed Protein on Different Surface Chemistries.

    Science.gov (United States)

    Thyparambil, Aby A; Wei, Yang; Latour, Robert A

    2015-11-01

    The elution and/or denaturation of proteins from material surfaces by chemical excipients such as surfactants and denaturants is important for numerous applications including medical implant reprocessing, bioanalyses, and biodefense. The objective of this study was to develop and apply methods to quantitatively assess how surface chemistry and adsorption conditions influence the effectiveness of three commonly used surfactants (sodium dodecyl sulfate, n-octyl-β-d-glucoside, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and two denaturants (guanidium hydrochloride and urea) to elute protein (hen egg white lysozyme and bovine pancreatic ribonuclease A) from three different surface chemistries (silica glass, poly(methyl methacrylate), and high-density polyethylene). The structure and bioactivity of residual protein on the surface following elution were characterized using circular dichroism spectropolarimetry and enzyme assays to assess the extent of protein denaturation. Our results indicate that the denaturants were generally more effective than the surfactants in removing the adsorbed proteins from each type of surface. Also, the denaturing capacity of these excipients on the residual proteins on the surfaces was distinctly different from their influence on the proteins in solution and was unique for each of the adsorption conditions. Taken altogether, these results reveal that the effectiveness of surfactants and denaturants to elute and denature adsorbed protein is significantly influenced by surface chemistry and the conditions from which the protein was adsorbed. These results provide a basis for the selection, design, and further development of chemical agents for protein elution and surface decontamination. PMID:26449787

  13. THE EFFECTS OF SURFACE CHEMISTRY ON THE PROPERTIES OF PROTEINS CONFINED IN NANO-POROUS MATERIALS

    Energy Technology Data Exchange (ETDEWEB)

    Garrett, L. M.; O' Neill, H.

    2007-01-01

    The entrapment of proteins using the sol-gel route provides a means to retain its native properties and artifi cially reproduce the molecular crowding and confi nement experienced by proteins in the cell allowing investigation of the physico-chemical and structural properties of biomolecules at the biotic/abiotic interface. The biomolecules are spatially separated and ‘caged’ in the gel structure but solutes can freely permeate the matrix. Thus, properties such as the folding of ensembles of individual molecules can be examined in the absence of aggregation effects that can occur in solution studies. Green fl uorescent protein from Aequorea coerulescens was used as a model protein to examine the unfolding/re-folding properties of protein in silica gels. The recombinant protein was isolated and purifi ed from Escherichia coli extracts by cell lysis, three-phase partitioning, dialysis, and anion exchange chromatography. The purity of the protein was greater than 90% as judged by SDS PAGE gel analysis. Sol-gels were synthesized using tetramethylorthosilicate (TMOS) in combination with, methyltrimethoxyorthosilane (MTMOS), ethyltrimethoxyorthosilane (ETMOS), 3-aminopropyltriethoxysilane (APTES), and 3-glycidoxypropyltrimethoxysilane (GPTMS). The acid induced denaturation and renaturation of GFP was analyzed by UV-visible, fl uorescence, and circular dichroism (CD) spectroscopies. No renaturation was observed in gels that were made with TMOS only, and in the presence of APTES, MTMOS, and ETMOS. However, in gels that were made with GPTMS, the CD and UV-visible spectra indicated that the protein had refolded. The fl uorescence emission spectrum indicated that approximately 20% of fl uorescence had returned. This study highlights the importance of the surface chemistry of the silica gels for the refolding properties of the entrapped GFP. Future studies will investigate the effect of surface chemistry on the thermal and solvent stability of the entrapped protein.

  14. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    DEFF Research Database (Denmark)

    Bokoch, Michael P; Zou, Yaozhong; Rasmussen, Søren Gøgsig Faarup;

    2010-01-01

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation...... extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known...... conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive...

  15. Exhaustive comparison and classification of ligand-binding surfaces in proteins

    Science.gov (United States)

    Murakami, Yoichi; Kinoshita, Kengo; Kinjo, Akira R; Nakamura, Haruki

    2013-01-01

    Many proteins function by interacting with other small molecules (ligands). Identification of ligand-binding sites (LBS) in proteins can therefore help to infer their molecular functions. A comprehensive comparison among local structures of LBSs was previously performed, in order to understand their relationships and to classify their structural motifs. However, similar exhaustive comparison among local surfaces of LBSs (patches) has never been performed, due to computational complexity. To enhance our understanding of LBSs, it is worth performing such comparisons among patches and classifying them based on similarities of their surface configurations and electrostatic potentials. In this study, we first developed a rapid method to compare two patches. We then clustered patches corresponding to the same PDB chemical component identifier for a ligand, and selected a representative patch from each cluster. We subsequently exhaustively as compared the representative patches and clustered them using similarity score, PatSim. Finally, the resultant PatSim scores were compared with similarities of atomic structures of the LBSs and those of the ligand-binding protein sequences and functions. Consequently, we classified the patches into ∼2000 well-characterized clusters. We found that about 63% of these clusters are used in identical protein folds, although about 25% of the clusters are conserved in distantly related proteins and even in proteins with cross-fold similarity. Furthermore, we showed that patches with higher PatSim score have potential to be involved in similar biological processes. PMID:23934772

  16. Limitations of yeast surface display in engineering proteins of high thermostability.

    Science.gov (United States)

    Park, Sheldon; Xu, Yao; Stowell, Xiaoran Fu; Gai, Feng; Saven, Jeffery G; Boder, Eric T

    2006-05-01

    Engineering proteins that can fold to unique structures remains a challenge. Protein stability has previously been engineered via the observed correlation between thermal stability and eukaryotic secretion level. To explore the limits of an expression-based approach, variants of the highly thermostable three-helix bundle protein alpha3D were studied using yeast surface display. A library of alpha3D mutants was created to explore the possible correlation of protein stability and fold with expression level. Five efficiently expressed mutants were then purified and further studied biochemically. Despite their differences in stability, most mutants expressed at levels comparable with that of wild-type alpha3D. Two other related sequences (alpha3A and alpha3B) that form collapsed, stable molten globules but lack a uniquely folded structure were similarly expressed at high levels by yeast display. Together these observations suggest that the quality control system in yeast is unable to discriminate between well-folded proteins of high stability and molten globules. The present study, therefore, suggests that an optimization of the surface display efficiency on yeast may yield proteins that are thermally and chemically stable yet are poorly folded.

  17. THE SURFACE-MEDIATED UNFOLDING KINETICS OF GLOBULAR PROTEINS IS DEPENDENT ON MOLECULAR WEIGHT AND TEMPERATURE

    Energy Technology Data Exchange (ETDEWEB)

    Patananan, A.N.; Goheen, S.C.

    2008-01-01

    The adsorption and unfolding pathways of proteins on rigid surfaces are essential in numerous complex processes associated with biomedical engineering, nanotechnology, and chromatography. It is now well accepted that the kinetics of unfolding are characterized by chemical and physical interactions dependent on protein deformability and structure, as well as environmental pH, temperature, and surface chemistry. Although this fundamental process has broad implications in medicine and industry, little is known about the mechanism because of the atomic lengths and rapid time scales involved. Therefore, the unfolding kinetics of myoglobin, β-glucosidase, and ovalbumin were investigated by adsorbing the globular proteins to non-porous cationic polymer beads. The protein fractions were adsorbed at different residence times (0, 9, 10, 20, and 30 min) at near-physiological conditions using a gradient elution system similar to that in high-performance liquid chromatography. The elution profi les and retention times were obtained by ultraviolet/visible spectrophotometry. A decrease in recovery was observed with time for almost all proteins and was attributed to irreversible protein unfolding on the non-porous surfaces. These data, and those of previous studies, fi t a positively increasing linear trend between percent unfolding after a fi xed (9 min) residence time (71.8%, 31.1%, and 32.1% of myoglobin, β-glucosidase, and ovalbumin, respectively) and molecular weight. Of all the proteins examined so far, only myoglobin deviated from this trend with higher than predicted unfolding rates. Myoglobin also exhibited an increase in retention time over a wide temperature range (0°C and 55°C, 4.39 min and 5.74 min, respectively) whereas ovalbumin and β-glucosidase did not. Further studies using a larger set of proteins are required to better understand the physiological and physiochemical implications of protein unfolding kinetics. This study confi rms that surface

  18. Killing of Staphylococcus aureus by C-8-Methoxy Fluoroquinolones

    OpenAIRE

    Zhao, Xilin; Wang, Jian-Ying; Xu, Chen; Dong, Yuzhi; Zhou, Jianfeng; Domagala, John; Drlica, Karl

    1998-01-01

    C-8-methoxy fluoroquinolones were more lethal than C-8-bromine, C-8-ethoxy, and C-8-H derivatives for Staphylococcus aureus, especially when topoisomerase IV was resistant. The methoxy group also increased lethality against wild-type cells when protein synthesis was inhibited. These properties encourage refinement of C-8-methoxy fluoroquinolones to kill staphylococci.

  19. 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.

  20. Merozoite surface proteins in red blood cell invasion, immunity and vaccines against malaria

    Science.gov (United States)

    Beeson, James G.; Drew, Damien R.; Boyle, Michelle J.; Feng, Gaoqian; Fowkes, Freya J.I.; Richards, Jack S.

    2016-01-01

    Malaria accounts for an enormous burden of disease globally, with Plasmodium falciparum accounting for the majority of malaria, and P. vivax being a second important cause, especially in Asia, the Americas and the Pacific. During infection with Plasmodium spp., the merozoite form of the parasite invades red blood cells and replicates inside them. It is during the blood-stage of infection that malaria disease occurs and, therefore, understanding merozoite invasion, host immune responses to merozoite surface antigens, and targeting merozoite surface proteins and invasion ligands by novel vaccines and therapeutics have been important areas of research. Merozoite invasion involves multiple interactions and events, and substantial processing of merozoite surface proteins occurs before, during and after invasion. The merozoite surface is highly complex, presenting a multitude of antigens to the immune system. This complexity has proved challenging to our efforts to understand merozoite invasion and malaria immunity, and to developing merozoite antigens as malaria vaccines. In recent years, there has been major progress in this field, and several merozoite surface proteins show strong potential as malaria vaccines. Our current knowledge on this topic is reviewed, highlighting recent advances and research priorities. PMID:26833236

  1. Protein adsorption on a hydrophobic surface: a molecular dynamics study of lysozyme on graphite.

    Science.gov (United States)

    Raffaini, Giuseppina; Ganazzoli, Fabio

    2010-04-20

    Adsorption of human lysozyme on hydrophobic graphite is investigated through atomistic computer simulations with molecular mechanics (MM) and molecular dynamics (MD) techniques. The chosen strategy follows a simulation protocol proposed by the authors to model the initial and the final adsorption stage on a bare surface. Adopting an implicit solvent and considering 10 starting molecular orientations so that all the main sides of the protein can face the surface, we first carry out an energy minimization to investigate the initial adsorption stage, and then long MD runs of selected arrangements to follow the surface spreading of the protein maximizing its adsorption strength. The results are discussed in terms of the kinetics of surface spreading, the interaction energy, and the molecular size, considering both the footprint and the final thickness of the adsorbed protein. The structural implications of the final adsorption geometry for surface aggregation and nanoscale structural organization are also pointed out. Further MD runs are carried out in explicit water for the native structure and the most stable adsorption state to assess the local stability of the geometry obtained in implicit solvent, and to calculate the statistical distribution of the water molecules around the whole lysozyme and its backbone.

  2. The Surface of Ice in the presence of Antifreeze Proteins studied by Atomic Force Microscopy

    Science.gov (United States)

    Zepeda, Salvador; Orme, Christine; Yeh, Yin

    2002-03-01

    The surface of ice has been a topic of interest for centuries. In particular, the surface structure and properties have been explored with the advent of new surface techniques. Several groups have convincingly shown a surface transition layer to exist between the solid-vapor interface as well as the solid-liquid interface. In addition, the characteristics of this region may be directly correlated with growth morphologies of ice. Certain peptide molecules have the ability to significantly alter the growth morphology of an ice crystal. Do these molecules simply disrupt this transition region? Or do they anchor themselves deep into it reaching the bulk-ice phase? And is there a similar mechanism by which they function? We use AFM to study the morphological changes to the true ice surface due to the presence antifreeze proteins. We will discuss the implications of our results on the longstanding debate to the above questions.

  3. Templating Biomineralization: Surface Directed Protein Self-assembly and External Magnetic Field Stimulation of Osteoblasts

    Science.gov (United States)

    Ba, Xiaolan

    biomineralization is investigated by SEM, GIXRD and energy dispersive X-ray spectroscopy (EDXS). Gene expression during the exposure of SMF is also studies by RT-PCR. The results indicated that exposure to SMF induces osteoblasts to produce larger quantities of HA, with higher degree of crystalline order. The controlling and understanding of protein on the surface is of great interest in biomedical application such as implant medicine, biosensor design, food processing, and chromatographic separations. The adsorbed protein onto the surface significantly determines the performance of biomaterials in a biological environment. Recent studies have suggested that the preservation of the native secondary structure of protein adsorbed is essential for biological application. In order to manipulate protein adsorption and design biocompatible materials, the mechanisms underlying protein-surface interactions, especially how surface properties of materials induce conformational changes of adsorbed proteins, needs to be well understood. Here we demonstrated that even though SPS is a necessary condition, it is not sufficient. We show that low substrate conductivity as well as proper salt concentration are also critical in sustained protein adsorption continuously. These factors allow one to pattern regions of different conducting properties and for the first time patterns physiologically relevant protein structures. Here we show that we can achieve patterned biomineralized regimes, both with plasma proteins in a simple and robust manner without additional functionalization or application of electrochemical gradients. Since the data indicate that the patterns just need to differ in electrical conductivity, rather than surface chemistry, we propose that the creation of transient image charges, due to incomplete charge screening, may be responsible for sustain the driving force for continual protein absorption.

  4. Protein microarrays based on polymer brushes prepared via surface-initiated atom transfer radical polymerization.

    Science.gov (United States)

    Barbey, Raphael; Kauffmann, Ekkehard; Ehrat, Markus; Klok, Harm-Anton

    2010-12-13

    Polymer brushes represent an interesting platform for the development of high-capacity protein binding surfaces. Whereas the protein binding properties of polymer brushes have been investigated before, this manuscript evaluates the feasibility of poly(glycidyl methacrylate) (PGMA) and PGMA-co-poly(2-(diethylamino)ethyl methacrylate) (PGMA-co-PDEAEMA) (co)polymer brushes grown via surface-initiated atom transfer radical polymerization (SI-ATRP) as protein reactive substrates in a commercially available microarray system using tantalum-pentoxide-coated optical waveguide-based chips. The performance of the polymer-brush-based protein microarray chips is assessed using commercially available dodecylphosphate (DDP)-modified chips as the benchmark. In contrast to the 2D planar, DDP-coated chips, the polymer-brush-covered chips represent a 3D sampling volume. This was reflected in the results of protein immobilization studies, which indicated that the polymer-brush-based coatings had a higher protein binding capacity as compared to the reference substrates. The protein binding capacity of the polymer-brush-based coatings was found to increase with increasing brush thickness and could also be enhanced by copolymerization of 2-(diethylamino)ethyl methacrylate (DEAEMA), which catalyzes epoxide ring-opening of the glycidyl methacrylate (GMA) units. The performance of the polymer-brush-based microarray chips was evaluated in two proof-of-concept microarray experiments, which involved the detection of biotin-streptavidin binding as well as a model TNFα reverse assay. These experiments revealed that the use of polymer-brush-modified microarray chips resulted not only in the highest absolute fluorescence readouts, reflecting the 3D nature and enhanced sampling volume provided by the brush coating, but also in significantly enhanced signal-to-noise ratios. These characteristics make the proposed polymer brushes an attractive alternative to commercially available, 2D microarray

  5. Ambient DESI and LESA-MS Analysis of Proteins Adsorbed to a Biomaterial Surface Using In-Situ Surface Tryptic Digestion

    Science.gov (United States)

    Rao, Wei; Celiz, Adam D.; Scurr, David J.; Alexander, Morgan R.; Barrett, David A.

    2013-12-01

    The detection and identification of proteins adsorbed onto biomaterial surfaces under ambient conditions has significant experimental advantages but has proven to be difficult to achieve with conventional measuring technologies. In this study, we present an adaptation of desorption electrospray ionization (DESI) and liquid extraction surface analysis (LESA) mass spectrometry (MS) coupled with in-situ surface tryptic digestion to identify protein species from a biomaterial surface. Cytochrome c, myoglobin, and BSA in a combination of single and mixture spots were printed in an array format onto Permanox slides, followed by in-situ surface digestion and detection via MS. Automated tandem MS performed on surface peptides was able to identify the proteins via MASCOT. Limits of detection were determined for DESI-MS and a comparison of DESI and LESA-MS peptide spectra characteristics and sensitivity was made. DESI-MS images of the arrays were produced and analyzed with imaging multivariate analysis to automatically separate peptide peaks for each of the proteins within a mixture into distinct components. This is the first time that DESI and LESA-MS have been used for the in-situ detection of surface digested proteins on biomaterial surfaces and presents a promising proof of concept for the use of ambient MS in the rapid and automated analysis of surface proteins.

  6. The surface protein Shr of Streptococcus pyogenes binds heme and transfers it to the streptococcal heme-binding protein Shp

    OpenAIRE

    Lei Benfang; Liu Mengyao; Zhu Hui

    2008-01-01

    Abstract Background The heme acquisition machinery in Streptococcus pyogenes is believed to consist of the surface proteins, Shr and Shp, and heme-specific ATP-binding cassette transporter HtsABC. Shp has been shown to rapidly transfer its heme to the lipoprotein component, HtsA, of HtsABC. The function of Shr and the heme source of Shp have not been established. Results The objective of this study was to determine whether Shr binds heme and is a heme source of Shp. To achieve the objective, ...

  7. Intracellular protein delivery by hollow mesoporous silica capsules with a large surface hole

    International Nuclear Information System (INIS)

    We prepared cell membrane-permeable hollow mesoporous silica capsules (HMSCs) by a simple new method. CTAB micellar assembly in cholesterol emulsion gave rise to a novel capsular morphology of the HMSC particles. The HMSCs consisted of mesostructured silica walls with a large surface hole (25–50 nm) and the average particle dimension was 100–300 nm. They exhibited high surface areas of up to 719.3 m2 g−1 and a mesoporous range of pores of 2.4–2.7 nm. The surface-functionalized HMSCs could also be prepared by a similar co-condensation method using tetraethoxysilane with various organoalkoxysilane precursors in the presence of cholesterol. These organically modified HMSCs could be further modified on demand. For example, a carboxy-functionalized HMSC could be surface-functionalized by a green fluorescent 5-aminofluorescein (AFL) through an amidation reaction to afford a fluorescent AFL–HMSC. The hollow capsular morphology of the HMSCs with a large surface hole enabled us to develop very efficient intracellular delivery systems for membrane-impermeable ions, molecules, and various functional proteins. Non-covalent sequestration and delivery of proteins as well as covalent linkage of fluorescent molecules on the silica surface are effective for this system. The highly negatively charged green fluorescent probe mag-fluo-4 could be intracellularly delivered into HeLa cells by HMSC without any difficulty. The HMSCs could also effectively transport large functional proteins such as antibodies into HeLa cells. The efficiency of protein delivery by HMSC seems to be 3–22-fold higher than that of mesoporous silica nanospheres (MSNs) based on confocal laser scanning microscopy (CLSM) analysis. (paper)

  8. UV- Killed Staphylococcus aureus Enhances Adhesion and Differentiation of Osteoblasts on Bone-associated Biomaterials

    OpenAIRE

    Somayaji, Shankari N.; Huet, Yvette M.; Gruber, Helen E.; Hudson, Michael C

    2010-01-01

    Titanium alloys (Ti) are the preferred material for orthopaedic applications. However, very often, these metallic implants loosen over a long period and mandate revision surgery. For implant success, osteoblasts must adhere to the implant surface and deposit a mineralized extracellular matrix. Here, we utilized UV-killed Staphylococcus aureus as a novel osteoconductive coating for Ti surfaces. S. aureus expresses surface adhesins capable of binding to bone and biomaterials directly. Furthermo...

  9. Synthesis of hepatitis B virus surface protein derivates in yeast S. cerevisiae

    OpenAIRE

    Bulavaitė, Aistė; Sabaliauskaitė, Rasa; Staniulis, Juozas; Sasnauskas, Kęstutis

    2006-01-01

    HBV surface proteins PreS1[13–59]-S, PreS1[20–59]-S, PreS1[30–59]-S, PreS1[40–59]-S, PreS1[50–59]-S, PreS1[90–119]-S were produced in S.cerevisiae and purified. Electron microscopy suggested spherical virus-like particle formation for all the proteins except PreS1[90–119]-S. The PreS1[90–119] sequence was demonstrated to decrease protein solubility. Proteins are suitable for Tupaia primary hepatocyte binding investigations, diagnostic products and vaccine candidate development. Hepatito B ...

  10. Construction and evaluation of novel fusion proteins for targeted delivery of micro particles to cellulose surfaces.

    Science.gov (United States)

    Lewis, William; Keshavarz-Moore, Eli; Windust, John; Bushell, Donna; Parry, Neil

    2006-07-01

    The use of IgG antibodies and fragments has been limited to specific sectors of the biotechnology industry due to the high cost of producing large batches of product necessary for alternative applications. A novel class of Camelid antibodies, known as V(HH) offer a more economical opportunity to meet a wider application in industry. In this study, we report the evaluation of four llama V(HH)-cellulose binding domain fusion proteins displaying varying formats of V(HH) and CBD domains. Proteins were characterized in a targeted particle delivery system as a method of delivering agents such as perfume to laundry in the wash cycle. Fusion proteins were shown to be stable at high pH and in the presence of a detergent base. They were also shown to bind effectively to both the designated antigen, the azo-dye reactive-red 6 (either conjugated to BSA or attached to coacervate microparticles), and cellulose. Binding strength differences were observed between the different fusion protein formats using surface plasmon resonance. The effect of key laundry ingredients was also studied. Combining the fusion proteins and particles into a delivery and deposition study generated clear microscopy evidence for bifunctionality. Confirmation of this was validated by GC-MS analysis of retained fragrance. This research, reporting the construction and characterization of a variety of fusion proteins, illustrates that the single multidomain fusion protein route offers a new technology for successful targeted delivery of encapsulated benefit agents. Furthermore, the potential to modify or select for proteins to recognize a wide range of surfaces is also possible. PMID:16673421

  11. Modulating protein behaviors on responsive surface by external electric fields: A molecular dynamics study

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Yun, E-mail: xieyunxx@gdpu.edu.cn; Pan, Yufang; Zhang, Rong; Liang, Ying; Li, Zhanchao

    2015-01-30

    Graphical abstract: The adsorption of Cyt c on phosphorylcholine self-assembled monolayers (N atoms of the choline groups are colored in blue while the P atoms of the phosphate groups in orange). - Highlights: • PC-SAM could sensitively adjust its charge distribution to applied electric fields. • Adsorption of Cyt c on the PC-SAM is promoted or retarded as the charge distribution of the SAM changes. • Orientations of Cyt c on the PC-SAM are regulated by the structural changes of the SAM. • The structural changes of the SAM cause little deformation in Cyt c. - Abstract: Molecular dynamics simulations were employed to investigate the modulation of protein behaviors on the electrically responsive zwitterionic phosphorylcholine self-assembled monolayers (PC-SAMs). Results show that PC-SAMs could sensitively respond to the applied electric fields and exhibit three states with different charge distributions, namely both the negatively charged phosphate groups and the positively charged choline groups are exposed to the solution in the absence of electric fields (state 1), phosphate groups exposed in the presence of positive electric fields (state 2), and choline groups exposed in the presence of negative electric fields (state 3). Under state 1, the adsorption of Cyt c on the PC-SAM is reversible and the orientations of Cyt c are randomly distributed. Under state 2, the adsorption of Cyt c is enhanced due to the electrostatic attractions between the exposed phosphate groups and the positively charged protein; when adsorbed on the PC-SAMs, Cyt c tends to adopt the orientation with the heme plane perpendicular to the surface plane, and the percentage of this orientation increases as the field strength rises up. Under state 3, the adsorption of Cyt c is retarded because of the electrostatic repulsions between the exposed choline groups and the protein; however, if the gaps between PC chains are large enough, Cyt c could insert into the PC-SAM and access the

  12. Analysis of Pseudomonas aeruginosa Cell Envelope Proteome by Capture of Surface-Exposed Proteins on Activated Magnetic Nanoparticles

    OpenAIRE

    Davide Vecchietti; Dario Di Silvestre; Matteo Miriani; Francesco Bonomi; Mauro Marengo; Alessandra Bragonzi; Lara Cova; Eleonora Franceschi; Pierluigi Mauri; Giovanni Bertoni

    2012-01-01

    We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen Pseudomonas aeruginosa. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedde...

  13. Staphylococcus aureus triggered reactive arthritis.

    OpenAIRE

    Siam, A R; M. Hammoudeh

    1995-01-01

    OBJECTIVES--To report two patients who developed reactive arthritis in association with Staphylococcus aureus infection. METHODS--A review of the case notes of two patients. RESULTS--Two adult female patients have developed sterile arthritis in association with Staph aureus infection. The first patient has had two episodes of arthritis; the first followed olecranon bursitis, the second followed infection of a central venous catheter used for dialysis. The second patient developed sterile arth...

  14. Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases.

    Science.gov (United States)

    Zeke, András; Bastys, Tomas; Alexa, Anita; Garai, Ágnes; Mészáros, Bálint; Kirsch, Klára; Dosztányi, Zsuzsanna; Kalinina, Olga V; Reményi, Attila

    2015-11-01

    Mitogen-activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less-characterized disordered regions. We used a structurally consistent model on kinase-docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under-explored part of the human proteome and applied experimental tools specifically tailored to detect low-affinity protein-protein interactions for their validation in vitro and in cell-based assays. The combined computational and experimental approach enabled the identification of many novel MAPK-docking motifs that were elusive for other large-scale protein-protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase-mediated partnerships evolved over time. The analysis suggests that most human MAPK-binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK-binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles. PMID:26538579

  15. Human Sperm Interaction with Staphylococcus aureus: A Molecular Approach

    Directory of Open Access Journals (Sweden)

    Sonia Gupta

    2012-01-01

    Full Text Available Sperm immobilization factor (SIF causing 100% immobilization of spermatozoa isolated from Staphylococcus aureus when characterized using LC-MS (Liquid chromatography-mass spectrometry showed that this 20 kDa protein had peptide sequence similarity with hsp-70 protein. It was found to completely (100% inhibit Mg++ ATPase activity of spermatozoa at concentration of 100 μg mL−1. Sperm samples treated with SIF also showed reduction in calcium ionophore-induced acrosome reaction as compared to control samples (treated with calcium ionophore alone. Binding studies of FITC labelled SIF with spermatozoa using fluorescent microscopy showed binding of SIF to the surface of spermatozoa indicating the presence of SIF binding receptor. The receptor was extracted by 3M NaCl and purified by gel permeation chromatography. Characterization of the receptor by MALDI-TOF (Matrix-assisted laser desorption ionization-time of flight indicated that the receptor shared sequence similarity with MHC class II antigen. A calorimetric study showed that the receptor moiety on spermatozoa was specific for the purified ligand as binding of the receptor to ligand was enthalpically (−11.9 kJ mole−1 as well as entropically (21.53 J mole−1 K−1 favored resulting in the Gibb's free energy of −18.57 kJ mole−1.

  16. Display of Peptides and Proteins on the Surface of Bacteriophage λ

    Science.gov (United States)

    Sternberg, Nat; Hoess, Ronald H.

    1995-02-01

    The display of peptides or proteins on the surface of viruses is an important technology for studying peptides or proteins and their interaction with other molecules. Here we describe a display vehicle based on bacteriophage λ that incorporates a number of features distinct from other currently used display systems. Fusions of peptides or protein domains have been made to the amino terminus of the 11-kDa D protein of the λ capsid. These fusions assemble onto the viral capsid and appear to be accessible to ligand interactions, based on the ability of a monoclonal antibody to recognize an epitope fused to the D protein on phage heads. To produce large D fusion display libraries and yet avoid the cumbersome task of cloning many fragments into λ DNA, we have used the Cre-loxP site-specific recombination system in vivo to incorporate plasmids encoding the D fusions into the phage genome. Finally, we show that D fusion proteins can be added in vitro to phage lacking D protein and be assembled onto the viral capsid.

  17. Layers: A molecular surface peeling algorithm and its applications to analyze protein structures

    Science.gov (United States)

    Karampudi, Naga Bhushana Rao; Bahadur, Ranjit Prasad

    2015-11-01

    We present an algorithm ‘Layers’ to peel the atoms of proteins as layers. Using Layers we show an efficient way to transform protein structures into 2D pattern, named residue transition pattern (RTP), which is independent of molecular orientations. RTP explains the folding patterns of proteins and hence identification of similarity between proteins is simple and reliable using RTP than with the standard sequence or structure based methods. Moreover, Layers generates a fine-tunable coarse model for the molecular surface by using non-random sampling. The coarse model can be used for shape comparison, protein recognition and ligand design. Additionally, Layers can be used to develop biased initial configuration of molecules for protein folding simulations. We have developed a random forest classifier to predict the RTP of a given polypeptide sequence. Layers is a standalone application; however, it can be merged with other applications to reduce the computational load when working with large datasets of protein structures. Layers is available freely at http://www.csb.iitkgp.ernet.in/applications/mol_layers/main.

  18. Tumor suppressor protein SMAR1 modulates the roughness of cell surface: combined AFM and SEM study

    Directory of Open Access Journals (Sweden)

    Mamgain Hitesh

    2009-10-01

    Full Text Available Abstract Background Imaging tools such as scanning electron microscope (SEM and atomic force microscope (AFM can be used to produce high-resolution topographic images of biomedical specimens and hence are well suited for imaging alterations in cell morphology. We have studied the correlation of SMAR1 expression with cell surface smoothness in cell lines as well as in different grades of human breast cancer and mouse tumor sections. Methods We validated knockdown and overexpression of SMAR1 using RT-PCR as well as Western blotting in human embryonic kidney (HEK 293, human breast cancer (MCF-7 and mouse melanoma (B16F1 cell lines. The samples were then processed for cell surface roughness studies using atomic force microscopy (AFM and scanning electron microscopy (SEM. The same samples were used for microarray analysis as well. Tumors sections from control and SMAR1 treated mice as well as tissues sections from different grades of human breast cancer on poly L-lysine coated slides were used for AFM and SEM studies. Results Tumor sections from mice injected with melanoma cells showed pronounced surface roughness. In contrast, tumor sections obtained from nude mice that were first injected with melanoma cells followed by repeated injections of SMAR1-P44 peptide, exhibited relatively smoother surface profile. Interestingly, human breast cancer tissue sections that showed reduced SMAR1 expression exhibited increased surface roughness compared to the adjacent normal breast tissue. Our AFM data establishes that treatment of cells with SMAR1-P44 results into increase in cytoskeletal volume that is supported by comparative gene expression data showing an increase in the expression of specific cytoskeletal proteins compared to the control cells. Altogether, these findings indicate that tumor suppressor function of SMAR1 might be exhibited through smoothening of cell surface by regulating expression of cell surface proteins. Conclusion Tumor suppressor

  19. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.; Liu, Corey W.; Nygaard, Rie; Rosenbaum, Daniel M.; Fung, Juan José; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Puglisi, Joseph D.; Weis, William I.; Pardo, Leonardo; Prosser, R. Scott; Mueller, Luciano; Kobilka, Brian K. (Stanford-MED); (Toronto); (BMS); (UAB, Spain)

    2010-01-14

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

  20. Role of surface chemistry in protein remodeling at the cell-material interface.

    Directory of Open Access Journals (Sweden)

    Virginia Llopis-Hernández

    Full Text Available BACKGROUND: The cell-material interaction is a complex bi-directional and dynamic process that mimics to a certain extent the natural interactions of cells with the extracellular matrix. Cells tend to adhere and rearrange adsorbed extracellular matrix (ECM proteins on the material surface in a fibril-like pattern. Afterwards, the ECM undergoes proteolytic degradation, which is a mechanism for the removal of the excess ECM usually approximated with remodeling. ECM remodeling is a dynamic process that consists of two opposite events: assembly and degradation. METHODOLOGY/PRINCIPAL FINDINGS: This work investigates matrix protein dynamics on mixed self-assembled monolayers (SAMs of -OH and -CH(3 terminated alkanethiols. SAMs assembled on gold are highly ordered organic surfaces able to provide different chemical functionalities and well-controlled surface properties. Fibronectin (FN was adsorbed on the different surfaces and quantified in terms of the adsorbed surface density, distribution and conformation. Initial cell adhesion and signaling on FN-coated SAMs were characterized via the formation of focal adhesions, integrin expression and phosphorylation of FAKs. Afterwards, the reorganization and secretion of FN was assessed. Finally, matrix degradation was followed via the expression of matrix metalloproteinases MMP2 and MMP9 and correlated with Runx2 levels. We show that matrix degradation at the cell material interface depends on surface chemistry in MMP-dependent way. CONCLUSIONS/SIGNIFICANCE: This work provides a broad overview of matrix remodeling at the cell-material interface, establishing correlations between surface chemistry, FN adsorption, cell adhesion and signaling, matrix reorganization and degradation. The reported findings improve our understanding of the role of surface chemistry as a key parameter in the design of new biomaterials. It demonstrates the ability of surface chemistry to direct proteolytic routes at the cell

  1. Protein Analysis by Ambient Ionization Mass Spectrometry Using Trypsin-Immobilized Organosiloxane Polymer Surfaces.

    Science.gov (United States)

    Dulay, Maria T; Eberlin, Livia S; Zare, Richard N

    2015-12-15

    In the growing field of proteomic research, rapid and simple protein analysis is a crucial component of protein identification. We report the use of immobilized trypsin on hybrid organic-inorganic organosiloxane (T-OSX) polymers for the on-surface, in situ digestion of four model proteins: melittin, cytochrome c, myoglobin, and bovine serum albumin. Tryptic digestion products were sampled, detected, and identified using desorption electrospray ionization mass spectrometry (DESI-MS) and nanoDESI-MS. These novel, reusable T-OSX arrays on glass slides allow for protein digestion in methanol:water solvents (1:1, v/v) and analysis directly from the same polymer surface without the need for sample preparation, high temperature, and pH conditions typically required for in-solution trypsin digestions. Digestion reactions were conducted with 2 μL protein sample droplets (0.35 mM) at incubation temperatures of 4, 25, 37, and 65 °C and digestion reaction times between 2 and 24 h. Sequence coverages were dependent on the hydrophobicity of the OSX polymer support and varied by temperature and digestion time. Under the best conditions, the sequence coverages, determined by DESI-MS, were 100% for melittin, 100% for cytochrome c, 90% for myoglobin, and 65% for bovine serum albumin. PMID:26567450

  2. Impact of surface coating and food-mimicking media on nanosilver-protein interaction

    Energy Technology Data Exchange (ETDEWEB)

    Burcza, Anna, E-mail: anna.burcza@mri.bund.de; Gräf, Volker; Walz, Elke; Greiner, Ralf [Max Rubner-Institute, Department of Food Technology and Bioprocess Engineering (Germany)

    2015-11-15

    The application of silver nanoparticles (AgNPs) in food contact materials has recently become a subject of dispute due to the possible migration of silver in nanoform into foods and beverages. Therefore, the analysis of the interaction of AgNPs with food components, especially proteins, is of high importance in order to increase our knowledge of the behavior of nanoparticles in food matrices. AgPURE™ W10 (20 nm), an industrially applied nanomaterial, was compared with AgNPs of similar size frequently investigated for scientific purposes differing in the surface capping agent (spherical AgNP coated with either PVP or citrate). The interactions of the AgNPs with whey proteins (BSA, α-lactalbumin and β-lactoglobulin) at different pH values (4.2, 7 or 7.4) were investigated using surface plasmon resonance, SDS-PAGE, and asymmetric flow field-flow fractionation. The data obtained by the three different methods correlated well. Besides the nature of the protein and the nanoparticle coating, the environment was shown to affect the interaction significantly. The strongest interaction was obtained with BSA and AgNPs in an acidic environment. Neutral and slightly alkaline conditions however, seemed to prevent the AgNP-protein interaction almost completely. Furthermore, the interaction of whey proteins with AgPURE™ W10 was found to be weaker compared to the interaction with the other two AgNPs under all conditions investigated.

  3. Effects of Proteins from Culture Medium on Surface Property of Silanes- Functionalized Magnetic Nanoparticles

    Directory of Open Access Journals (Sweden)

    Chen ZP

    2008-01-01

    Full Text Available Abstract Monodisperse magnetic nanoparticles (MNPs were synthesized by thermal decomposition of iron-oleate and functionalized with silanes bearing various functional groups such as amino group (NH2, short-chain poly(ethylene glycol (PEG, and carboxylic group (COOH. Then, silanes-functionalized magnetic nanoparticles (silanes-MNPs were incubated in cell culture medium plus fetal calf serum to investigate the effects of proteins from culture medium on surface property of MNPs. Zeta potential measurements showed that although surface charges of silanes-MNPs were different, they exhibited negative charges at neutral pH and approximate isoelectric points after they were incubated in cell culture medium. The reason was that silanes-MNPs could easily adsorb proteins from culture medium via non-covalent binding, resulting in the formation of protein-silanes-MNPs conjugates. Moreover, silanes-MNPs with various functional groups had different adsorption capacity to proteins, as confirmed by Coomassie blue fast staining method. The in vitro cell experiments showed that protein-silanes-MNPs had higher cellular uptake by cancer cells than silanes-MNPs.

  4. Enzyme directed formation of un-natural side-chains for covalent surface attachment of proteins.

    Science.gov (United States)

    Cho, Hwayoung; Jaworski, Justyn

    2014-10-01

    The covalent immobilization of proteins onto surfaces is an essential aspect of several fields of research, including proteomics, sensing, heterogeneous biocatalysis, and more broadly biotechnology. Site-specific, covalent attachment of proteins has been achieved in recent years by the use of expanded genetic codes to produce proteins with controlled placement of un-natural amino acids bearing bio-orthogonal functional groups. Unfortunately, the complexity of developing such systems is impractical for most laboratories; hence, a less complicated approach to generating un-natural amino acid side-chains has been employed. Utilizing a straightforward reaction with formylglycine generating enzyme, we use the site-specific modification of engineered proteins to yield un-natural amino acid side-chains for protein immobilization. Using this approach, we demonstrate the controlled immobilization of various enzymes onto a variety of amine coated surfaces. Our results reveal reusability of the immobilized enzymes via this strategy, and furthermore, we find the activity of the immobilized enzymes to remain even after a month of use indicating significant stability of the linkage.

  5. Impact of surface coating and food-mimicking media on nanosilver-protein interaction

    International Nuclear Information System (INIS)

    The application of silver nanoparticles (AgNPs) in food contact materials has recently become a subject of dispute due to the possible migration of silver in nanoform into foods and beverages. Therefore, the analysis of the interaction of AgNPs with food components, especially proteins, is of high importance in order to increase our knowledge of the behavior of nanoparticles in food matrices. AgPURE™ W10 (20 nm), an industrially applied nanomaterial, was compared with AgNPs of similar size frequently investigated for scientific purposes differing in the surface capping agent (spherical AgNP coated with either PVP or citrate). The interactions of the AgNPs with whey proteins (BSA, α-lactalbumin and β-lactoglobulin) at different pH values (4.2, 7 or 7.4) were investigated using surface plasmon resonance, SDS-PAGE, and asymmetric flow field-flow fractionation. The data obtained by the three different methods correlated well. Besides the nature of the protein and the nanoparticle coating, the environment was shown to affect the interaction significantly. The strongest interaction was obtained with BSA and AgNPs in an acidic environment. Neutral and slightly alkaline conditions however, seemed to prevent the AgNP-protein interaction almost completely. Furthermore, the interaction of whey proteins with AgPURE™ W10 was found to be weaker compared to the interaction with the other two AgNPs under all conditions investigated

  6. Cell Surface Proteins in S. Pneumoniae, S. Mitis and S. Oralis

    Directory of Open Access Journals (Sweden)

    R Hakenbeck

    2011-06-01

    Full Text Available Background and objectives: Streptococcus pneumoniae, a major human pathogen, is closely related to the commensal species S. mitis and S. oralis. S. pneumoniae surface proteins are implicated in virulence and host interaction of this species, but many of them have recently been detected in S. mitis B6 in silico. We tested for the presence of such genes usinga set of eight S. mitis and eleven S. oralis strains from different geographic locations.Materials and Methods: An oligonucleotide microarray was designed based on the genomes of S. pneumoniae R6 and TIGR4 as well as S. mitis B6 to include 63 cell surface proteins. The S. pneumoniae genes encoding neuraminidases, hyaluronidase and pneumolysin were also included. In addition to comparative genomic hybridization experiments, homologues were identified in silico in the genome of S. oralis Uo5.Results and Conclusions: The results document that many S. pneumoniae related surface proteins are ubiquitously present among the Mitis group of streptococci. All 19 samples hybridized with the pavA probe representing a gene important for adherence and invasion of S. pneumoniae. Only eight genes were not recognized in any strain, including the S. pneumoniae PcpC gene as the only virulence gene of the S. pneumoniae core genome.The fact that only 12 out of 26 genes present in the S. oralis Uo5 genome could be detected by microarray analysis confirms the sequence variation of surface components.

  7. Synthetic Peptide Immunogens Elicit Polyclonal and Monoclonal Antibodies Specific for Linear Epitopes in the D Motifs of Staphylococcus aureus Fibronectin-Binding Protein, Which Are Composed of Amino Acids That Are Essential for Fibronectin Binding

    OpenAIRE

    Huesca, Mario; Sun, Qing; Peralta, Robert; Shivji, Gulnar M.; Sauder, Daniel N.; McGavin, Martin J.

    2000-01-01

    A fibronectin (Fn)-binding adhesin of Staphylococcus aureus contains three tandem 37- or 38-amino-acid motifs (D1, D2, and D3), which function to bind Fn. Plasma from patients with S. aureus infections contain antibodies that preferentially recognize ligand induced binding sites in the D motifs and do not inhibit Fn binding (F. Casolini, L. Visai, D. Joh, P. G. Conaldi, A. Toniolo, M. Höök, and P. Speziale, Infect. Immun. 66:5433–5442, 1998). To eliminate the influence of Fn binding on antibo...

  8. Reverse Line Blot Assay for Direct Identification of Seven Streptococcus agalactiae Major Surface Protein Antigen Genes

    OpenAIRE

    Zhao, Zuotao; Kong, Fanrong; Gilbert, Gwendolyn L.

    2006-01-01

    We developed a multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB) to detect the genes encoding members of the family of variable surface-localized proteins of Streptococcus agalactiae (group B streptococcus [GBS]), namely, Bca (Cα), Rib, Epsilon (Epsilon/Alp1/Alp5), Alp2, Alp3, and Alp4, and the immunoglobulin A binding protein, Bac (Cβ). We used the assay to identify these genes in a collection of well-characterized GBS isolates and reference strains. The results showed tha...

  9. 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.

  10. 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.

  11. The SaeRS Two-Component System of Staphylococcus aureus

    Science.gov (United States)

    Liu, Qian; Yeo, Won-Sik; Bae, Taeok

    2016-01-01

    In the Gram-positive pathogenic bacterium Staphylococcus aureus, the SaeRS two-component system (TCS) plays a major role in controlling the production of over 20 virulence factors including hemolysins, leukocidins, superantigens, surface proteins, and proteases. The SaeRS TCS is composed of the sensor histidine kinase SaeS, response regulator SaeR, and two auxiliary proteins SaeP and SaeQ. Since its discovery in 1994, the sae locus has been studied extensively, and its contributions to staphylococcal virulence and pathogenesis have been well documented and understood; however, the molecular mechanism by which the SaeRS TCS receives and processes cognate signals is not. In this article, therefore, we review the literature focusing on the signaling mechanism and its interaction with other global regulators. PMID:27706107

  12. The posttranslocational chaperone lipoprotein PrsA is involved in both glycopeptide and oxacillin resistance in Staphylococcus aureus.

    Science.gov (United States)

    Jousselin, Ambre; Renzoni, Adriana; Andrey, Diego O; Monod, Antoinette; Lew, Daniel P; Kelley, William L

    2012-07-01

    Understanding in detail the factors which permit Staphylococcus aureus to counteract cell wall-active antibiotics is a prerequisite to elaborating effective strategies to prolong the usefulness of these drugs and define new targets for pharmacological intervention. Methicillin-resistant S. aureus (MRSA) strains are major pathogens of hospital-acquired and community-acquired infections and are most often treated with glycopeptides (vancomycin and teicoplanin) because of their resistance to most penicillins and a limited arsenal of clinically proven alternatives. In this study, we examined PrsA, a lipid-anchored protein of the parvulin PPIase family (peptidyl-prolyl cis/trans isomerase) found ubiquitously in all Gram-positive species, in which it assists posttranslocational folding at the outer surface of the cytoplasmic membrane. We show by both genetic and biochemical assays that prsA is directly regulated by the VraRS two-component sentinel system of cell wall stress. Disruption of prsA is tolerated by S. aureus, and its loss results in no detectable overt macroscopic changes in cell wall architecture or growth rate under nonstressed growth conditions. Disruption of prsA leads, however, to notable alterations in the sensitivity to glycopeptides and dramatically decreases the resistance of COL (MRSA) to oxacillin. Quantitative transcriptional analysis reveals that prsA and vraR are coordinately upregulated in a panel of stable laboratory and clinical glycopeptide-intermediate S. aureus (GISA) strains compared to their susceptible parents. Collectively, our results point to a role for prsA as a facultative facilitator of protein secretion or extracellular folding and provide a framework for understanding why prsA is a key element of the VraRS-mediated cell wall stress response. PMID:22526301

  13. Methicillin-resistant Staphylococcus aureus in central Iowa wildlife.

    Science.gov (United States)

    Wardyn, Shylo E; Kauffman, Lin K; Smith, Tara C

    2012-10-01

    Livestock and pets have been identified as carriers of Staphylococcus aureus; however, the role of wild animals as a reservoir of S. aureus strains has not yet been examined. We conducted a pilot study to determine the prevalence of methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in 37 species of wild animals rehabilitated at a university clinic. Nasal, wing, wound, and cloacal swabs were collected. Of 114 animals, seven (6.1%) were MSSA-positive and three (2.6%) were MRSA-positive. The MRSA isolates were obtained from two eastern cottontail rabbits (Sylvilagus floridanus) and a Lesser Yellowlegs (Tringa flavipes), a migratory shorebird. Antibiotic resistance testing of the MRSA isolates revealed that two were additionally resistant to tetracycline and erythromycin, and the third isolate was also resistant to erythromycin, clindamycin, and levofloxacin. All three isolates were positive for the Panton-Valentine leukocidin (PVL) gene. Sequence typing of the staphylococcal protein A (spa) region revealed one MRSA isolate to be t002, whereas the other two MRSA isolates were found to be t008. Our results suggest that S. aureus, including MRSA, is being carried by wild animals, although at a low prevalence with the limited number of animals tested. Additional studies are needed to determine how this may impact human health. PMID:23060511

  14. Protein-repellent silicon nitride surfaces: UV-induced formation of oligoethylene oxide monolayers.

    Science.gov (United States)

    Rosso, Michel; Nguyen, Ai T; de Jong, Ed; Baggerman, Jacob; Paulusse, Jos M J; Giesbers, Marcel; Fokkink, Remko G; Norde, Willem; Schroën, Karin; van Rijn, Cees J M; Zuilhof, Han

    2011-03-01

    The grafting of polymers and oligomers of ethylene oxide onto surfaces is widely used to prevent nonspecific adsorption of biological material on sensors and membrane surfaces. In this report, we show for the first time the robust covalent attachment of short oligoethylene oxide-terminated alkenes (CH(3)O(CH(2)CH(2)O)(3)(CH(2))(11)-(CH═CH(2)) [EO(3)] and CH(3)O(CH(2)CH(2)O)(6)(CH(2))(11)-(CH═CH(2)) [EO(6)]) from the reaction of alkenes onto silicon-rich silicon nitride surfaces at room temperature using UV light. Reflectometry is used to monitor in situ the nonspecific adsorption of bovine serum albumin (BSA) and fibrinogen (FIB) onto oligoethylene oxide coated silicon-rich silicon nitride surfaces (EO(n)-Si(x)N(4), x > 3) in comparison with plasma-oxidized silicon-rich silicon nitride surfaces (SiO(y)-Si(x)N(4)) and hexadecane-coated Si(x)N(4) surfaces (C(16)-Si(x)N(4)). A significant reduction in protein adsorption on EO(n)-Si(x)N(4) surfaces was achieved, adsorption onto EO(3)-Si(x)N(4) and EO(6)-Si(x)N(4) were 0.22 mg m(-2) and 0.08 mg m(-2), respectively. The performance of the obtained EO(3) and EO(6) layers is comparable to those of similar, highly protein-repellent monolayers formed on gold and silver surfaces. EO(6)-Si(x)N(4) surfaces prevented significantly the adsorption of BSA (0.08 mg m(-2)). Atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), X-ray reflectivity and static water contact angle measurements were employed to characterize the modified surfaces. In addition, the stability of EO(6)-Si(x)N(4) surfaces in phosphate-buffered saline solution (PBS) and alkaline condition (pH 10) was studied. Prolonged exposure of the surfaces to PBS solution for 1 week or alkaline condition for 2 h resulted in only minor degradation of the ethylene oxide moieties and no oxidation of the Si(x)N(4) substrates was observed. Highly stable antifouling coatings on Si(x)N(4) surfaces significantly broaden the application potential of silicon

  15. 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 obse