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Ubiquitin-fusion degradation pathway: A new strategy for inducing CD8 cells specific for mycobacterial HSP65  

International Nuclear Information System (INIS)

The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8+ T cells. In this study, we exploited UPS to induce CD8+ T cells specific for mycobacterial HSP65 (mHSP65), one of the leading vaccine candidates against infection with Mycobacterium tuberculosis. A chimeric DNA termed pU-HSP65 encoding a fusion protein between murine ubiquitin and mHSP65 was constructed, and C57BL/6 (B6) mice were immunized with the DNA using gene gun bombardment. Mice immunized with the chimeric DNA acquired potent resistance against challenge with the syngeneic B16F1 melanoma cells transfected with the mHSP65 gene (HSP65/B16F1), compared with those immunized with DNA encoding only mHSP65. Splenocytes from the former group of mice showed a higher grade of cytotoxic activity against HSP65/B16F1 cells and contained a larger number of granzyme B- or IFN-?-producing CD8+ T cells compared with those from the latter group of mice

2008-01-25

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Mycobacterial Hsp65 potentially cross-reacts with autoantibodies of diabetes sera and also induces (in vitro) cytokine responses relevant to diabetes mellitus.  

Science.gov (United States)

Diabetes mellitus is a multifactorial disease and its incidence is increasing worldwide. Among the two types of diabetes, type-2 accounts for about 90% of all diabetic cases, whereas type-1 or juvenile diabetes is less prevalent and presents with humoral immune responses against some of the autoantigens. We attempted to test whether the sera of type-1 diabetes patients cross-react with mycobacterial heat shock protein 65 (Hsp65) due to postulated epitope homologies between mycobacterial Hsp65 and an important autoantigen of type-1 diabetes, glutamic acid decarboxylase-65 (GAD65). In our study, we used either recombinant mycobacterial Hsp65 protein or synthetic peptides corresponding to some of the potential epitopes of mycobacterial Hsp65 that are shared with GAD65 or human Hsp60, and a control peptide sourced from mycobacterial Hsp65 which is not shared with GAD65, Hsp60 and other autoantigens of type-1 diabetes. The indirect ELISA results indicated that both type-1 diabetes and type-2 diabetes sera cross-react with conserved mycobacterial Hsp65 peptides and recombinant mycobacterial Hsp65 protein but do not do so with the control peptide. Our results suggest that cross-reactivity of mycobacterial Hsp65 with autoantibodies of diabetes sera could be due to the presence of significantly conserved peptides between mycobacterial Hsp65 and human Hsp60 rather than between mycobacterial Hsp65 and GAD65. The treatment of human peripheral blood mononuclear cells (PBMCs) with recombinant mycobacterial Hsp65 protein or the synthetic peptides resulted in a significant increase in the secretion of cytokines such as IL-1?, IL-8, IL-6, TNF-? and IL-10. Taken together, these findings point towards a dual role for mycobacterial Hsp65: in inducing autoimmunity and in inflammation, the two cardinal features of diabetes mellitus. PMID:24056978

Rani, Pittu Sandhya; Babajan, Banaganapalli; Tulsian, Nikhil K; Begum, Mahabubunnisa; Kumar, Ashutosh; Ahmed, Niyaz

2013-11-01

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Description of a new Mycobacterium intracellulare pattern of PCR restriction enzyme analysis of hsp65 gene.  

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This work comprises 9 pulmonary nontuberculous mycobateria isolates obtained from sputum of 4 different patients from Brazil. The sequencing and phylogenetic analysis allowed their accurate identification as Mycobacterium intracellulare. We report a mutation at position 453 creating a new HaeIII cutting site and, therefore, a new PRA-hsp65 M. intracellulare profile. PMID:24655570

Caldas, Paulo Cesar de Souza; Campos, Carlos Eduardo Dias; Dos Reis, Lusiano Motta; Ferreira, Nicole Victor; de Carvalho, Luciana Distásio; da Silva, Mariza Villas Boas; Medeiros, Reginalda Ferreira de Melo; Montes, Fátima Cristina Onofre Fandinho; Ramos, Jesus Pais

2014-06-01

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Ten tandem repeats of ?-hCG 109-118 enhance immunogenicity and anti-tumor effects of ?-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65  

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The ?-subunit of human chorionic gonadotropin (?-hCG) is secreted by many kinds of tumors and it has been used as an ideal target antigen to develop vaccines against tumors. In view of the low immunogenicity of this self-peptide,we designed a method based on isocaudamer technique to repeat tandemly the 10-residue sequence X of ?-hCG (109-118), then 10 tandemly repeated copies of the 10-residue sequence combined with ?-hCG C-terminal 37 peptides were fused to mycobacterial heat-shock protein 65 to construct a fusion protein HSP65-X10-?hCGCTP37 as an immunogen. In this study, we examined the effect of the tandem repeats of this 10-residue sequence in eliciting an immune by comparing the immunogenicity and anti-tumor effects of the two immunogens, HSP65-X10-?hCGCTP37 and HSP65-?hCGCTP37 (without the 10 tandem repeats). Immunization of mice with the fusion protein HSP65-X10-?hCGCTP37 elicited much higher levels of specific anti-?-hCG antibodies and more effectively inhibited the growth of Lewis lung carcinoma (LLC) in vivo than with HSP65-?hCGCTP37, which should suggest that HSP65-X10-?hCGCTP37 may be an effective protein vaccine for the treatment of ?-hCG-dependent tumors and multiple tandem repeats of a certain epitope are an efficient method to overcome the low immunogenicity of self-peptide antigens

2006-07-14

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Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery.  

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In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system. PMID:16445866

Coelho-Castelo, A A M; Trombone, A P; Rosada, R S; Santos, R R; Bonato, V L D; Sartori, A; Silva, C L

2006-01-01

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hsp65 PCR-restriction enzyme analysis (PRA) for identification of mycobacteria in the clinical laboratory / PCR e análise de padrões de restrição do gene hsp65 (PRA) para identificação de micobactérias no laboratório clínico  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in portuguese Mais de 70 espécies de micobactérias já foram definidas e algumas delas podem causar enfermidade em humanos, especialmente em pacientes imunocomprometidos. A identificação de espécie, na maioria dos laboratórios clínicos, se baseia em características fenotípicas e testes bioquímicos e resultados def [...] initivos só são obtidos após duas a quatro semanas. Métodos rápidos de identificação reduzem o tempo necessário para o diagnóstico e podem antecipar a instituição do tratamento específico, aumentando as chances de sucesso. A análise de padrões de restrição do gene hsp65 amplificado por PCR (PRA) foi utilizada como método rápido de identificação em 103 isolamentos clínicos. Os padrões de bandas foram interpretados por comparação com tabelas publicadas e padrões disponíveis em um site de Internet (http://www.hospvd.ch:8005). Resultados concordantes de PRA e identificação bioquímica foram obtidos em 76 de 83 isolamentos (91,5%). Os resultados de 20 isolamentos não puderam ser comparados porque a identificação fenotípica ou por PRA foi inconclusiva. Os resultados deste trabalho mostram que PRA pode ser útil para identificação de rotina de micobactérias por ser um método acurado, rápido e mais econômico do que a identificação convencional. Abstract in english More than 70 species of mycobacteria have been defined, and some can cause disease in humans, especially in immunocompromised patients. Species identification in most clinical laboratories is based on phenotypic characteristics and biochemical tests and final results are obtained only after two to f [...] our weeks. Quick identification methods, by reducing time for diagnosis, could expedite institution of specific treatment, increasing chances of success. PCR restriction-enzyme analysis (PRA) of the hsp65 gene was used as a rapid method for identification of 103 clinical isolates. Band patterns were interpreted by comparison with published tables and patterns available at an Internet site (http://www.hospvd.ch:8005). Concordant results of PRA and biochemical identification were obtained in 76 out of 83 isolates (91.5%). Results from 20 isolates could not be compared due to inconclusive PRA or biochemical identification. The results of this work showed that PRA could improve identification of mycobacteria in a routine setting because it is accurate, fast, and cheaper than conventional phenotypic identification.

Carolina Feher da, SILVA; Suely Yoko Mizuka, UEKI; Débora de Cássia Pires, GEIGER; Sylvia Cardoso, LEÃO.

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Sequencing of hsp65 Gene for Identification of Mycobacterium Species Isolated from Environmental and Clinical Sources in Rio de Janeiro, Brazil? †  

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This study evaluated the biodiversity of 28 clinical and 24 environmental Mycobacterium isolates from Rio de Janeiro, Brazil, by using hsp65 sequences, with the aim of contributing to a better understanding of the genetic diversity and usefulness of this marker. An extensive phylogenetic analysis was performed. The nucleotide diversity was similar between clinical (0.06508) and environmental (0.06221) isolates.

2008-01-01

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Identification and Characterization of Protective T Cells in hsp65 DNA-Vaccinated and Mycobacterium tuberculosis-Infected Mice  

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Immunization by intramuscular injection of plasmid DNA expressing mycobacterial 65-kDa heat shock protein (hsp65) protects mice against challenge with virulent Mycobacterium tuberculosis H37Rv. During infection or after immunization, CD4+/CD8? and CD8+/CD4? hsp65-reactive T cells increased equally in spleens. During infection, the majority of these cells were weakly CD44 positive (CD44lo) and produced interleukin 4 (IL-4) whereas after immunization the majority were highly CD44 positive (...

1998-01-01

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DETECÇÃO DO COMPLEXO Mycobacterium tuberculosis NO LEITE PELA REAÇÃO EM CADEIA DA POLIMERASE SEGUIDA DE ANÁLISE DE RESTRIÇÃO DO FRAGMENTO AMPLIFICADO (PRA DETECTION OF Mycobacterium tuberculosis COMPLEX BY PCR-RESTRICTION FRAGMENT LENGTH POLYMORFISM ANALYSIS OF THE HSP65 GENE  

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Full Text Available Mycobacterium bovis é membro do complexo Mycobacterium tuberculosis (MTBC, grupo este composto por espécies com grande homologia genética. É o agente etiológico da tuberculose bovina, importante zoonose transmissível ao homem, principalmente através da inalação do bacilo e/ou pelo consumo de leite e derivados não-pasteurizados provenientes de vacas tuberculosas. O objetivo deste estudo foi padronizar a identificação de micobactérias do complexo M. tuberculosis presentes no leite, por metodologia molecular. Fez-se a extração de DNA diretamente do leite contaminado e realizou-se a identificação molecular pela reação em cadeia da polimerase seguida de análise de restrição do fragmento amplificado (PRA. Utilizaram-se inhagens de referência e leite cru artificialmente contaminado com M. bovis IP. Um fragmento de 441pb do gene hsp65 foi amplificado, tratado com BstEII e HaeIII e empregou-se o perfil de restrição enzimática obtido para identificar o complexo M. tuberculosis no leite. Com a PRA foi possível detectar com especificidade e sensibilidade a presença de M. bovis em até 10 UFC/mL de leite. A metodologia padronizada poderá auxiliar os métodos microbiológicos e bioquímicos tradicionalmente usados na identificação do bacilo em alimentos suspeitos de contaminação, como, por exemplo, o leite proveniente de animais suspeitos de infecção por M. bovis.

Palavras-chaves: Análise de perfil de restrição enzimática (PRA, complexo Mycobacterium tuberculosis, leite, Mycobacterium bovis, limite de detecção (PCR. Mycobacterium bovis is a member of the M. tuberculosis complex, a group composed by species with high genetic homology. The pathogen is the etiological agent of bovine tuberculosis, an important zoonosis that is mainly transmitted by inhalation of infectious droplet nuclei or by ingestion of milk and crude milk derivative products from tuberculosis cows. The definitive identification of M. bovis, up to species level, is time consuming and difficult. In this work, the objective was to standardize a polymerase chain reaction followed by an enzyme restriction analysis in order to identify the M. tuberculosis complex in milk, without a microbiological isolation step. Reference strains and raw milk seeded with M. Bovis, were used as the starting material.  A 441pb fragment of the hsp65 gene was amplified and digested by two restriction enzymes BstEII and HaeIII. The obtained profile was used to identify the M. tuberculosis complex in milk. The minimum limit of detection of M. bovis in milk was 10CFU/mL. PRA methodology proved to be a specific and sensible method. It can be used to assist the microbiological and biochemical methods commonly used to identifying the bacilli in clinical samples, as milk 

Key word: Detection limit (PRA, Mycobacterium tuberculosis complex, milk Mycobacterium bovis, Restriction Enzyme Analysis (PCR,

Joab Trajano Silva

2008-12-01

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Humoral response to hsp 65 and hsp 70 in cerebrospinal fluid in Parkinson's disease.  

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Parkinson's disease (PD) is an age-related neurodegenerative movement disorder of unknown etiology. In PD immune abnormalities were reported, but the cause of such abnormalities has not been resolved. Recently, the increased proportion of gamma delta + T cells out of all T cells has been found in patients with PD. Heat shock proteins (hsps) could be targets for gamma delta + T lymphocytes. We examined serum and CSF of patients with PD, age-matched patients with other non-inflammatory neurological diseases (OND old), young patients with other non-inflammatory neurological diseases (OND young), and donors of blood (DB). Antibodies were detected using the enzyme-linked immunosorbent assay. The plates were coated with recombinant mycobacterial hsp 65 and hsp 70. The present study showed that the mean ELISA ratio of CSF from patients with PD was significantly greater than that of CSF from patients with OND old (tested against IgG anti-hsp 65 and IgG anti-hsp 70) and OND young (tested against IgG anti-hsp 70). There was no difference between the mean ELISA ratio of sera from patients with PD, OND old and OND young (tested against IgG anti-hsp 65 and IgG anti-hsp 70). The significance of hsps immunity is not completely clear. Increased hsps expression, which is induced by stress, provides cells with protection against the environmental insults. Alternatively, the antibodies may be present as a consequence of prior infections. PMID:8836974

Fiszer, U; Fredrikson, S; Cz?onkowska, A

1996-07-01

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Th1 polarized response induced by intramuscular DNA-HSP65 immunization is preserved in experimental atherosclerosis  

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Full Text Available We previously reported that a DNA vaccine constructed with the heat shock protein (HSP65 gene from Mycobacterium leprae (DNA-HSP65 was protective and also therapeutic in experimental tuberculosis. By the intramuscular route, this vaccine elicited a predominant Th1 response that was consistent with its protective efficacy against tuberculosis. It has been suggested that the immune response to Hsp60/65 may be the link between exposure to microorganisms and increased cardiovascular risk. Additionally, the high cholesterol levels found in atherosclerosis could modulate host immunity. In this context, we evaluated if an atherogenic diet could modulate the immune response induced by the DNA-HSP65 vaccine. C57BL/6 mice (4-6 animals per group were initially submitted to a protocol of atherosclerosis induction and then immunized by the intramuscular or intradermal route with 4 doses of 100 µg DNA-HSP65. On day 150 (15 days after the last immunization, the animals were sacrificed and antibodies and cytokines were determined. Vaccination by the intramuscular route induced high levels of anti-Hsp65 IgG2a antibodies, but not anti-Hsp65 IgG1 antibodies and a significant production of IL-6, IFN-g and IL-10, but not IL-5, indicating a Th1 profile. Immunization by the intradermal route triggered a mixed pattern (Th1/Th2 characterized by synthesis of anti-Hsp65 IgG2a and IgG1 antibodies and production of high levels of IL-5, IL-6, IL-10, and IFN-g. These results indicate that experimentally induced atherosclerosis did not affect the ability of DNA-HSP65 to induce a predominant Th1 response that is potentially protective against tuberculosis.

D.M. Fonseca

2007-11-01

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Sequencing of hsp65 Distinguishes among Subsets of the Mycobacterium avium Complex  

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The Mycobacterium avium complex consists of epidemiologically distinct subsets. The classification of these subsets is complicated by a number of factors, including the ambiguous results obtained with phenotypic and genetic assays and the recent appreciation that human and avian strains appear to be distinct. In previous work, sequencing based on a 441-bp portion of the hsp65 gene has proven to efficiently classify isolates within the Mycobacterium genus but provides low resolution for distin...

Turenne, Christine Y.; Semret, Makeda; Cousins, Debby V.; Collins, Desmond M.; Behr, Marcel A.

2006-01-01

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Ub Combination Enhanced Cellular Immune Response Elicited by HSP65 DNA Vaccine against Mycobacterium tuberculosis  

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Full Text Available  This study observed the immune response induced by a HSP65 DNA vaccine fused with UbGR against Mycobacterium tuberculosis. BALB/c mice were inoculated with HSP65 DNA vaccine, UbGR-fused HSP65 DNA vaccine (Ub-GR-HSP65 and blank vector respectively. HSP65 DNA vaccine elicited a Thl-polarized immune response. The Thl-type cytokine (IFN-? and proliferative T cell responses from spleen were improved significantly in UbGR-HSP65 group, compared with those in HSP65 DNA vaccine group. Furthermore, this fusion DNA vaccine also led to an increased ratio of IgG2ato IgGl and the cytotoxicity of T cells. IFN-? intracellular staining of splenocytes indicated that UbGR-HSP65 fusion DNA vaccine could activate CD4+ and CD8+ T cells, with much higher CD8+ T cells. Thus, this study demonstrated that the UbGR fusion could improve HSP65-specific cellular immune responses, which is helpful to protect against TB infection.

Qingmin Wang

2013-08-01

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Recurrent nontuberculous mycobacterial endophthalmitis: a diagnostic conundrum  

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Objective To report a case of recurrent nontuberculous mycobacterial endophthalmitis in the context of neurotrophic keratopathy secondary to herpes zoster ophthalmicus that had an atypical presentation and complex course, and highlights the challenges of causative organism identification and therapeutic interventions in this condition. Methods A retrospective chart review was conducted to determine the visual outcomes of the patient. Results A 68-year-old pseudophakic male with long-standing neurotrophic keratopathy and perforated descemetocele managed with cyanoacrylate glue and a contact bandage lens in the left eye, began experiencing recurrent episodes of endophthalmitis after undergoing a penetrating keratoplasty. Several therapeutic procedures including an anterior chamber washout, two pars plana vitrectomies, explantation of the posterior chamber intraocular lens and capsular bag, and multiple intravitreal antimicrobial injections, were performed to which he has ultimately responded favorably, with no signs of infection to date and stable visual acuity. The causative organism of his recurrent infections was initially identified as Mycobacterium abscessus through biochemical testing and 16S ribosomal ribonucleic acid gene sequencing; however, repeat polymerase chain reaction (PCR) and sequencing of the 65 kDa heat shock protein (hsp65) gene for experimental purposes confirmed the accurate identification of the organism to be Mycobacterium chelonae. Given the greater reliability of PCR and sequencing of the hsp65 gene over traditional biochemical tests and culture techniques, M. chelonae was likely the infectious agent all along, and the organism was originally misidentified on the basis of less accurate tests. Conclusion Recurrent atypical mycobacterial endophthalmitis requires expedient identification and management to prevent poor visual outcomes. Standard biochemical testing can identify the causative organism but is limited by the inability to distinguish between nontuberculous species reliably. We recommend the use of PCR in conjunction with sequencing of the hsp65 gene for reliable differentiation of M. chelonae and M. abscessus in atypical mycobacterial ocular infections. Minimum inhibitory concentration antibiotic susceptibility tests on cultured strains are the best guide to antibiotic selection, given the rapidly rising resistance to antimicrobials in atypical mycobacterial species.

Venkateswaran, Nandini; Yeaney, Gabrielle; Chung, Mina; Hindman, Holly B

2014-01-01

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Tissue distribution of DNA-Hsp65/TDM-loaded PLGA microspheres and uptake by phagocytic cells.  

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This study aimed to demonstrate that microspheres, used as delivery vehicle of DNA-Hsp65/TDM [plasmid DNA encoding heat shock protein 65 (Hsp65) coencapsulated with trehalose dimycolate (TDM) into PLGA microspheres], are widely spread among several organs after intramuscular administration in BALB/c mice. In general, we showed that these particles were phagocytosed by antigen presenting cells, such as macrophages and dendritic cells. Besides, it was demonstrated herein that draining lymph node cells presented a significant increase in the number of cells expressing costimulatory molecules (CD80 and CD86) and MHC class II, and also that the administration of the DNA-Hsp65/TDM and vector/TDM formulations resulted in the up-regulation of CD80, CD86 and MHC class II expression when compared to control formulations (vector/TDM and empty). Regarding the intracellular trafficking we observed that following phagocytosis, the microspheres were not found in the late endosomes and/or lysosomes, until 15 days after internalization, and we suggest that these constructions were hydrolysed in early compartments. Overall, these data expand our knowledge on PLGA [poly (lactic-co-glycolic acid)] microspheres as gene carriers in vaccination strategies, as well as open perspectives for their potential use in clinical practice. PMID:17880727

Trombone, Ana Paula F; Silva, Celio L; Almeida, Luciana P; Rosada, Rogerio S; Lima, Karla M; Oliver, Constance; Jamur, Maria C; Coelho-Castelo, Arlete A M

2007-01-01

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Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis.  

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In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-? but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis. PMID:22983180

Rocha, C D; Trombone, A P F; Lorenzi, J C C; Almeida, L P; Gembre, A F; Padilha, E; Ramos, S G; Silva, C L; Coelho-Castelo, A A M

2012-12-01

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Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis  

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In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-? but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

Rocha, C.D.; Trombone, A.P.F.; Lorenzi, J.C.C.; Almeida, L.P.; Gembre, A.F.; Padilha, E.; Ramos, S.G.; Silva, C.L.; Coelho-Castelo, A.A.M.

2012-01-01

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The Role of M. leprae Hsp65 Protein and Peptides in the Pathogenesis of Uveitis  

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Experimental autoimmune uveitis (EAU) is a well established model for immune-mediated organ-specific disease. Our group has recently shown that the M. leprae Hsp65 aggravated the uveitis in mice; in the present study, we evaluated the action of M. leprae??K409A mutant protein and the synthetic peptides Leader pep and K409A pep (covering amino acids residues 352–371 of WT and K409A proteins of M. leprae Hsp65, resp.) on the pathogenesis of EAU. Mice received the 161–180 IRBP peptide an...

Commodaro, Alessandra Gonc?alves; Marengo, Eliana Blini; Peron, Jean Pierre S.; Brandao, Wesley; Arslanian, Christina; Melo, Robson Lopes; Baldon, Estevam J.; Belfort, Rubens; Sant Anna, Osvaldo Augusto; Rizzo, Luiz Vicente

2012-01-01

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Mycobacterium tuberculosis Chaperonin 60.1 Is a More Potent Cytokine Stimulator than Chaperonin 60.2 (Hsp 65) and Contains a CD14-Binding Domain  

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Much attention has focused on the Mycobacterium tuberculosis molecular chaperone chaperonin (Cpn) 60.2 (Hsp 65) in the pathology of tuberculosis because of its immunogenicity and ability to directly activate human monocytes and vascular endothelial cells. However, M. tuberculosis is one of a small group of bacteria that contain multiple genes encoding Cpn 60 proteins. We have now cloned and expressed both M. tuberculosis proteins and report that the novel chaperonin 60, Cpn 60.1, is a more po...

2001-01-01

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Administration of M. leprae Hsp65 Interferes with the Murine Lupus Progression  

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The heat shock protein [Hsp] family guides several steps during protein synthesis, are abundant in prokaryotic and eukaryotic cells, and are highly conserved during evolution. The Hsp60 family is involved in assembly and transport of proteins, and is expressed at very high levels during autoimmunity or autoinflammatory phenomena. Here, the pathophysiological role of the wild type [WT] and the point mutated K409A recombinant Hsp65 of M. leprae in an animal model of Systemic Lupus Erythematosus...

Marengo, Eliana B.; Moraes, Luciana V.; Faria, Marcella; Fernandes, Beatriz L.; Carvalho, Luciana V.; Tambourgi, Denise V.; Rizzo, Luiz V.; Portaro, Fernanda C. V.; Camargo, Anto?nio Carlos M.; Sant Anna, Osvaldo A.

2008-01-01

 
 
 
 
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Genus-specific polymerase chain reaction for the mycobacterial dnaJ gene and species-specific oligonucleotide probes.  

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Identification of tuberculous and nontuberculous mycobacteria by biochemical methods is a long-term process that takes up to 8 weeks for completion and requires expertise to interpret the results. In order to detect and differentiate the major pathogenic mycobacterial species, we developed genus-specific primers that amplify the dnaJ gene from the broad spectrum of mycobacterial species and determined the nucleotide sequences within the dnaJ genes from 19 mycobacterial species (Mycobacterium ...

Takewaki, S.; Okuzumi, K.; Ishiko, H.; Nakahara, K.; Ohkubo, A.; Nagai, R.

1993-01-01

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Protection against tuberculosis by a single intranasal administration of DNA-hsp65 vaccine complexed with cationic liposomes  

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Full Text Available Abstract Background The greatest challenges in vaccine development include optimization of DNA vaccines for use in humans, creation of effective single-dose vaccines, development of delivery systems that do not involve live viruses, and the identification of effective new adjuvants. Herein, we describe a novel, simple technique for efficiently vaccinating mice against tuberculosis (TB. Our technique consists of a single-dose, genetic vaccine formulation of DNA-hsp65 complexed with cationic liposomes and administered intranasally. Results We developed a novel and non-toxic formulation of cationic liposomes, in which the DNA-hsp65 vaccine was entrapped (ENTR-hsp65 or complexed (COMP-hsp65, and used to immunize mice by intramuscular or intranasal routes. Although both liposome formulations induced a typical Th1 pattern of immune response, the intramuscular route of delivery did not reduce the number of bacilli. However, a single intranasal immunization with COMP-hsp65, carrying as few as 25 ?g of plasmid DNA, leads to a remarkable reduction of the amount of bacilli in lungs. These effects were accompanied by increasing levels of IFN-? and lung parenchyma preservation, results similar to those found in mice vaccinated intramuscularly four times with naked DNA-hsp65 (total of 400 ?g. Conclusion Our objective was to overcome the significant obstacles currently facing DNA vaccine development. Our results in the mouse TB model showed that a single intranasal dose of COMP-hsp65 elicited a cellular immune response that was as strong as that induced by four intramuscular doses of naked-DNA. This formulation allowed a 16-fold reduction in the amount of DNA administered. Moreover, we demonstrated that this vaccine is safe, biocompatible, stable, and easily manufactured at a low cost. We believe that this strategy can be applied to human vaccines to TB in a single dose or in prime-boost protocols, leading to a tremendous impact on the control of this infectious disease.

Silva Célio L

2008-07-01

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The Role of M. leprae Hsp65 Protein and Peptides in the Pathogenesis of Uveitis  

Science.gov (United States)

Experimental autoimmune uveitis (EAU) is a well established model for immune-mediated organ-specific disease. Our group has recently shown that the M. leprae Hsp65 aggravated the uveitis in mice; in the present study, we evaluated the action of M. leprae??K409A mutant protein and the synthetic peptides Leader pep and K409A pep (covering amino acids residues 352–371 of WT and K409A proteins of M. leprae Hsp65, resp.) on the pathogenesis of EAU. Mice received the 161–180 IRBP peptide and B. pertussis toxin followed by the intraperitoneal inoculation of K409A protein or the Leader pep or K409A pep. The Leader pep aggravated the disease, but mice receiving the K409A pep did not develop the disease and presented an increase in IL-10 levels by spleen cells and a decrease in the percentage of CD4+ IFN-?+ T cells. Moreover, animals receiving the Leader pep presented the highest scores of the disease associated with increase percentage of CD4+ IFN-?+ T cells. These results would contribute to understanding of the pathogenesis of EAU and support the concept that immune responses to Hsp are of potential importance in exacerbating, perpetuating, or even controlling organ-restricted autoimmune diseases, and it is discussed the irreversibility of autoimmune syndromes.

Commodaro, Alessandra Goncalves; Marengo, Eliana Blini; Peron, Jean Pierre S.; Brandao, Wesley; Arslanian, Christina; Melo, Robson Lopes; Baldon, Estevam J.; Belfort, Rubens; Sant'Anna, Osvaldo Augusto; Rizzo, Luiz Vicente

2012-01-01

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Protection against tuberculosis by a single intranasal administration of DNA-hsp65 vaccine complexed with cationic liposomes  

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Abstract Background The greatest challenges in vaccine development include optimization of DNA vaccines for use in humans, creation of effective single-dose vaccines, development of delivery systems that do not involve live viruses, and the identification of effective new adjuvants. Herein, we describe a novel, simple technique for efficiently vaccinating mice against tuberculosis (TB). Our technique consists of a single-dose, genetic vaccine formulation of DNA-hsp65 complexe...

Rosada Rogério S; Torre Lucimara; Frantz Fabiani G; Pf, Trombone Ana; Zárate-Bladés Carlos R; Fonseca Denise M; Rm, Souza Patri?cia; Brandão Izaíra T; Masson Ana P; Soares Édson G; Ramos Simone G; Faccioli Lúcia H; Silva Célio L; Ha, Santana Maria; Am, Coelho-castelo Arlete

2008-01-01

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Administration of Mycobacterium leprae rHsp65 Aggravates Experimental Autoimmune Uveitis in Mice  

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The 60kDa heat shock protein family, Hsp60, constitutes an abundant and highly conserved class of molecules that are highly expressed in chronic-inflammatory and autoimmune processes. Experimental autoimmune uveitis [EAU] is a T cell mediated intraocular inflammatory disease that resembles human uveitis. Mycobacterial and homologous Hsp60 peptides induces uveitis in rats, however their participation in aggravating the disease is poorly known. We here evaluate the effects of the Mycobacterium ...

Marengo, Eliana B.; Commodaro, Alessandra Gonc?alves; Peron, Jean Pierre S.; Moraes, Luciana V.; Portaro, Fernanda C. V.; Belfort, Rubens; Rizzo, Luiz Vicente; Sant Anna, Osvaldo Augusto

2009-01-01

26

Mutational and Phylogenetic Analyses of the Mycobacterial mbt Gene Cluster ?§  

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The mycobactin siderophore system is present in many Mycobacterium species, including M. tuberculosis and other clinically relevant mycobacteria. This siderophore system is believed to be utilized by both pathogenic and nonpathogenic mycobacteria for iron acquisition in both in vivo and ex vivo iron-limiting environments, respectively. Several M. tuberculosis genes located in a so-called mbt gene cluster have been predicted to be required for the biosynthesis of the core scaffold of mycobacti...

Chavadi, Sivagami Sundaram; Stirrett, Karen L.; Edupuganti, Uthamaphani R.; Vergnolle, Olivia; Sadhanandan, Gigani; Marchiano, Emily; Martin, Che; Qiu, Wei-gang; Soll, Clifford E.; Quadri, Luis E. N.

2011-01-01

27

Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus.  

Science.gov (United States)

Mycobacterium abscessus is an emerging rapidly growing mycobacterium (RGM) causing a pseudotuberculous lung disease to which patients with cystic fibrosis (CF) are particularly susceptible. We report here its complete genome sequence. The genome of M. abscessus (CIP 104536T) consists of a 5,067,172-bp circular chromosome including 4920 predicted coding sequences (CDS), an 81-kb full-length prophage and 5 IS elements, and a 23-kb mercury resistance plasmid almost identical to pMM23 from Mycobacterium marinum. The chromosome encodes many virulence proteins and virulence protein families absent or present in only small numbers in the model RGM species Mycobacterium smegmatis. Many of these proteins are encoded by genes belonging to a "mycobacterial" gene pool (e.g. PE and PPE proteins, MCE and YrbE proteins, lipoprotein LpqH precursors). However, many others (e.g. phospholipase C, MgtC, MsrA, ABC Fe(3+) transporter) appear to have been horizontally acquired from distantly related environmental bacteria with a high G+C content, mostly actinobacteria (e.g. Rhodococcus sp., Streptomyces sp.) and pseudomonads. We also identified several metabolic regions acquired from actinobacteria and pseudomonads (relating to phenazine biosynthesis, homogentisate catabolism, phenylacetic acid degradation, DNA degradation) not present in the M. smegmatis genome. Many of the "non mycobacterial" factors detected in M. abscessus are also present in two of the pathogens most frequently isolated from CF patients, Pseudomonas aeruginosa and Burkholderia cepacia. This study elucidates the genetic basis of the unique pathogenicity of M. abscessus among RGM, and raises the question of similar mechanisms of pathogenicity shared by unrelated organisms in CF patients. PMID:19543527

Ripoll, Fabienne; Pasek, Sophie; Schenowitz, Chantal; Dossat, Carole; Barbe, Valérie; Rottman, Martin; Macheras, Edouard; Heym, Beate; Herrmann, Jean-Louis; Daffé, Mamadou; Brosch, Roland; Risler, Jean-Loup; Gaillard, Jean-Louis

2009-01-01

28

Molecular mimicry between HSP 65 of Mycobacterium leprae and cytokeratin 10 of the host keratin; role in pathogenesis of leprosy.  

Science.gov (United States)

Mycobacteria are known to induce autoimmune response in the host. Anti-host keratrin antibodies (AkAbs) might be responsible for the autoimmune phenomena in leprosy patients as majority of leprosy lesions are manifested in the skin and occurrence of keratosis is not an uncommon feature. The aim of this study was to find out the level of AkAbs in leprosy patients across the spectrum and to explore its correlation with the clinical manifestation of the disease. Further, mimicking epitopes of keratin and Mycobacterium leprae components were characterized. We screened 140 leprosy patients (27 BT, 28 BL, 41 LL, 25 T1R, 19 ENL), 74 healthy controls (HC) and 3 psoriasis patients as positive control. Highest AkAbs level was observed in the psoriasis patients followed by T1R, LL, BL, ENL, TT/BT. AkAbs level was significantly (pMLSA) induce higher level of AkAbs. The percentage of FoxP3(+) expressing Treg cells (total CD4(+)CD25(+)FoxP3(+) andCD4(+)CD25(+hi)FoxP3(+)) in splenocytes and lymph nodes of hyperimmunized mice were declined in comparison to control mice. Further, it was found that this autoimmune response can be adoptively transferred in naïve mice by splenocytes and lymph node cells as well as T cells. Comparative molecular characterization between keratin and MLSA noted a cross-reactivity/similarity between these two antigens. The cross-reactive protein of keratin was found to be in molecular weight range ?74-51kDa and at pI 4.5 while the cross-reactive protein of MLSA was found to be in molecular weight ?65kDa and at pI 4-4.5. Cross-reactive protein of keratin and MLSA was identified and characterized by MALDI-TOF/TOF analysis and Mascot software. It was found that the keratin (host protein) which reacted with anti-M. leprae sera is cytokeratin-10 and MLSA which reacted with anti-keratin sera is heat shock protein 65 (HSP 65). Seven B-cell epitopes of cytokeratin-10 and HSP 65 was found to be similar by multiple sequence alignment using ClustalW server and out of which 6 B-cell epitopes were found to be on the surface of HSP 65. In conclusion, our study provides evidence for the existence of molecular mimicry between cytokeratin-10 of keratin (host protein) and 65kDa HSP (groEL2) of M. leprae. Presence of heightened CMI response of leprosy patients to keratin and positive correlation of AkAbs level with number of lesions of leprosy patients showed the clinical evidence for its role in the pathogenesis in leprosy. PMID:23121977

Singh, Itu; Yadav, Asha Ram; Mohanty, Keshar Kunja; Katoch, Kiran; Bisht, Deepa; Sharma, Prashant; Sharma, Bhawna; Gupta, U D; Sengupta, Utpal

2012-01-01

29

Identification of Mycobacterium using the EF-Tu encoding (tuf) gene and the tmRNA encoding (ssrA) gene.  

Science.gov (United States)

The partial nucleotide sequences encoding the elongation factor Tu (tuf gene) (652 bp) and transfer-mRNA (tmRNA or ssrA gene) (340 bp) were determined to assess the suitability of these two genes as phylogenetic markers for the classification of mycobacteria, and thus as alternative target molecules for identifying mycobacteria. A total of 125 reference strains of the genus Mycobacterium and 74 clinical isolates were amplified by PCR and sequenced. Phylogenies of the two genes constructed by the neighbour-joining method were created and compared to a concatenated tree of 16S rDNA, hsp65, sodA and rpoB genes. The phylogenetic trees revealed the overall natural relationships among Mycobacterium species. The tmRNA phylogeny was similar to that of 16S rDNA, with low resolving power. The tuf gene provided better resolution of each mycobacterial species, with a phylogeny close to that of hsp65. However, none of these methods differentiated between the members of the Mycobacterium tuberculosis complex or the subspecies of the Mycobacterium avium complex. The correct identification of clinical isolates confirms the interest of these genes, especially tuf. It is suggested from these findings that tmRNA might be useful as another housekeeping gene in a polyphyletic approach to Mycobacterium species, but not as a first-line marker of species. tuf gene analysis suggests that this gene could be used effectively for phylogenetic analysis and to identify mycobacteria. PMID:17644709

Mignard, Sophie; Flandrois, Jean-Pierre

2007-08-01

30

Induction of early-response genes KC and JE by mycobacterial lipoarabinomannans: regulation of KC expression in murine macrophages by Lsh/Ity/Bcg (candidate Nramp).  

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The murine chromosome 1 gene Lsh/Ity/Bcg (candidate Nramp) regulates macrophage activation for antimicrobial activity against Salmonella typhimurium, Leishmania donovani, and Mycobacterium spp. To determine early events in the activation pathway, the ability of mycobacterial lipoarabinomannan (LAM) to induce early gene (KC and JE) expression in macrophages from susceptible (S) C57BL/10ScSn (Lshs) and congenic resistant (R) B10.L-Lshr mice was investigated. Stimulation with 1.8 microgram of ar...

Roach, T. I.; Chatterjee, D.; Blackwell, J. M.

1994-01-01

31

Mycobacterial lymphadenitis.  

Science.gov (United States)

The plan for cervical lymph node biopsy should include special maneuvers for recognition of patients with lymphadenitis due to atypical mycobacteria, since these children need extensive operations. The diagnosis should be suspected in children less than 3 yr old with lymphadenopathy present for several months and no exposure to cats (or with negative cat scratch skin tests). Wide local excision of all visibly involved nodes is recommended; acid-fast touch preparations should be done and interpreted during operation in any suspicious case. Limited operations should be avoided in children with mycobacterial lymphadenitis. The illness may be more common than previously suspected. PMID:7175650

Harris, B H; Webb, H W; Wilkinson, A H; Santelices, A A

1982-10-01

32

Identification and Characterization of the Genes Involved in Glycosylation Pathways of Mycobacterial Glycopeptidolipid Biosynthesis  

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Glycopeptidolipids (GPLs) are major components present on the outer layers of the cell walls of several nontuberculous mycobacteria. GPLs are antigenic molecules and have variant oligosaccharides in mycobacteria such as Mycobacterium avium. In this study, we identified four genes (gtf1, gtf2, gtf3, and gtf4) in the genome of Mycobacterium smegmatis. These genes were independently inactivated by homologous recombination in M. smegmatis, and the structures of GPLs from each gene disruptant were...

Miyamoto, Yuji; Mukai, Tetsu; Nakata, Noboru; Maeda, Yumi; Kai, Masanori; Naka, Takashi; Yano, Ikuya; Makino, Masahiko

2006-01-01

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High-Resolution Phenotypic Profiling Defines Genes Essential for Mycobacterial Growth and Cholesterol Catabolism  

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The pathways that comprise cellular metabolism are highly interconnected, and alterations in individual enzymes can have far-reaching effects. As a result, global profiling methods that measure gene expression are of limited value in predicting how the loss of an individual function will affect the cell. In this work, we employed a new method of global phenotypic profiling to directly define the genes required for the growth of Mycobacterium tuberculosis. A combination of high-density mutagen...

Griffin, Jennifer E.; Gawronski, Jeffrey D.; Dejesus, Michael A.; Ioerger, Thomas R.; Akerley, Brian J.; Sassetti, Christopher M.

2011-01-01

34

Method to integrate multiple plasmids into the mycobacterial chromosome.  

Science.gov (United States)

In order to create a system in which two independent plasmids can be integrated into a mycobacterial chromosome, a mycobacterial plasmid was constructed containing the phage attachment site attP from the mycobacteriophage L5 genome and additionally containing the bacterial attachment site, attB. This plasmid will integrate into the mycobacterial chromosome via recombination of the plasmid-borne attP site with the chromosomal attB site in the presence of a mycobacterial vector carrying the L5 integrase (int) gene. The integrated plasmid has a plasmid-borne attB site that is preserved and will accept the integration of additional mycobacterial plasmids containing the L5 attP site. This system should be useful in the construction of novel mycobacterial strains. In particular, this system provides a method by which several recombinant antigens or reporter constructs can be sequentially inserted into a mycobacterial strain and subsequently tested. PMID:14718555

Saviola, Beatrice; Bishai, William R

2004-01-01

35

Evidence for three separate genes encoding the proteins of the mycobacterial antigen 85 complex.  

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The secreted Mycobacterium bovis BCG antigen 85 complex, which is known to bind to human fibronectin, consists of three closely related cross-reacting antigens. Amino-terminal sequence analysis of the purified proteins showed distinct differences. Data are presented to show that the three components are produced by individual cells, which indicates that three separate genes are involved.

Wiker, H. G.; Sletten, K.; Nagai, S.; Harboe, M.

1990-01-01

36

Mutation Analysis of Mycobacterial rpoB Genes and Rifampin Resistance Using Recombinant Mycobacterium smegmatis  

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Rifampin is a major drug used to treat leprosy and tuberculosis. The rifampin resistance of Mycobacterium leprae and Mycobacterium tuberculosis results from a mutation in the rpoB gene, encoding the ? subunit of RNA polymerase. A method for the molecular determination of rifampin resistance in these two mycobacteria would be clinically valuable, but the relationship between the mutations and susceptibility to rifampin must be clarified before its use. Analyses of mutations responsible for ri...

Nakata, Noboru; Kai, Masanori; Makino, Masahiko

2012-01-01

37

Downregulation of TACO gene transcription restricts mycobacterial entry/survival within human macrophages.  

Science.gov (United States)

Recent reports have indicated that cholesterol-dependent association of tryptophan-aspartate containing coat protein (TACO) plays a crucial role in the entry/survival of Mycobacterium tuberculosis within human macrophages. Keeping this in view, the present study explored whether the molecules that have the ability to downregulate TACO gene transcription could also restrict entry/survival of mycobacteria within human macrophages. The study revealed that chenodeoxycholic acid (CDCA), either alone or in combination with retinoic acid (RA), had the inherent capacity to downregulate TACO gene transcription in a dose-dependent fashion. This result was in conformity with the existence of a functional FXR/RXR binding site analyzed in the regulatory region of the TACO gene. Furthermore, we demonstrate that the entry and intracellular survival of M. tuberculosis is significantly restricted in THP-1 macrophages exposed to CDCA/RA. On the basis of these findings, we propose that the CDCA/RA-dependent pathway may open a new possibility for the treatment of tuberculosis. PMID:16040207

Anand, Paras K; Kaul, Deepak

2005-09-01

38

Zebrafish embryo screen for mycobacterial genes involved in the initiation of granuloma formation reveals a newly identified ESX-1 component  

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The hallmark of tuberculosis (TB) is the formation of granulomas, which are clusters of infected macrophages surrounded by additional macrophages, neutrophils and lymphocytes. Although it has long been thought that granulomas are beneficial for the host, there is evidence that mycobacteria also promote the formation of these structures. In this study, we aimed to identify new mycobacterial factors involved in the initial stages of granuloma formation. We exploited the zebrafish embryo Mycobac...

Stoop, Esther J. M.; Schipper, Tim; Rosendahl Huber, Sietske K.; Nezhinsky, Alexander E.; Verbeek, Fons J.; Gurcha, Sudagar S.; Besra, Gurdyal S.; Vandenbroucke-grauls, Christina M. J. E.; Bitter, Wilbert; Sar, Astrid M.

2011-01-01

39

Comparative analysis of mycobacterial truncated hemoglobin promoters and the groEL2 promoter in free-living and intracellular mycobacteria.  

Science.gov (United States)

The success of Mycobacterium tuberculosis depends on its ability to withstand and survive the hazardous environment inside the macrophages that are created by reactive oxygen intermediates, reactive nitrogen intermediates, severe hypoxia, low pH, and high CO(2) levels. Therefore, an effective detoxification system is required for the pathogen to persist in vivo. The genome of M. tuberculosis contains a new family of hemoproteins named truncated hemoglobin O (trHbO) and truncated hemoglobin N (trHbN), encoded by the glbO and glbN genes, respectively, important in the survival of M. tuberculosis in macrophages. Mycobacterial heat shock proteins are known to undergo rapid upregulation under stress conditions. The expression profiles of the promoters of these genes were studied by constructing transcriptional fusions with green fluorescent protein and monitoring the promoter activity in both free-living and intracellular milieus at different time points. Whereas glbN showed an early response to the oxidative and nitrosative stresses tested, glbO gave a lasting response to lower concentrations of both stresses. At all time points and under all stress conditions tested, groEL2 showed higher expression than both trHb promoters and expression of both promoters showed an increase while inside the macrophages. Real-time PCR analysis of trHb and groEL2 mRNAs showed an initial upregulation at 24 h postinfection. The presence of the glbO protein imparted an increased survival to M. smegmatis in THP-1 differentiated macrophages compared to that imparted by the glbN and hsp65 proteins. The comparative upregulation shown by both trHb promoters while grown inside macrophages indicates the importance of these promoters for the survival of M. tuberculosis in the hostile environment of the host. PMID:22773641

Joseph, Sunil V; Madhavilatha, G K; Kumar, R Ajay; Mundayoor, Sathish

2012-09-01

40

Mycobacterial Phylogenomics: An Enhanced Method for Gene Turnover Analysis Reveals Uneven Levels of Gene Gain and Loss among Species and Gene Families  

Science.gov (United States)

Species of the genus Mycobacterium differ in several features, from geographic ranges, and degree of pathogenicity, to ecological and host preferences. The recent availability of several fully sequenced genomes for a number of these species enabled the comparative study of the genetic determinants of this wide lifestyle diversity. Here, we applied two complementary phylogenetic-based approaches using information from 19 Mycobacterium genomes to obtain a more comprehensive view of the evolution of this genus. First, we inferred the phylogenetic relationships using two new approaches, one based on a Mycobacterium-specific amino acid substitution matrix and the other on a gene content dissimilarity matrix. Then, we utilized our recently developed gain-and-death stochastic models to study gene turnover dynamics in this genus in a maximum-likelihood framework. We uncovered a scenario that differs markedly from traditional 16S rRNA data and improves upon recent phylogenomic approaches. We also found that the rates of gene gain and death are high and unevenly distributed both across species and across gene families, further supporting the utility of the new models of rate heterogeneity applied in a phylogenetic context. Finally, the functional annotation of the most expanded or contracted gene families revealed that the transposable elements and the fatty acid metabolism-related gene families are the most important drivers of gene content evolution in Mycobacterium.

Librado, Pablo; Vieira, Filipe G.; Sanchez-Gracia, Alejandro; Kolokotronis, Sergios-Orestis; Rozas, Julio

2014-01-01

 
 
 
 
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Validation of pncA Gene Sequencing in Combination with the Mycobacterial Growth Indicator Tube Method To Test Susceptibility of Mycobacterium tuberculosis to Pyrazinamide  

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Pyrazinamide is important in the treatment of tuberculosis. Unfortunately, the diagnosis of pyrazinamide resistance is hampered by technical difficulties. We hypothesized that mutation analysis combined with the mycobacterial growth indicator tube (MGIT) phenotypic method would be a good predictor of pyrazinamide resistance. We prospectively analyzed 1,650 M. tuberculosis isolates referred to our tuberculosis reference laboratory in 2008 and 2009. In our laboratory, the MGIT 960 system was us...

Simons, Sami O.; Ingen, Jakko; Laan, Tridia; Mulder, Arnout; Dekhuijzen, P. N. Richard; Boeree, Martin J.; Soolingen, Dick

2012-01-01

42

Quinolones for mycobacterial infections.  

Science.gov (United States)

The fluoroquinolones (FQs) are important agents for the treatment of mycobacterial infections. In leprosy, the use of FQs has enabled a dramatic shortening of formerly long and complicated therapy. Both animal and human studies support the inclusion of certain FQs as a cornerstone of leprosy therapy. In tuberculosis (TB), particularly in multidrug-resistant (MDR) infections, the place of the major antimycobacterial FQs is less clear as there is widespread resistance to these agents in areas of the world in which MDR-TB and extensively drug-resistant (XDR)-TB are prevalent, particularly in Southeast Asia. The place of the newly developed FQ-related diarylquinoline compound known as bedaquiline in the treatment of drug-resistant TB is unclear; however, human studies suggest that it might be effective for this indication. PMID:23643393

Rubinstein, Ethan; Keynan, Yoav

2013-07-01

43

Molecular characterization of the mycobacterial heparin-binding hemagglutinin, a mycobacterial adhesin  

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Although it generally is accepted that the interaction of Mycobacterium tuberculosis with alveolar macrophages is a key step in the pathogenesis of tuberculosis, interactions with other cell types, especially epithelial cells, also may be important. In this study we describe the molecular characterization of a mycobacterial heparin-binding hemagglutinin (HBHA), a protein that functions as an adhesin for epithelial cells. The structural gene was cloned from M. tuberculosis and bacillus Calmett...

Menozzi, Franco D.; Bischoff, Rainer; Fort, Emmanuelle; Brennan, Michael J.; Locht, Camille

1998-01-01

44

A seven-gene, multilocus, genus-wide approach to the phylogeny of mycobacteria using supertrees.  

Science.gov (United States)

This is the first study that estimates mycobacterial phylogeny using the maximum-likelihood method (PhyML-aLRT) on a seven-gene concatenate (hsp65, rpoB, 16S rRNA, smpB, sodA, tmRNA and tuf) and the super distance matrix (SDM) supertree method. Two sets of sequences were studied: a complete seven gene sequence set (set R, type strains of 87 species) and an incomplete set (set W, 132 species) with some missing data. Congruencies were computed by using the consense program (phylip package). The evolution rate of each gene was determined, as was the evolution rate of each strain for a given gene. Maximum-likelihood trees resulting from concatenation of the R and W sets resulted in a similar phylogeny, usually showing an early separation between slow-growing (SG) and rapidly growing (RG) mycobacteria. The SDM tree for the W set resulted in a different phylogeny. The separation of SG and RG was still evident, but it was located later in the nodes. The SG were therefore positioned as a subgroup of RG. Maximum-likelihood phylogenetic reconstruction was less affected by increasing the number of strains (with incomplete data), but did seem to cushion the variability of the evolution rate (ER), whereas the SDM method seemed to be more accurate and took into account both the differing ER values and the incomplete data. With regard to ER, it was observed that the 16S rRNA gene was the gene that displayed the slowest evolution, whereas smpB was the most rapidly evolving gene. Surprisingly, these two genes alone accurately separated the SG from the RG on the basis of their ER values. This study focused on the differences in ER between genes and in some cases linked the ER to the phenotypic classification of the mycobacteria. PMID:18523191

Mignard, Sophie; Flandrois, Jean-Pierre

2008-06-01

45

Construction and application of mycobacterial reporter transposons.  

Science.gov (United States)

The transposon Tn5367, which is a derivative of the mycobacterial insertion sequence IS1096, was modified by introducing novel genes to produce reporter transposons which can be used to generate transposon insertion libraries containing mycobacterial gene or operon fusions. A plasmid that is temperature-sensitive for replication in mycobacteria was used to deliver promoterless lacZ or aph reporter genes to Mycobacterium smegmatis as transcriptional (lacZ), or translational ('aph) fusions. Mutants containing lacZ produced varying intensities of blue colour on indicator media. This reporter activity could be used as a quantitative measure of promoter strength. Mutants displaying varying levels of resistance to kanamycin were obtained by transpositional insertion of the 'aph reporter lacking a promoter, ribosome binding site and start codon to form functionally active translational fusions. Finally, inclusion of the R6Kgamma origin within Tn5367 allowed transposon insertions to be rescued in an Escherichia coli host strain permissive for the replication of this origin. This study demonstrates that transcriptional and translational reporter derivatives of Tn5367 are functional, and they supplement the growing range of molecular tools available for the study of mycobacteria. PMID:10925203

Machowski, E E; McAdam, R A; Derbyshire, K M; Mizrahi, V

2000-07-25

46

Identification of Mycobacterial ? Factor Binding Sites by Chromatin Immunoprecipitation Assays?  

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Mycobacterium tuberculosis and Mycobacterium bovis are responsible for infections that cause a substantial amount of death, suffering, and loss around the world. Still, relatively little is known about the mechanisms of gene expression in these bacteria. Here, we used genome-wide location assays to identify direct target genes for mycobacterial ? factors. Chromatin immunoprecipitation assays were performed with M. bovis BCG for Myc-tagged proteins expressed using an anhydrotetracycline-induc...

Rodrigue, Se?bastien; Brodeur, Joe?lle; Jacques, Pierre-e?tienne; Gervais, Alain L.; Brzezinski, Ryszard; Gaudreau, Luc

2007-01-01

47

Murine T-lymphocyte activation by mycobacterial antigens  

International Nuclear Information System (INIS)

There has been renewed interest in the diagnosis of tuberculosis and other mycobacterial infections in the United States. Effective immunity to mycobacterial infections, as well as diagnosis by the skin test, involves T-cells rather than antibodies. Studies currently underway use the new technologies of monoclonal antibodies and recombinant DNA to define better mycobacterial antigens for T-cell activation, in the hope of identifying species specific antigens. Lymph node cells from mice sensitized to Mycobacterium intracellulare and Mycobacterium avium were assayed for activation by mycobacterial fractions, and cell lines and clones were generated. Comparing BALB/c and B10 mice indicated better responses to M. avium sonicate by B10 mice. A recombinant gene product containing a M. intracellulare peptide was assayed with lymph node cells and indicated excellent T-cell stimulation in BALB/c lymph node cells and cell lines. However, assays using B10 T-cell clones have yet to detect responders to the recombinant protein. Future studies using synthetic epitopes produced by recombinant DNA techniques and defined by monoclonal antibodies are necessary for the identification of reactive T-cell epitopes that are potentially species specific. 4 refs, 7 figs, 1 tab

1991-07-01

48

Phenotypic heterogeneity in mycobacterial stringent response  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background A common survival strategy of microorganisms subjected to stress involves the generation of phenotypic heterogeneity in the isogenic microbial population enabling a subset of the population to survive under stress. In a recent study, a mycobacterial population of M. smegmatis was shown to develop phenotypic heterogeneity under nutrient depletion. The observed heterogeneity is in the form of a bimodal distribution of the expression levels of the Green Fluorescent Protein (GFP as reporter with the gfp fused to the promoter of the rel gene. The stringent response pathway is initiated in the subpopulation with high rel activity. Results In the present study, we characterise quantitatively the single cell promoter activity of the three key genes, namely, mprA, sigE and rel, in the stringent response pathway with gfp as the reporter. The origin of bimodality in the GFP distribution lies in two stable expression states, i.e., bistability. We develop a theoretical model to study the dynamics of the stringent response pathway. The model incorporates a recently proposed mechanism of bistability based on positive feedback and cell growth retardation due to protein synthesis. Based on flow cytometry data, we establish that the distribution of GFP levels in the mycobacterial population at any point of time is a linear superposition of two invariant distributions, one Gaussian and the other lognormal, with only the coefficients in the linear combination depending on time. This allows us to use a binning algorithm and determine the time variation of the mean protein level, the fraction of cells in a subpopulation and also the coefficient of variation, a measure of gene expression noise. Conclusions The results of the theoretical model along with a comprehensive analysis of the flow cytometry data provide definitive evidence for the coexistence of two subpopulations with overlapping protein distributions.

Bose Indrani

2011-01-01

49

The essential mycobacterial genes, fabG1 and fabG4, encode 3-oxoacyl-thioester reductases that are functional in yeast mitochondrial fatty acid synthase type 2  

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Mycobacterium tuberculosis represents a severe threat to human health worldwide. Therefore, it is important to expand our knowledge of vital mycobacterial processes, such as that effected by fatty acid synthase type 2 (FASII), as well as to uncover novel ones. Mycobacterial FASII undertakes mycolic acid biosynthesis, which relies on a set of essential enzymes, including 3-oxoacyl-AcpM reductase FabG1/Rv1483. However, the M. tuberculosis genome encodes four additional FabG homologs, designated...

Gurvitz, Aner

2009-01-01

50

TNF-? Modulates TLR2-Dependent Responses During Mycobacterial Infection.  

Science.gov (United States)

Multifunctional roles of tumor necrosis factor-alpha (TNF-?) during the mycobacterial pathogenesis make it an important molecule to understand and to examine the course of infection. Identification and analysis of TNF-? response can largely contribute to determine the potential host mediators for therapeutic intervention against tuberculosis. The current chapter describes several methods to assess the ability of TNF-? signaling to modulate toll-like receptor (TLR)2 signaling, another key player in mycobacterial infection and its responses. Experiments involving neutralizing antibodies, antagonists, pharmacological inhibitors, and siRNA-mediated gene silencing are discussed in this chapter to establish the role of TNF-? signaling. The widely used protein and mRNA analysis readouts like enzyme-linked immunosorbent assay (ELISA), immunoblotting, fluorescence-activated cell sorting (FACS), and quantitative real-time RT-PCR are useful to estimate and confirm the mediators involved in TNF-? and TLR2 signaling. PMID:24788179

Holla, Sahana; Trinath, Jamma; Balaji, Kithiganahalli Narayanaswamy

2014-01-01

51

Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors  

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Mycobacterium bovis Bacillus Calmette-Guérin (BCG) as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261) and Mycobacteria spp. ?-antigen promo...

Joan Joseph; Raquel Fernández-Lloris; Elías Pezzat; Saubi, Narc S.; Pere-Joan Cardona; Beatriz Mothe; Josep Maria Gatell

2010-01-01

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Differential expression of iron-, carbon-, and oxygen-responsive mycobacterial genes in the lungs of chronically infected mice and tuberculosis patients  

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Pathogenetic processes that facilitate the entry, replication, and persistence of Mycobacterium tuberculosis (MTB) in the mammalian host likely include the regulated expression of specific sets of genes at different stages of infection. Identification of genes that are differentially expressed in vivo would provide insights into host-pathogen interactions in tuberculosis (TB); this approach might be particularly valuable for the study of human TB, where experimental opportunities are limited....

Timm, Juliano; Post, Frank A.; Bekker, Linda-gail; Walther, Gabriele B.; Wainwright, Helen C.; Manganelli, Riccardo; Chan, Wai-tsing; Tsenova, Liana; Gold, Benjamin; Smith, Issar; Kaplan, Gilla; Mckinney, John D.

2003-01-01

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Mycobacterial mutants with defective control of phagosomal acidification.  

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The pathogenesis of mycobacterial infection is associated with an ability to interfere with maturation of the phagosomal compartment after ingestion by macrophages. Identification of the mycobacterial components that contribute to this phenomenon will allow rational design of novel approaches to the treatment and prevention of tuberculosis. Microarray-based screening of a transposon library was used to identify mutations that influence the fate of Mycobacterium bovis bacille Calmette-Guérin (BCG) following uptake by macrophages. A screen based on bacterial survival during a 3-d infection highlighted genes previously implicated in growth of Mycobacterium tuberculosis in macrophages and in mice, together with a number of other virulence genes including a locus encoding virulence-associated membrane proteins and a series of transporter molecules. A second screen based on separation of acidified and non-acidified phagosomes by flow cytometry identified genes involved in mycobacterial control of early acidification. This included the KefB potassium/proton antiport. Mutants unable to control early acidification were significantly attenuated for growth during 6-d infections of macrophages. Early acidification of the phagosome is associated with reduced survival of BCG in macrophages. A strong correlation exists between genes required for intracellular survival of BCG and those required for growth of M. tuberculosis in mice. In contrast, very little correlation exists between genes required for intracellular survival of BCG and those that are up-regulated during intracellular adaptation of M. tuberculosis. This study has identified targets for interventions to promote immune clearance of tuberculosis infection. The screening technologies demonstrated in this study will be useful to the study of pathogenesis in many other intracellular microorganisms. PMID:16322769

Stewart, Graham R; Patel, Janisha; Robertson, Brian D; Rae, Aaron; Young, Douglas B

2005-11-01

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Mycobacterial mutants with defective control of phagosomal acidification.  

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Full Text Available The pathogenesis of mycobacterial infection is associated with an ability to interfere with maturation of the phagosomal compartment after ingestion by macrophages. Identification of the mycobacterial components that contribute to this phenomenon will allow rational design of novel approaches to the treatment and prevention of tuberculosis. Microarray-based screening of a transposon library was used to identify mutations that influence the fate of Mycobacterium bovis bacille Calmette-Guérin (BCG following uptake by macrophages. A screen based on bacterial survival during a 3-d infection highlighted genes previously implicated in growth of Mycobacterium tuberculosis in macrophages and in mice, together with a number of other virulence genes including a locus encoding virulence-associated membrane proteins and a series of transporter molecules. A second screen based on separation of acidified and non-acidified phagosomes by flow cytometry identified genes involved in mycobacterial control of early acidification. This included the KefB potassium/proton antiport. Mutants unable to control early acidification were significantly attenuated for growth during 6-d infections of macrophages. Early acidification of the phagosome is associated with reduced survival of BCG in macrophages. A strong correlation exists between genes required for intracellular survival of BCG and those required for growth of M. tuberculosis in mice. In contrast, very little correlation exists between genes required for intracellular survival of BCG and those that are up-regulated during intracellular adaptation of M. tuberculosis. This study has identified targets for interventions to promote immune clearance of tuberculosis infection. The screening technologies demonstrated in this study will be useful to the study of pathogenesis in many other intracellular microorganisms.

2005-11-01

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Identification by 16S rRNA Gene Analyses of a Potential Novel Mycobacterial Species as an Etiological Agent of Canine Leproid Granuloma Syndrome  

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PCR amplifications of the 16S rRNA gene were performed on 46 specimens obtained from 43 dogs with canine leproid granuloma syndrome to help determine its etiology. Sequence capture PCR was applied to 37 paraffin-embedded specimens from 37 dogs, and nested PCR was attempted on DNA from 9 fresh tissue specimens derived from 3 of the 37 aforementioned dogs and from an additional 6 dogs. Molecular analyses of the paraffin-embedded tissues and fresh tissue specimen analyses were performed at separ...

Hughes, M. S.; James, G.; Ball, N.; Scally, M.; Malik, R.; Wigney, D. I.; Martin, P.; Chen, S.; Mitchell, D.; Love, D. N.

2000-01-01

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Inhibitors of UDP-galactopyranose mutase thwart mycobacterial growth.  

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Galactofuranose (Galf) residues are fundamental components of the cell wall of mycobacteria. A key enzyme, UDP-galactopyranose mutase (UGM), that participates in Galf incorporation mediates isomerization of UDP-Galf from UDP-galactopyranose (UDP-Galp). UGM is of special interest as a therapeutic target because the gene encoding it is essential for mycobacterial viability and there is no comparable enzyme in humans. We used structure-activity relationships and molecular design to devise UGM inhibitors. From a focused library of synthetic aminothiazoles, several compounds that block the UGM from Klebsiella pneumoniae or Mycobacterium tuberculosis were identified. These inhibitors block the growth of M. smegmatis. PMID:18447352

Dykhuizen, Emily C; May, John F; Tongpenyai, Aimon; Kiessling, Laura L

2008-05-28

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Mycobacterial species as case-study of comparative genome analysis  

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The genus Mycobacterium represents more than 120 species including important pathogens of human and cause major public health problems and illnesses. Further, with more than 100 genome sequences from this genus, comparative genome analysis can provide new insights for better understanding the evolutionary events of these species and improving drugs, vaccines, and diagnostics tools for controlling Mycobacterial diseases. In this present study we aim to outline a comparative genome analysis of fourteen Mycobacterial genomes: M. avium subsp. paratuberculosis Kâ??10, M. bovis AF2122/97, M. bovis BCG str. Pasteur 1173P2, M. leprae Br4923, M. marinum M, M. sp. KMS, M. sp. MCS, M. tuberculosis CDC1551, M. tuberculosis F11, M. tuberculosis H37Ra, M. tuberculosis H37Rv, M. tuberculosis KZN 1435 , M. ulcerans Agy99,and M. vanbaalenii PYRâ??1, For this purpose a comparison has been done based on their length of genomes, GC content, number of genes in different data bases (Genbank, Refseq, and Prodigal). The BLAST matrix of these genomes has been figured to give a lot of information about the similarity between species in a simple scheme. As a result of multiple genome analysis, the pan and core genome have been defined for twelve Mycobacterial species. We have also introduced the genome atlas of the reference strain M. tuberculosis H37Rv which can give a good overview of this genome. And for examining the phylogenetic relationships among these bacteria, a phylogenic tree has been constructed from 16S rRNA gene for tuberculosis and non tuberculosis Mycobacteria to understand the evolutionary events of these species.

Zakham, F.; Belayachi, L.

2011-01-01

58

CD36 deficiency attenuates experimental mycobacterial infection  

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Full Text Available Abstract Background Members of the CD36 scavenger receptor family have been implicated as sensors of microbial products that mediate phagocytosis and inflammation in response to a broad range of pathogens. We investigated the role of CD36 in host response to mycobacterial infection. Methods Experimental Mycobacterium bovis Bacillus Calmette-Guérin (BCG infection in Cd36+/+ and Cd36-/- mice, and in vitro co-cultivation of M. tuberculosis, BCG and M. marinum with Cd36+/+ and Cd36-/-murine macrophages. Results Using an in vivo model of BCG infection in Cd36+/+ and Cd36-/- mice, we found that mycobacterial burden in liver and spleen is reduced (83% lower peak splenic colony forming units, p Cd36-/- animals. Intracellular growth of all three mycobacterial species was reduced in Cd36-/- relative to wild type Cd36+/+ macrophages in vitro. This difference was not attributable to alterations in mycobacterial uptake, macrophage viability, rate of macrophage apoptosis, production of reactive oxygen and/or nitrogen species, TNF or interleukin-10. Using an in vitro model designed to recapitulate cellular events implicated in mycobacterial infection and dissemination in vivo (i.e., phagocytosis of apoptotic macrophages containing mycobacteria, we demonstrated reduced recovery of viable mycobacteria within Cd36-/- macrophages. Conclusions Together, these data indicate that CD36 deficiency confers resistance to mycobacterial infection. This observation is best explained by reduced intracellular survival of mycobacteria in the Cd36-/- macrophage and a role for CD36 in the cellular events involved in granuloma formation that promote early bacterial expansion and dissemination.

Min-Oo Gundula

2010-10-01

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The essential mycobacterial genes, fabG1 and fabG4, encode 3-oxoacyl-thioester reductases that are functional in yeast mitochondrial fatty acid synthase type 2.  

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Mycobacterium tuberculosis represents a severe threat to human health worldwide. Therefore, it is important to expand our knowledge of vital mycobacterial processes, such as that effected by fatty acid synthase type 2 (FASII), as well as to uncover novel ones. Mycobacterial FASII undertakes mycolic acid biosynthesis, which relies on a set of essential enzymes, including 3-oxoacyl-AcpM reductase FabG1/Rv1483. However, the M. tuberculosis genome encodes four additional FabG homologs, designated FabG2-FabG5, whose functions have hitherto not been characterized in detail. Of the four candidates, FabG4/Rv0242c was recently shown to be essential for the survival of M. bovis BCG. The present work was initiated by assessing the suitability of yeast oar1Delta mutant cells lacking mitochondrial 3-oxoacyl-ACP reductase activity to act as a surrogate system for expressing FabG1/MabA directed to the mitochondria. Mutant yeast cells producing this targeted FabG1 variant were essentially wild type for all of the chronicled phenotype characteristics, including respiratory growth on glycerol medium, cytochrome assembly and lipoid acid production. This indicated that within the framework of de novo fatty acid biosynthesis in yeast mitochondria, FabG1 was able to act on shorter (C(4)) acyl substrates than was previously proposed (C(8-20)) during mycolic acid biosynthesis in M. tuberculosis. Thereafter, FabG2-FabG5 were expressed as mitochondrial proteins in the oar1Delta strain, and FabG4 was found to complement the mutant phenotype and contain high levels of 3-oxoacyl-thioester reductase activity. Hence, like FabG1, FabG4 is also an essential, physiologically functional 3-oxoacyl-thioester reductase, albeit the latter's involvement in mycobacterial FASII remains to be explored. PMID:19685079

Gurvitz, Aner

2009-10-01

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Molecular characterization of the mycobacterial heparin-binding hemagglutinin, a mycobacterial adhesin.  

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Although it generally is accepted that the interaction of Mycobacterium tuberculosis with alveolar macrophages is a key step in the pathogenesis of tuberculosis, interactions with other cell types, especially epithelial cells, also may be important. In this study we describe the molecular characterization of a mycobacterial heparin-binding hemagglutinin (HBHA), a protein that functions as an adhesin for epithelial cells. The structural gene was cloned from M. tuberculosis and bacillus Calmette-Guérin, and the sequence was found to be identical between the two species. The calculated Mr was smaller than the observed Mr when analyzed by SDS/PAGE. This difference can be attributed to the Lys/Pro-rich repeats that occur at the C-terminal end of the protein and to a putative carbohydrate moiety. Glycosylation of HBHA appears to protect the protein from proteolytic degradation, which results in the removal of the C-terminal Lys/Pro-rich region responsible for binding of HBHA to sulfated carbohydrates. Evidence suggests that glycosylation is also important for HBHA-mediated hemagglutination and for certain immunologic properties of the protein. Finally, the absence of a signal peptide in the coding region of HBHA raises the possibility that this protein is not secreted via the general secretion pathway. PMID:9770536

Menozzi, F D; Bischoff, R; Fort, E; Brennan, M J; Locht, C

1998-10-13

 
 
 
 
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Pulmonary mycobacterial infections associated with neoplasia.  

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Patients with cancer are at increased risk for disease caused by mycobacteria when there is immunosuppression resulting from the underlying disease or its treatment. Pulmonary disease is usual with Mycobacterium tuberculosis or with mycobacteria other than M tuberculosis (MOTT) and atypical presentations with extrapulmonary dissemination occur frequently. No clinical features reliably distinguish between disease caused by M tuberculosis or MOTT. The incidence of M tuberculosis infection depends on a history of prior exposure in patients and on patterns of disease within hospitals and the surrounding community. Infection with different species of MOTT reflects their environmental prevalence. Diagnosis of mycobacterial infection can be clinically challenging and must be pursued aggressively. Despite recent improvements in clinical and laboratory methods, diagnosis of mycobacterial infection may take weeks. Recent increases in the incidence of M tuberculosis infection and the emergence of drug-resistant strains require heightened alertness to its diagnosis and careful epidemiological control measures to prevent continued spread of this contagion. M tuberculosis with routine antimicrobial susceptibility responds well to conventional therapy when initiated early. Prophylaxis of tuberculin positive patients is effective and should be started before immunosuppressive therapy. Guidelines for therapy of MOTT depend on the species isolated but remains poorly defined in most cases. There are several new compounds that may be useful for treatment of drug-resistant species of MOTT. New methods for the rapid diagnosis, speciation, and epidemiological investigation of mycobacterial infection are being developed, and some are available for clinical application. Nonetheless, the timely diagnosis of mycobacterial disease in patients with cancer remains a challenge of increasing clinical importance. PMID:1439320

Brown, S T; Almenoff, P L

1992-06-01

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Clinical agreement in pulmonary nontuberculous mycobacterial disease  

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Peter J Nolan Department of Internal Medicine, Toowoomba General Hospital, Toowoomba, Queensland, Australia Purpose: To consider the clinical agreement among respiratory and infectious disease physicians, working in a tertiary chest diseases center serving a population with a low incidence of pulmonary tuberculosis (<3/100,000/year), in the assessment of cases of pulmonary nontuberculous mycobacterial (NTM) lung disease. Method: A series of previously notified cases of NTM disease was abstrac...

2013-01-01

63

Functional Domains Present in the Mycobacterial Hemagglutinin, HBHA  

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Identification and characterization of mycobacterial adhesins and complementary host receptors required for colonization and dissemination of mycobacteria in host tissues are needed for a more complete understanding of the pathogenesis of diseases caused by these bacteria and for the development of effective vaccines. Previous investigations have demonstrated that a 28-kDa heparin-binding mycobacterial surface protein, HBHA, can agglutinate erythrocytes and promote mycobacterial aggregation i...

Delogu, Giovanni; Brennan, Michael J.

1999-01-01

64

Foundations of antibiotic resistance in bacterial physiology: the mycobacterial paradigm.  

Science.gov (United States)

The intrinsic resistance of Mycobacterium tuberculosis and related pathogens to most common antibiotics limits chemotherapeutic options to treat tuberculosis and other mycobacterial diseases. Resistance has traditionally been attributed to the unusual multi-layer cell envelope that functions as an effective barrier to the penetration of antibiotics. Recent insights into mechanisms that neutralize the toxicity of antibiotics in the cytoplasm have revealed systems that function in synergy with the permeability barrier to provide intrinsic resistance. Here, we highlight the growing pool of information about internal, antibiotic-responsive regulatory proteins and corresponding resistance genes, and present new concepts that rationalize how they might have evolved. Pharmaceutical inhibition of these intrinsic systems could make many previously available antibiotics active against M. tuberculosis. PMID:16759863

Nguyen, Liem; Thompson, Charles J

2006-07-01

65

Method to integrate multiple plasmids into the mycobacterial chromosome  

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In order to create a system in which two independent plasmids can be integrated into a mycobacterial chromosome, a mycobacterial plasmid was constructed containing the phage attachment site attP from the mycobacteriophage L5 genome and additionally containing the bacterial attachment site, attB. This plasmid will integrate into the mycobacterial chromosome via recombination of the plasmid-borne attP site with the chromosomal attB site in the presence of a mycobacterial vector carrying the L5 ...

Saviola, Beatrice; Bishai, William R.

2004-01-01

66

Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis  

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Full Text Available Abstract Background mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease. Results We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 ?g of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c+, CD11b+ and CD19+ cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO production through Toll-like receptor 7 (TLR7. Conclusions Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.

Silva Aristóbolo M

2010-10-01

67

Mycobacterial P1-Type ATPases Mediate Resistance to Zinc Poisoning in Human Macrophages  

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Mycobacterium tuberculosis thrives within macrophages by residing in phagosomes and preventing them from maturing and fusing with lysosomes. A parallel transcriptional survey of intracellular mycobacteria and their host macrophages revealed signatures of heavy metal poisoning. In particular, mycobacterial genes encoding heavy metal efflux P-type ATPases CtpC, CtpG, and CtpV, and host cell metallothioneins and zinc exporter ZnT1, were induced during infection. Consistent with this pattern of ...

Botella, He?le?ne; Peyron, Pascale; Levillain, Florence; Poincloux, Renaud; Poquet, Yannick; Brandli, Ire?ne; Wang, Chuan; Tailleux, Ludovic; Tilleul, Sylvain; Charrie?re, Guillaume M.; Waddell, Simon j; Foti, Maria; Lugo-villarino, Geanncarlo; Gao, Qian; Maridonneau-parini, Isabelle

2011-01-01

68

Immunopathologic Effects of Tumor Necrosis Factor Alpha in Murine Mycobacterial Infection Are Dose Dependent  

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In experimental mycobacterial infection, tumor necrosis factor alpha (TNF-?) is required for control of bacillary growth and the protective granulomatous response, but may cause immunopathology. To directly examine the positive and detrimental effects of this cytokine, a murine model was used in which different amounts of TNF-? were delivered to the site of infection. Mice with a disruption in the TNF-? gene (TNF-KO) or wild-type mice were infected with low or high doses of recombinant Myc...

Bekker, Linda-gail; Moreira, Andre L.; Bergtold, Amy; Freeman, Sherry; Ryffel, Bernard; Kaplan, Gilla

2000-01-01

69

[Mendelian susceptibility to mycobacterial disease: a case report of disseminated infection due to Mycobacterium avium].  

Science.gov (United States)

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare genetic syndrome that predisposes patients to infections caused by weakly virulent mycobacterial species, such as bacillus Calmette-Guérin (BCG) vaccines and nontuberculous environmental mycobacteria in children free of classical immunodeficiencies. This syndrome consists of impaired antimycobacterial immunity (axis IL12/INF-?) constituting a new immune deficiency and outlining its major role in mycobacterial immunity. We report a new case of MSMD through the observation of a young girl with a disseminated infection due to Mycobacterium avium. The molecular defect was 2 autosomal recessive mutations of the IL12R?1 gene (gene encoding for the ?1 chain of the IL12 receptor) leading to the absence of the IL12 receptor on the activated T lymphocytes' surface. IL-12RB1 deficiency is the most common genetic etiology of MSMD. Today, there are 6 MSMD-causing genes, leading to 13 distinct genetic disorders. The clinical phenotype differs between patients. The description of the molecular and immunological basis of this syndrome has allowed us to explain the pathophysiology of antimycobacterial immunity and is essential to understanding and managing these diseases. PMID:23726680

Darleguy, A; Bost-Bru, C; Pagnier, A; Plantaz, D; Piolat, C; Nugues, F; Picard, C

2013-07-01

70

Detection of mycobacterial antigens in leprosy serum immune complex.  

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The antigens from immune complexes of sera from patients with mycobacterial diseases were released by sodium dodecyl sulfate. The antigenic activity of the released proteins was tested by agar gel diffusion and immunoelectrophoresis. This simple method provided direct evidence for the presence of mycobacterial antigens in the immune complexes of sera from patients with leprosy and tuberculosis.

1986-01-01

71

Mycobacterial disease in renal allograft recipients  

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Full Text Available Implication for health policy/practice/research/medical education:Solid organ transplant recipients have impaired cell-mediated immunity, and are at increased risk of mycobacterial infection. Mycobacterium tuberculosis infection (TB has a high mortality rate among this population. The diagnosis of tuberculosis in solid organ transplant recipients is a big challenge and needs rapid and accurate modalities. These patients have 3.8 time greater risk of developing extra-pulmonary TB than general population. High index of suspicion and applying with invasive diagnostic procedure are needed for diagnosis of TB in renal transplanted patients.

Ardalan Mohammad-Reza

2013-06-01

72

Computational genomics-proteomics and Phylogeny analysis of twenty one mycobacterial genomes (Tuberculosis & non Tuberculosis strains  

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Full Text Available Abstract Background The genus Mycobacterium comprises different species, among them the most contagious and infectious bacteria. The members of the complex Mycobacterium tuberculosis are the most virulent microorganisms that have killed human and other mammals since millennia. Additionally, with the many different mycobacterial sequences available, there is a crucial need for the visualization and the simplification of their data. In this present study, we aim to highlight a comparative genome, proteome and phylogeny analysis between twenty-one mycobacterial (Tuberculosis and non tuberculosis strains using a set of computational and bioinformatics tools (Pan and Core genome plotting, BLAST matrix and phylogeny analysis. Results Considerably the result of pan and core genome Plotting demonstrated that less than 1250 Mycobacterium gene families are conserved across all species, and a total set of about 20,000 gene families within the Mycobacterium pan-genome of twenty one mycobacterial genomes. Viewing the BLAST matrix a high similarity was found among the species of the complex Mycobacterium tuberculosis and less conservation is found with other slow growing pathogenic mycobacteria. Phylogeny analysis based on both protein conservation, as well as rRNA clearly resolve known relationships between slow growing mycobacteria. Conclusion Mycobacteria include important pathogenic species for human and animals and the Mycobacterium tuberculosis complex is the most cause of death of the humankind. The comparative genome analysis could provide a new insight for better controlling and preventing these diseases.

Zakham Fathiah

2012-08-01

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Distribution of lactoferrin and 60/65 kDa heat shock protein in normal and inflamed human intestine and liver.  

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Immunisation against the mycobacterial heat shock protein (hsp-65) has been proposed to lead to production of autoantibodies against human lactoferrin. Such antibodies occur in ulcerative colitis and in primary sclerosing cholangitis. This study analysed the distribution of hsp-65 and lactoferrin in biopsy specimens from patients with inflammatory bowel disease and primary sclerosing cholangitis and studied whether immunisation against mycobacterial hsp-65 resulted in production of antilactof...

Peen, E.; Enestro?m, S.; Skogh, T.

1996-01-01

74

Genetic determinants of susceptibility to Mycobacterial infections: IRF8, a new kid on the block.  

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Genetic and population studies suggest that onset, progression and ultimate outcome of infection with Mycobacteria, including the agent of tuberculosis Mycobacterium tuberculosis, are strongly influenced by genetic factors. Family-based and case-control linkage and association studies have suggested a complex genetic component for susceptibility to tuberculosis. On the other hand, patients with inborn errors in the IL12/IFN? circuit may develop disseminated mycobacterial infections following perinatal BCG vaccination. The study of such MSMD (Mendelian Susceptibility to Mycobacterial Diseases) patients has provided much insight into innate and acquired immune defenses against mycobacteria. Parallel genetic analyses in mouse models of mycobacterial infections have also indicated complex genetic control, and have provided candidate genes for parallel testing in humans. Recently, mutations in human IRF8 were discovered and shown to cause two distinct forms of a novel primary immunodeficiency and associated susceptibility to mycobacteria. Autosomal recessive IRF8 deficiency is caused by mutation K108E and associated with severe disease with complete depletion of monocytes and dendritic cells. Mutation T80A causes autosomal dominant IRF8 deficiency and a milder form of the disease with selective loss of a subset of dendritic cells. These findings have established that IRF8 is required for ontogeny of the myeloid lineage and for host response to mycobacteria. The ongoing study of the IRF8 transcriptome has shown promise for the identification of IRF8 dependent pathways that play a critical role in host defense against mycobacteria in particular, and against intracellular pathogens in general. PMID:23468103

Salem, S; Gros, P

2013-01-01

75

Recurrent pulmonary aspergillosis and mycobacterial infection in an unsplenectomized patient with type 1 Gaucher disease  

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Background The clinical presentation of Gaucher disease (GD), an inherited lysosomal storage disorder caused by the deficient activity of the lysosomal enzyme glucocerebrosidase, is highly variable, and three clinical types are distinguished based upon the presence of neurologic symptoms. Thrombocytopenia, anemia, hepatosplenomegaly, and bone manifestations are the most typical signs of GD type 1 (GD1). Case presentation We present the case of an unsplenectomized man suffering from heterozygous GD1 with mutations of c.1226A>G (N370S) and RecNci I (L444P, A456P, and V460V) in the GBA1 gene, who developed recurrent pulmonary aspergillosis caused by Aspergillus fumigatus and a mycobacterial infection caused by Mycobacterium avium. Despite long-lasting therapy of both aspergillosis (including antifungal drugs and surgery), and the mycobacterial infection (triple therapy with rifampicin, ethambutol, and clarithromycin), recurrent positivity for M. avium and A. fumigatus was detected. Conclusions Symptomatic lung involvement and an increased susceptibility to pulmonary infections are uncommon in GD and, if present, are often associated with more severe disease manifestations. To our knowledge, this is the first published report on the association of GD and pulmonary aspergillosis and mycobacterial infection. It illustrates the increased susceptibility of untreated GD patients to opportunistic pulmonary infections and ineffective eradication of these infections despite adequate therapy.

Lorenz, Fryderyk; Kleinotiene, Grazina; Bulanda, Agnieszka; Markuszewska-Kuczynska, Alicja; Raistenskis, Juozas; Klimkowska, Monika

2014-01-01

76

Aaptamines, marine spongean alkaloids, as anti-dormant mycobacterial substances.  

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A new aaptamine class alkaloid, designated 2-methoxy-3-oxoaaptamine (1), together with seven known aaptamines (2-8) were isolated from a marine sponge of Aaptos sp. as anti-mycobacterial substances against active and dormant bacilli. The chemical structure of 1 was determined on the basis of spectroscopic analysis. Compound 1 was anti-mycobacterial against Mycobacterium smegmatis in both active growing and dormancy-inducing hypoxic conditions with a minimum inhibitory concentration (MIC) of 6.25 ?g/ml, and compounds 2, 5, 6, and 7 showed anti-mycobacterial activities under hypoxic condition selectively, with MIC values of 1.5-6.25 ?g/ml. PMID:24414399

Arai, Masayoshi; Han, Chisu; Yamano, Yoshi; Setiawan, Andi; Kobayashi, Motomasa

2014-04-01

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Pulmonary Nontuberculous Mycobacterial Infection in Congenital Contractural Arachnodactyly  

Science.gov (United States)

SUMMARY Congenital contractural arachnodactyly (CCA) is caused by mutations within fibrillin-2 (FBN2), which is crucial for microfibril structure. Affected individuals may have contractures, chest wall deformities, scoliosis, abnormal ear folding and elongated limbs. We describe a novel FBN2 mutation in a woman with CCA who also has pulmonary nontuberculous mycobacterial infection. The population with pulmonary nontuberculous mycobacterial infections shares phenotypic features with CCA, such as elongated body habitus, scoliosis and pectus deformities. While it is unlikely that FBN2 defects account for susceptibility to nontuberculous mycobacterial infection in the majority of cases, the overlap between these two diseases suggests some shared pathophysiology.

Paulson, Michelle L.; Olivier, Kenneth N.; Holland, Steven M.

2013-01-01

78

Eurasian wild boar response to skin-testing with mycobacterial and non-mycobacterial antigens.  

Science.gov (United States)

Eurasian wild boar (Sus scrofa) are able to maintain bovine tuberculosis (bTB) in the wild and are most probably able to transmit the disease to other species, thus acting as a true wildlife reservoir. Translocation of wild boar is a common practice in European countries. Therefore, identifying effective tools for bTB diagnosis in living wild boar is crucial for the implementation of control measures. We describe for the first time the sex and origin related differences in the skin-test response to mycobacterial antigens (bPPD and aPPD) and to a non-mycobacterial antigen (PHA, a plant derived mitogen) in wild and farmed wild boar, and used a small sample of known M. bovis infected wild boar to establish whether skin-testing is an option for bTB diagnosis in living wild boar. The highest skinfold increase response was detected at the PHA injection site, evidencing that the PHA test could be useful in monitoring cell mediated immunity (CMI) in wild boar populations. A clear age-increasing trend of the PHA response indicated that age should be taken into account when measuring CMI in wild boar. Origin related differences in the response against mycobacterial antigens could reflect differential exposure to mycobacterial antigens. Skin testing in BCG immunized wild boar showed low sensitivity (43-57%), while the sensitivity was good in the culture positive controls (75-100%), depending on the reading criterion. Specificity improved when the criterion was a response to bPPD larger than 2 mm and bPPD response larger than aPPD response (77%). Although a limited sample, our results indicated the potential of skin test as a bTB diagnostic tool in Eurasian wild boar. However, handling of wild boar is dangerous, specificity is low, and more effort is needed in order to define the sensitivity of this technique. PMID:20633938

Jaroso, R; Vicente, J; Fernandez-de-Mera, I G; Aranaz, A; Gortazar, C

2010-09-01

79

CT of tuberculosis and nontuberculous mycobacterial infections.  

Science.gov (United States)

Radiologic manifestations of pulmonary tuberculosis (TB) can vary according to several host factors, including prior exposure to TB, age, and underlying immune status. Although chest radiography is the mainstay in the evaluation of pulmonary TB, CT generally is required to detect fine lesions overlooked on chest radiographs, to define equivocal lesions, or to evaluate complications. High-resolution CT is useful in understanding the pathologic process of the disease and in determining disease activity in selected cases. This article describes the characteristic CT findings of various forms of pulmonary TB and nontuberculous mycobacterial infection according to immune status of the patients, and assesses the role of CT in the diagnosis and management of pulmonary TB. PMID:11813821

Goo, Jin Mo; Im, Jung-Gi

2002-01-01

80

Selective enrichment and detection of mycobacterial DNA in paucibacillary specimens  

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A major challenge for tuberculosis control is mycobacterial detection in paucibacillary disease, particularly in pediatric, extrapulmonary and smear-negative pulmonary infections. We developed a simple and efficient DNA extraction and real-time quantitative PCR (qPCR) protocol for mycobacterial detection and quantification in paucibacillary specimens. The method was refined using an in vitro model mimicking blood specimens which are characterized by the presence of numerous qPCR inhibitors. M...

2006-01-01

 
 
 
 
81

Mycobacterial Mutants with Defective Control of Phagosomal Acidification  

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The pathogenesis of mycobacterial infection is associated with an ability to interfere with maturation of the phagosomal compartment after ingestion by macrophages. Identification of the mycobacterial components that contribute to this phenomenon will allow rational design of novel approaches to the treatment and prevention of tuberculosis. Microarray-based screening of a transposon library was used to identify mutations that influence the fate of Mycobacterium bovis bacille Calmette-Guérin ...

Stewart, Graham R.; Patel, Janisha; Robertson, Brian D.; Rae, Aaron; Young, Douglas B.

2005-01-01

82

MENDELIAN SUSCEPTIBILITY TO MYCOBACTERIAL DISEASE IN EGYPTIAN CHILDREN  

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Background: Tuberculosis remains a major health problem in developing countries especially with the emergence of multidrug resistant strains. Mendelian Susceptibility to Mycobacterial Disease (MSMD) is a rare disorder with impaired immunity against mycobacterial pathogens. Reported MSMD etiologies highlight the crucial role of the Interferon gamma /Interleukin 12 (IFN-g/ IL-12) axis and the phagocyte respiratory burst axis.

Nermeen Galal; Jeannette Boutros; Aisha Marsafy; Xiao-Fei Kong; Jacqueline Feinberg; Jean-Laurent Casanova; Stéphanie Boisson-Dupuis; Jacinta Bustamante

2012-01-01

83

First case of nontuberculous mycobacterial lung disease caused by Mycobacterium marseillense in a patient with systemic lupus erythematosus.  

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Mycobacterium marseillense was designated as a new species within Mycobacterium avium complex. We report the first case of M. marseillense lung disease in a patient with systemic lupus erythematosus. All serial isolates were identified as M. marseillense by multilocus sequence analysis, based on hsp65, 16S-23S rRNA internal transcribed spacer, and 16S rRNA fragments. PMID:24768296

Kim, Su-Young; Yoo, Hongseok; Jeong, Byeong-Ho; Jeon, Kyeongman; Ha, Young Eun; Huh, Hee Jae; Ki, Chang-Seok; Lee, Nam Yong; Shin, Sung Jae; Koh, Won-Jung

2014-07-01

84

Clinical agreement in pulmonary nontuberculous mycobacterial disease  

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Full Text Available Peter J Nolan Department of Internal Medicine, Toowoomba General Hospital, Toowoomba, Queensland, Australia Purpose: To consider the clinical agreement among respiratory and infectious disease physicians, working in a tertiary chest diseases center serving a population with a low incidence of pulmonary tuberculosis (<3/100,000/year, in the assessment of cases of pulmonary nontuberculous mycobacterial (NTM lung disease. Method: A series of previously notified cases of NTM disease was abstracted and anonymously presented to a cohort of seven respiratory and infectious disease physicians. Their individual decisions to notify, treat, and follow the cases was evaluated and compared using the intraclass correlation coefficient. Results: A wide range was demonstrated in the diagnostic and management decision triage of each case by the physicians participating in the study. Clinical agreement on the likelihood of disease was limited, with an intraclass correlation coefficient of 0.394. Indication to notify the case to the state registry was linked to the clinical intent to initiate a treatment program. Conclusion: There appears to be limited agreement on the clinical significance of NTM isolates from pulmonary specimens among this cohort of experienced clinicians. If this trend is generalizable to a wider population of respiratory and infectious disease physicians, the number of notified and treated cases of disease is likely to be an underestimate of the true burden of disease in the general population. Keywords: diagnostic certainty, Kappa index, intraclass correlation coefficient, lung disease

Nolan PJ

2013-11-01

85

Inhibitors Selective for Mycobacterial Versus Human Proteasomes  

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Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis. Given that the proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-one compounds kill non-replicating M.?tuberculosis and act as selective suicide-substrate inhibitors of the M.?tuberculosis proteasome by cyclocarbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose hydrogen bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-one compounds to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homologue.

Lin, G.; Li, D; Sorio de Carvalho, L; Deng, H; Tao, H; Vogt, G; Wu, K; Schneider, J; Chidawanyika, T; et. al.

2009-01-01

86

Functional domains present in the mycobacterial hemagglutinin, HBHA.  

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Identification and characterization of mycobacterial adhesins and complementary host receptors required for colonization and dissemination of mycobacteria in host tissues are needed for a more complete understanding of the pathogenesis of diseases caused by these bacteria and for the development of effective vaccines. Previous investigations have demonstrated that a 28-kDa heparin-binding mycobacterial surface protein, HBHA, can agglutinate erythrocytes and promote mycobacterial aggregation in vitro. In this study, further molecular and biochemical analysis of HBHA demonstrates that it has three functional domains: a transmembrane domain of 18 amino acids residing near the N terminus, a large domain of 81 amino acids consistent with an alpha-helical coiled-coil region, and a Lys-Pro-Ala-rich C-terminal domain that mediates binding to proteoglycans. Using His-tagged recombinant HBHA proteins and nickel chromatography we demonstrate that HBHA polypeptides which contain the coiled-coil region form multimers. This tendency to oligomerize may be responsible for the induction of mycobacterial aggregation since a truncated N-terminal HBHA fragment containing the coiled-coil domain promotes mycobacterial aggregation. Conversely, a truncated C-terminal HBHA fragment which contains Lys-Pro-Ala-rich repeats binds to the proteoglycan decorin. These results indicate that HBHA contains at least three distinct domains which facilitate intercalation into surface membranes, promote bacterium-bacterium interactions, and mediate the attachment to sulfated glycoconjugates found in host tissues. PMID:10601202

Delogu, G; Brennan, M J

1999-12-01

87

Mycobacterial infections in a large Virginia hospital, 2001-2009  

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Full Text Available Abstract Background In areas where both tuberculosis (TB and nontuberculous mycobacteria (NTM are prevalent, descriptive studies of the clinical features of individual mycobacteria are needed to inform clinical triage. Methods We queried the University of Virginia Clinical Data Repository for all mycobacterial infections from 2001-2009. Results Of 494 mycobacterial infections in 467 patients there were 22 species. Patients with pulmonary Tb were more likely to be reported as immigrants (p M. kansasii, M. xenopi, and M. fortuitum were more likely than MAC to have cavities. There were at least 83 patients that met criteria for NTM lung disease and these were caused by 9 species. M. abscessus infection was associated with cystic fibrosis and M. xenopi infection was associated with male gender. Conclusions In our center mycobacterial infections were common and of diverse species. Immigrant status, cavities, and effusion were associated with TB vs. NTM.

Scully Kenneth W

2011-05-01

88

Novel STAT1 Alleles in Otherwise Healthy Patients with Mycobacterial Disease  

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The transcription factor signal transducer and activator of transcription-1 (STAT1) plays a key role in immunity against mycobacterial and viral infections. Here, we characterize three human STAT1 germline alleles from otherwise healthy patients with mycobacterial disease. The previously reported L706S, like the novel Q463H and E320Q alleles, are intrinsically deleterious for both interferon gamma (IFNG)–induced gamma-activating factor–mediated immunity and interferon alpha (IFNA)–induced interferon-stimulated genes factor 3–mediated immunity, as shown in STAT1-deficient cells transfected with the corresponding alleles. Their phenotypic effects are however mediated by different molecular mechanisms, L706S affecting STAT1 phosphorylation and Q463H and E320Q affecting STAT1 DNA-binding activity. Heterozygous patients display specifically impaired IFNG-induced gamma-activating factor–mediated immunity, resulting in susceptibility to mycobacteria. Indeed, IFNA-induced interferon-stimulated genes factor 3–mediated immunity is not affected, and these patients are not particularly susceptible to viral disease, unlike patients homozygous for other, equally deleterious STAT1 mutations recessive for both phenotypes. The three STAT1 alleles are therefore dominant for IFNG-mediated antimycobacterial immunity but recessive for IFNA-mediated antiviral immunity at the cellular and clinical levels. These STAT1 alleles define two forms of dominant STAT1 deficiency, depending on whether the mutations impair STAT1 phosphorylation or DNA binding.

Jouanguy, Emmanuelle; Vogt, Guillaume; Feinberg, Jacqueline; Prochnicka-Chalufour, Ada; Casrouge, Armanda; Yang, Kun; Soudais, Claire; Fieschi, Claire; Santos, Orchidee Filipe; Bustamante, Jacinta; Picard, Capucine; de Beaucoudrey, Ludovic; Emile, Jean-Francois; Arkwright, Peter D; Schreiber, Robert D; Rolinck-Werninghaus, Claudia; Rosen-Wolff, Angela; Magdorf, Klaus; Roesler, Joachim; Casanova, Jean-Laurent

2006-01-01

89

[Genetic susceptibility to infectious diseases: immunogenetical approaches to mycobacterial infections and subacute sclerosing panencephalitis].  

Science.gov (United States)

Genetic susceptibility to infectious diseases can be explained by nucleotide alteration (mutation, polymorphism, etc.) of genes encoding molecules involved in the entry of or the immune response to microorganisms. We have conducted studies on host genetic factors for the development of mycobacterial infections and subacute sclerosing panencephalitis (SSPE) in the past decade. First, we identified autosomal dominant IFN-gamma receptor deficiency as a predominant genetic basis of patients with bacille Calmette-Guérin osteomyelitis in Japan. Second, by gene-based association analysis of 21 candidate genes, it was suggested that genetic variants of IL-12 receptor beta1 gene (IL12RB1) confer genetic susceptibility to tuberculosis, and are associated with the progression of the disease in Japanese. Third, we demonstrated that variants of several genes encoding molecules associated with innate immunity (MxA and TLR3 genes) and acquired immunity (IL4 and programmed cell death 1 [PD1] genes) were associated with the development of SSPE. Immunogenetical approaches to infectious diseases would help us to evaluate the risk for disease development and progression, individualize prevention and treatment strategies, and create new therapies. PMID:20549906

Kusuhara, Koichi

2010-06-01

90

Mycobacterial survival strategies in the phagosome: Defense against host stresses  

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Infections with Mycobacterium tuberculosis (Mtb) remain a major cause of disease and death in humans. Among the factors that contribute to Mtb’s success as a pathogen is its ability to withstand potentially bactericidal host defenses and to resist elimination by an activated immune system. This resistance to killing by the host is in part due to the low permeability of the mycobacterial cell envelope for many toxic molecules. In addition, it depends upon the detoxification of reactive oxyge...

Ehrt, Sabine; Schnappinger, Dirk

2009-01-01

91

Clinical Latency and Reactivation of AIDS-Related Mycobacterial Infections  

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The immune mechanisms associated with the evolution from latent to clinically active mycobacterial coinfection in human immunodeficiency virus type 1 (HIV-1)-infected humans remain poorly understood. Previous work has demonstrated that macaques infected with simian immunodeficiency virus (SIVmac) can develop persistent Mycobacterium bovis BCG coinfection and a fatal SIV-related tuberculosis-like disease by 4 months after BCG inoculation. In the present study, SIVmac-infected monkeys that deve...

Shen, Yun; Shen, Ling; Sehgal, Prabhat; Huang, Dan; Qiu, Liyou; Du, George; Letvin, Norman L.; Chen, Zheng W.

2004-01-01

92

Acquired predisposition to mycobacterial disease due to autoantibodies to IFN-?  

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Genetic defects in the IFN-? response pathway cause unique susceptibility to intracellular pathogens, particularly mycobacteria, but are rare and do not explain mycobacterial disease in the majority of affected patients. We postulated that acquired defects in macrophage activation by IFN-? may cause a similar immunological phenotype and thus explain the occurrence of disseminated intracellular infections in some patients without identifiable immune deficiency. Macrophage activation in respo...

Kampmann, Beate; Hemingway, Cheryl; Stephens, Alick; Davidson, Robert; Goodsall, Anna; Anderson, Suzanne; Nicol, Mark; Scho?lvinck, Elisabeth; Relman, David; Waddell, Simon; Langford, Paul; Sheehan, Brian; Semple, Lynn; Wilkinson, Katalin A.; Wilkinson, Robert J.

2005-01-01

93

Insights into early mycobacterial pathogenesis from the zebrafish  

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Here we discuss the application of the zebrafish as a relatively new model host for the study of mycobacterial pathogenesis. Recent advances in our understanding of host-mycobacteria interactions from the zebrafish include insights into the role of the innate immune system in both controlling and facilitating infection. Analysis in the zebrafish has revealed that innate macrophages restrict initial bacterial growth, but also convey infecting bacteria into the granuloma, which serves as a plac...

Lesley, Robin; Ramakrishnan, Lalita

2008-01-01

94

A Sir2-Like Protein Participates in Mycobacterial NHEJ  

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In eukaryotic cells, repair of DNA double-strand breaks (DSBs) by the nonhomologous end-joining (NHEJ) pathway is critical for genome stability. In contrast to the complex eukaryotic repair system, bacterial NHEJ apparatus consists of only two proteins, Ku and a multifunctional DNA ligase (LigD), whose functional mechanism has not been fully clarified. We show here for the first time that Sir2 is involved in the mycobacterial NHEJ repair pathway. Here, using tandem affinity purification (TAP)...

Li, Zhongdao; Wen, Jikai; Lin, Yaning; Wang, Shihua; Xue, Peng; Zhang, Zhiping; Zhou, Ying; Wang, Xiao; Sui, Li; Bi, Li-jun; Zhang, Xian-en

2011-01-01

95

Antimicrobial activity of clofazimine is not dependent on mycobacterial C-type phospholipases.  

Science.gov (United States)

We have used a phospholipase C (PLC)-deletion mutant (plcABC) of the H37Rv strain of Mycobacterium tuberculosis (MTB), as well as a plcA-insertion mutant of Mycobacterium smegmatis, to investigate the possible involvement of PLCs in clofazimine-mediated inhibition of mycobacterial K(+) transport and growth. Inactivation of the PLCs of MTB and insertion of the plcA gene into M. smegmatis resulted in a substantial reduction and increase in hydrolysis of phosphatidylcholine (PC), respectively. However, both the mutant and wild-type strains of MTB and M. smegmatis were equally sensitive to the inhibitory effects of clofazimine on K(+) uptake and growth. These observations demonstrate that the PLCs of MTB are not involved in the antimicrobial activity of clofazimine. PMID:15117926

Bopape, M C; Steel, H C; Cockeran, R; Matlola, N M; Fourie, P B; Anderson, R

2004-06-01

96

Validating subcellular localization prediction tools with mycobacterial proteins  

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Full Text Available Abstract Background The computational prediction of mycobacterial proteins' subcellular localization is of key importance for proteome annotation and for the identification of new drug targets and vaccine candidates. Several subcellular localization classifiers have been developed over the past few years, which have comprised both general localization and feature-based classifiers. Here, we have validated the ability of different bioinformatics approaches, through the use of SignalP 2.0, TatP 1.0, LipoP 1.0, Phobius, PA-SUB 2.5, PSORTb v.2.0.4 and Gpos-PLoc, to predict secreted bacterial proteins. These computational tools were compared in terms of sensitivity, specificity and Matthew's correlation coefficient (MCC using a set of mycobacterial proteins having less than 40% identity, none of which are included in the training data sets of the validated tools and whose subcellular localization have been experimentally confirmed. These proteins belong to the TBpred training data set, a computational tool specifically designed to predict mycobacterial proteins. Results A final validation set of 272 mycobacterial proteins was obtained from the initial set of 852 mycobacterial proteins. According to the results of the validation metrics, all tools presented specificity above 0.90, while dispersion sensitivity and MCC values were above 0.22. PA-SUB 2.5 presented the highest values; however, these results might be biased due to the methodology used by this tool. PSORTb v.2.0.4 left 56 proteins out of the classification, while Gpos-PLoc left just one protein out. Conclusion Both subcellular localization approaches had high predictive specificity and high recognition of true negatives for the tested data set. Among those tools whose predictions are not based on homology searches against SWISS-PROT, Gpos-PLoc was the general localization tool with the best predictive performance, while SignalP 2.0 was the best tool among the ones using a feature-based approach. Even though PA-SUB 2.5 presented the highest metrics, it should be taken into account that this tool was trained using all proteins reported in SWISS-PROT, which includes the protein set tested in this study, either as a BLAST search or as a training model.

Niño Luis F

2009-05-01

97

Polyphenolic acetates : A newer anti-Mycobacterial therapeutic option  

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Full Text Available The objective of our research project was screening of various highly specific substrates of Acetoxy Drug: Protein Transacytylase (M.TAase for antimycobacterial activity. Mycobacterial culture was done in Middlebrook’s 7H9 media. Protein purification (Mycobacterial Tranacetylase, M.TAase was done by ion exchange chromatography and its demonstration was done on SDS- polyacrylamide gel electrophoresis (SDS-PAGE and western blot. Middlebrook’s 7H9 broth was procured from Becton Dickinson. CM-Sepharose, DEAE-Sepharose and Q-Sephharose were purchased from Amersham Pharmacia. Anti acetyl lysine polyclonal antibody was purchased from Cell Signaling. The Middlebrook 7H9 medium was used for M. smegmatis culture. The media was prepared according to the manufacturer’s instructions. The various Polyphenol acetate compounds were tested for their antimycobacterial activities. Minimal inhibitory concentrations (MIC were calculated by Alamar blue dye assay method. The GST protein was used as a receptor protein and purified Mycobacterial Glutamine Synthetase (GS as TAase for acetylation by DAMC. To demonstrate the TAase catalyzed acetylation of GST by DAMC, purified M.TAase (GS was preincubated with GST and DAMC followed by western blot using anti acetyl lysine antibody, which avidly react with the acetylated proteins. The growth pattern of M. smegmatis was diminished under the influence of various polyphenolic acetates (PA tested for their anti-mycobacterial activity. DAMC and DAMC-5-carboxylic acid was found to have MIC of 40?g/ml whereas DAMC-6-carboxylic acid was reported to have MIC value of 35?g/ml and for ellagic acid tetra acetate (EATA it was 60?g/ml. Previous work in our lab has led to discovery of a novel enzyme acetoxy drug: protein transacetylase (TAase, catalyzing transfer of acetyl group from various polyphenolic peracetate (PA to certain receptor proteins such as cytochromes P-450, NADPH cytochrome reductase, nitric oxide synthase (NOS has been established in various eukaryotic as well as prokaryotic sources. PA(s irreversible inhibitors of mammalian CYP linked MFO, possibly due to modification of cytochrome p- 450 by acetylation in a reaction catalysed by M.TAase (GS utilizing PA(s as a donor of acetyl groups. Accordingly, it was hypothesized that the CYP51 of mycobacteria involved in the cell wall sterol synthesis could possibly be modified by our PA(s through the novel unknown action of GS as transacetylase leading to the death of mycobacterial cell by way of acetylation catalyzed by acetoxy drug: protein transacetylase (M.TAase or GS.

Santram

2014-01-01

98

In vitro reconstitution of Mycobacterial ergothioneine biosynthesis.  

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Ergothioneine is a histidine-derived thiol of bacterial and fungal origin that has also been isolated from animal and human tissue. Recent findings point to critical functions of ergothioneine in human physiology, but its role in microbial life is poorly understood. This report describes the identification of the ergothioneine biosynthetic gene cluster from mycobacteria and in vitro reconstitution of this process using recombinant proteins from Mycobacterium smegmatis. The key reactions are catalyzed by a methyltransferase that transfers three methyl groups to the alpha-amino moiety of histidine and an iron(II)-dependent enzyme that catalyzes oxidative sulfurization of trimethylhistidine. A search for homologous genes indicated that ergothioneine production is a frequent trait among fungi, actinobacteria, and cyanobacteria but also occurs in numerous bacteroidetes and proteobacteria. PMID:20420449

Seebeck, Florian P

2010-05-19

99

Immune Responses to Mycobacterial Antigens in the Gambian Population: Implications for Vaccines and Immunodiagnostic Test Design  

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Recombinant immunodominant mycobacterial antigens are needed for the development of new vaccines and immunodiagnostic tools for use against tuberculosis. Ubiquitous exposure to mycobacteria in tropical countries could influence vaccine-induced immunity and the specificity of tuberculosis immunodiagnosis. For this study conducted in The Gambia, cellular immune responses to recombinant mycobacterial antigens were characterized in Mycobacterium bovis BCG-vaccinated and nonvaccinated infants, adu...

Vekemans, Johan; Ota, Martin O. C.; Sillah, Jackson; Fielding, Katherine; Alderson, Mark R.; Skeiky, Yasir A. W.; Dalemans, Wilfried; Mcadam, Keith P. W. J.; Lienhardt, Christian; Marchant, Arnaud

2004-01-01

100

Integrative immunoinformatics for Mycobacterial diseases in R platform.  

Science.gov (United States)

The sequencing of genomes of the pathogenic Mycobacterial species causing pulmonary and extrapulmonary tuberculosis, leprosy and other atypical mycobacterial infections, offer immense opportunities for discovering new therapeutics and identifying new vaccine candidates. Enhanced RV, which uses additional algorithms to Reverse Vaccinology (RV), has increased potential to reduce likelihood of undesirable features including allergenicity and immune cross reactivity to host. The starting point for MycobacRV database construction includes collection of known vaccine candidates and a set of predicted vaccine candidates identified from the whole genome sequences of 22 mycobacterium species and strains pathogenic to human and one non-pathogenic Mycobacterium tuberculosis H37Ra strain. These predicted vaccine candidates are the adhesins and adhesin-like proteins obtained using SPAAN at Pad > 0.6 and screening for putative extracellular or surface localization characteristics using PSORTb v.3.0 at very stringent cutoff. Subsequently, these protein sequences were analyzed through 21 publicly available algorithms to obtain Orthologs, Paralogs, BetaWrap Motifs, Transmembrane Domains, Signal Peptides, Conserved Domains, and similarity to human proteins, T cell epitopes, B cell epitopes, Discotopes and potential Allergens predictions. The Enhanced RV information was analysed in R platform through scripts following well structured decision trees to derive a set of nonredundant 233 most probable vaccine candidates. Additionally, the degree of conservation of potential epitopes across all orthologs has been obtained with reference to the M. tuberculosis H37Rv strain, the most commonly used strain in M. tuberculosis studies. Utilities for the vaccine candidate search and analysis of epitope conservation across the orthologs with reference to M. tuberculosis H37Rv strain are available in the mycobacrvR package in R platform accessible from the "Download" tab of MycobacRV webserver. MycobacRV an immunoinformatics database of known and predicted mycobacterial vaccine candidates has been developed and is freely available at http://mycobacteriarv.igib.res.in. PMID:24592289

Chaudhuri, Rupanjali; Kulshreshtha, Deepika; Raghunandanan, Muthukurussi Varieth; Ramachandran, Srinivasan

2014-03-01

 
 
 
 
101

Identification of the target protein of agelasine D, a marine sponge diterpene alkaloid, as an anti-dormant mycobacterial substance.  

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One of the major reasons for the wide epidemicity of tuberculosis and for the necessity for extensive chemotherapeutic regimens is that the causative agent, Mycobacterium tuberculosis, has an ability to become dormant. Therefore, new lead compounds that are anti-bacterial against M. tuberculosis in both active and dormant states are urgently needed. Marine sponge diterpene alkaloids, agelasines B, C, and D, from an Indonesian marine sponge of the genus Agelas were rediscovered as anti-dormant-mycobacterial substances. Based on the concept that the transformants over-expressing targets of antimicrobial substances confer drug resistance, strains resistant to agelasine D were screened from Mycobacterium smegmatis transformed with a genomic DNA library of Mycobacterium bovis BCG. Sequence analysis of the cosmids isolated from resistant transformants revealed that the responsible gene was located in the genome region between 3475.051 and 3502.901 kb. Further analysis of the transformants over-expressing the individual gene contained in this region indicated that BCG3185c (possibly a dioxygenase) might be a target of the molecule. Moreover, agelasine D was found to bind directly to recombinant BCG3185c protein (KD 2.42 ?m), based on surface plasmon resonance (SPR). This evidence strongly suggests that the BCG3185c protein is the major target of agelasine D, and that the latter is the anti-mycobacterial substance against dormant bacilli. PMID:24243718

Arai, Masayoshi; Yamano, Yoshi; Setiawan, Andi; Kobayashi, Motomasa

2014-01-01

102

Partial overlap of anti-mycobacterial, and anti-Saccharomyces cerevisiae mannan antibodies in Crohns disease  

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Full Text Available AIM: To test whether humoral immune reaction against mycobacteria may play a role in anti-Saccharomyces cerevisiae antibodies (ASCA generation in Crohn's disease (CD and/or whether it correlates with clinical subtypes.METHODS: The dominant ASCA epitope was detected by Galanthus nivalis lectin (GNL-binding assay. ASCA and IgG against mycobacterial lysates [M avium, M smegmatis, M chelonae, M bovis BCG, M avium ssp. paratuberculosis (MAP] or purified lipoarabinomannans (LAM were detected by ELISA. ASCA and anti-mycobacterial antibodies were affinity purified to assess cross-reactivities. Anti-mycobacterial IgG were induced by BCG-infection of mice.RESULTS: GNL bound to different extents to mycobacterial lysates, abundantly to purified mannose-capped (Man LAM from M tuberculosis, but not to uncapped LAM from M smegmatis. Fifteen to 45% of CD patients but only 0%-6% of controls were seropositive against different mycobacterial antigens. Anti-mycobacterial IgG correlated with ASCA (r = 0.37-0.64; P = 0.003-P < 0.001. ASCA-positivity and deficiency for mannan-binding lectin synergistically associated with anti-mycobacterial IgG. In some patients, anti-mycobacterial antibodies represent cross-reactive ASCA. Vice-versa, the predominant fraction of ASCA did not cross-react with mycobacteria. Finally, fistulizing disease associated with antibodies against M avium, M smegmatis and MAP (P = 0.024, 0.004 and 0.045, respectively.CONCLUSION: Similar to ASCA, seroreactivity against mycobacteria may define CD patients with complicated disease and a predisposition for immune responses against ubiquitous antigens. While in some patients anti-mycobacterial antibodies strongly cross-react with yeast mannan; these cross-reactive antibodies only represent a minor fraction of total ASCA. Thus, mycobacterial infection unlikely plays a role in ASCA induction.

Stefan Müller, Thomas Schaffer, Alain M Schoepfer, Annamarie Hilty, Thomas Bodmer, Frank Seibold

2008-06-01

103

Conditional depletion of KasA, a key enzyme of mycolic acid biosynthesis, leads to mycobacterial cell lysis.  

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Inhibition or inactivation of InhA, a fatty acid synthase II (FASII) enzyme, leads to mycobacterial cell lysis. To determine whether inactivation of other enzymes of the mycolic acid-synthesizing FASII complex also leads to lysis, we characterized the essentiality of two beta-ketoacyl-acyl carrier protein synthases, KasA and KasB, in Mycobacterium smegmatis. Using specialized transduction for allelic exchange, null kasB mutants, but not kasA mutants, could be generated in Mycobacterium smegmatis, suggesting that unlike kasB, kasA is essential. To confirm the essentiality of kasA, and to detail the molecular events that occur following depletion of KasA, we developed CESTET (conditional expression specialized transduction essentiality test), a genetic tool that combines conditional gene expression and specialized transduction. Using CESTET, we were able to generate conditional null inhA and kasA mutants. We studied the effects of depletion of KasA in M. smegmatis using the former strain as a reference. Depletion of either InhA or KasA led to cell lysis, but with different biochemical and morphological events prior to lysis. While InhA depletion led to the induction of an 80-kDa complex containing both KasA and AcpM, the mycobacterial acyl carrier protein, KasA depletion did not induce the same complex. Depletion of either InhA or KasA led to inhibition of alpha and epoxy mycolate biosynthesis and to accumulation of alpha'-mycolates. Furthermore, scanning electron micrographs revealed that KasA depletion resulted in the cell surface having a "crumpled" appearance, in contrast to the blebs observed on InhA depletion. Thus, our studies support the further exploration of KasA as a target for mycobacterial-drug development. PMID:16267284

Bhatt, Apoorva; Kremer, Laurent; Dai, Annie Z; Sacchettini, James C; Jacobs, William R

2005-11-01

104

Role of 5'-TGN-3' motif in the interaction of mycobacterial RNA polymerase with a promoter of 'extended -10' class.  

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In a systematic approach to understand the transcriptional machinery of mycobacteria, we had previously isolated and characterized mycobacterial promoter regions. In this study, we have investigated molecular interactions between mycobacterial RNA polymerase holoenzyme, reconstituted with different sigma subunits and the promoter element of the Mycobacterium tuberculosis gene pknH (Rv1266c), a representative of promoters belonging to the 'extended -10' class. In vitro transcription assays using the pknH promoter and reconstituted RNA polymerase holoenzyme demonstrated that transcription from the pknH promoter is specifically initiated by sigmaA, the principal sigma factor of mycobacteria. DNase I protection assay and deletion studies with the pknH promoter revealed that the minimal region required for optimal transcription carries the sequence from position -37 to position +6. Moreover, mutation in the TGN motif of the pknH promoter resulted in the loss of >75% of its activity. Binding of RNA polymerase with wild-type promoter as well as its TG- mutant revealed that the TGN motif is required for the transition from a close complex into an open complex. Further, it was observed that the presence of the TGN motif reduces the thermal energy required for the conversion of a close complex into an open complex, necessary for initiation of transcription. PMID:12900024

Agarwal, Nisheeth; Tyagi, Anil K

2003-08-01

105

Development of novel N-linked aminopiperidine-based mycobacterial DNA gyrase B inhibitors: scaffold hopping from known antibacterial leads.  

Science.gov (United States)

DNA gyrase of Mycobacterium tuberculosis (MTB) is a type II topoisomerase that ensures the regulation of DNA topology and has been genetically demonstrated to be a bactericidal drug target. We present the discovery and optimisation of a novel series of mycobacterial DNA gyrase inhibitors with a high degree of specificity towards the mycobacterial ATPase domain. Compound 5-fluoro-1-(2-(4-(4-(trifluoromethyl)benzylamino)piperidin-1-yl)ethyl)indoline-2,3-dione (17) emerged as the most potent lead, exhibiting inhibition of MTB DNA gyrase supercoiling assay with an IC50 (50% inhibitory concentration) of 3.6 ± 0.16 ?M, a Mycobacterium smegmatis GyrB IC50 of 10.6 ± 0.6 ?M, and MTB minimum inhibitory concentrations of 6.95 ?M and 10 ?M against drug-sensitive (MTB H37Rv) and extensively drug-resistant strains, respectively. Furthermore, the compounds did not show any signs of cardiotoxicity in zebrafish ether-à-go-go-related gene (zERG), and hence constitute a major breakthrough among the otherwise cardiotoxic N-linked aminopiperidine analogues. PMID:24434114

Jeankumar, Variam Ullas; Renuka, Janupally; Pulla, Venkat Koushik; Soni, Vijay; Sridevi, Jonnalagadda Padma; Suryadevara, Priyanka; Shravan, Morla; Medishetti, Raghavender; Kulkarni, Pushkar; Yogeeswari, Perumal; Sriram, Dharmarajan

2014-03-01

106

Mycobacterial culture of fine needle aspirate - A useful tool in diagnosing tuberculous lymphadenitis  

Directory of Open Access Journals (Sweden)

Full Text Available A prospective study was undertaken on suspected lymph node TB (LNTB patients, to evaluate the diagnostic utility of mycobacterial culture of fine needle aspirate (FNA, in comparison with the cytological examination and acid fast staining. Eighty percent of 157 aspirates studied were positive by cytological examination; 18% by ZN smear and 45% were positive by culture. Twelve aspirates which were negative by cytological features yielded positive mycobacterial cultures; four out of these were from HIV positive patients. Our observations suggest that supplementing FNA cytology with mycobacterial culture would increase the sensitivity of diagnosing LNTB; in addition to giving a highly specific diagnosis.

Kishore Reddy V

2008-01-01

107

[Novel vaccines against M. tuberculosis].  

Science.gov (United States)

CDC and ACET in U.S.A. reported that novel vaccines instead of BCG are required for the protection against infection of Mycobacterium tuberculosis worldwide. However, no novel vaccine for clinical use has not yet been developed in the world including U.S.A. and Europe. We have developed two novel tuberculosis (TB) vaccines; a DNA vaccine combination expressing mycobacterial heat shock protein 65 (HSP 65) and interleukin-12 (IL-12) by using the hemagglutinating virus of Japan (HVJ)-liposome (HSP 65 + IL-12/HVJ). A mouse IL-12 expression vector (mIL-12 DNA) encoding single-chain IL-12 proteins comorised of p40 and p35 subunits were constructed. In a mouse model, a single gene gun vaccination with the combination of HSP 65 DNA and mIL-12 DNA provided a remarkably high degree of protection against challenge with virulent Mycobacterium tuberculosis; bacterial numbers were 100 fold lower in the lungs compared to BCG-vaccinated mice. To explore the clinical use of the DNA vaccines, we evaluated HVJ-liposome encapsulated HAP 65 DNA and mIL-12 DNA (HSP 65 + mIL-12/ HVJ). The HVJ-liposome method improved the protective efficacy of the HSP 65 DNA vaccine compared to gene gun vaccination. This vaccine provide remarkable protective efficacy in mouse and guinea pig models, as compared to the current by available BCG vaccine. HSP 65 + IL-12/HVJ vaccine induced CD8+cytoxic T lymphocyte activity against HSP 65 antigen. Protective efficacy of this vaccine was associated with the emergence of IFN-gamma-secreting T cells and activation of proliferative T cells as well as CTL induction upon stimulation with the HSP 65 and antigens from M. tuberculosis. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis, to evaluate the HSP 65 + IL-12/HVJ vaccine. Vaccination with HSP 65 + IL-12/HVJ provided better protective efficacy as assessed by the Erythrocyte Sedimentation Rate, chest X-ray findings, and immune responses than BCG. Most importantly, HSP 65 + IL-12/HVJ resulted in an increased survival for over a year. This is the first report of successful DNA vaccination against M. tuberculosis in the monkey model. Novel TB vaccines using the monkey model will be discussed in this issue. The development of novel vaccines against tuberculosis was also studied in murine and cynomolgus monkey systems. Four distinct methods; DNA vaccination (1. plasmid, 2. adenovirus vector, 3. adenoassouated virus), 4. recombinant BCG, and 5. subunit (recombinant protein) were used for the development of novel vaccines. Genes (HSP 65 gene, IL-12 gene as well as Ag 85A-, 85B-, MPB51-gene) and IL-6 related genes (IL-6 gene + IL-6R gene +gp130 gene) were administered into the Balb/c mice infected (i.v. or intra-tracheal injection) with Mycobacterium tuberculosis (M. tuberculosis). Elimination of M. tuberculosis in lungs, liver, and spleen of these mice and survival were studied in these models. HSP 65 gene + IL-12 gene vaccination, or recombinant BCG (BA51 : Antigen 85B(-) + Antigen 85A(-) + MPB51-gene recombinant BCG) were more prophylactically efficient than parental BCG Tokyo vaccination. In contrast, IL-6 related genes vaccination using adenovirus vector showed therapeutic effect on M. tuberculosis infected mice. Cytotoxic T cells (CTL) activity against M. tuberculosis in the spleen cells from mice treated with IL-6 related genes vaccination were significantly augmented. Furthermore, NOD-SCID-PBL/hu mice treated with anti-IL-2 receptor beta-chain antibody provide an useful tool for analyzing in vivo human T cell immunity against tuberculosis. In conclusion, we demonstrate the development of a novel HVJ-liposome DNA vaccine encapsulating HSP 65 DNA plus IL-12 DNA. These results suggest that HSP 65 + IL-12/HVJ could be a promising candidate for a new tuberculosis DNA vaccine, which is superior to the currently available BCG vaccine. The goal of our study is to develop a new tuberculosis vaccine superior to BCG. To this aim, we believe that the protective efficacy and protective immune responses f

Okada, Masaji

2006-12-01

108

[Atypical mycobacterial infection after kidney transplant: two clinical cases].  

Science.gov (United States)

Infections are an important cause of morbidity and mortality during kidney transplant. In areas where tuberculosis is not endemic, Mycobacteria other than tuberculosis (MOOT), also known as 'atypical' Mycobacteria, are more frequently involved in mycobacterial infections than M. tuberculosis. The incidence of MOOT infection in renal transplant recipients ranges from 0.16 to 0.38 percent. This low rate of reported incidence is, however, often due to delay in diagnosis and lack of therapeutic protocols. Further difficulty is caused by the interaction of antimycobacterial drugs with the post-transplant immunosuppressive regimen, necessitating close monitoring of plasma concentrations and careful dose modification. We present two cases of Mycobacterium Chelonae infection in kidney transplant recipients which differ in both clinical presentation and pharmacological approach. PMID:23832441

Mele, Alessandra Antonia; Bilancio, G; Luciani, Remo; Bellizzi, Vincenzo; Palladino, Giuseppe

2013-01-01

109

Mycobacterial growth and bacterial contamination in the mycobacteria growth indicator tube and BACTEC 460 culture systems.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The BACTEC 460 system currently provides the most rapid detection of mycobacterial growth, but the system is radiometric and requires needles to inoculate specimens through the bottle's septum. The Mycobacteria Growth Indicator Tube (MGIT) system has a liquid medium, like the BACTEC system, and does not require needles when inoculating specimens. We compared mycobacterial growth from 510 specimens in the two systems. Average time to acid-fast bacillus (AFB) detection and identification to the...

Cornfield, D. B.; Beavis, K. G.; Greene, J. A.; Bojak, M.; Bondi, J.

1997-01-01

110

Cytodiagnosis of coexistent cryptococcal and mycobacterial lymphadenitis in a case of AIDS  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Multiple infections are a common feature of acquired immunodeficiency syndrome (AIDS), but coexistent infections at the same site are rare. In this report, we describe a 35-year-old human immunodeficiency virus infected male with coexistent cryptococcal and mycobacterial lymphadenitis. He presented with generalised lymphadenopathy. Fine needle aspiration cytology of enlarged cervical lymph node, aided by special stains, revealed coexistent cryptococcal and mycobacterial infection. Coexistent ...

Anvikar Arti; Gosavi Alka; Kulkarni Medha; Lanjewar D

2011-01-01

111

Color selection with a hygromycin-resistance-based Escherichia coli-mycobacterial shuttle vector.  

Science.gov (United States)

Hygromycin-resistance (HyR)-based Escherichia coli-mycobacterial shuttle plasmids have high efficiencies of transformation and a broad mycobacterial host range. We have introduced a lacZ alpha (encoding the alpha-polypeptide fragment of beta-galactosidase (beta Gal))-multiple cloning site cassette into a HyR-based shuttle vector to generate a plasmid with nine unique cloning sites and the added feature of beta Gal color selection in appropriate E. coli host strains. PMID:8529888

Howard, N S; Gomez, J E; Ko, C; Bishai, W R

1995-12-01

112

Mycobacterial Antigens Exacerbate Disease Manifestations in Mycobacterium tuberculosis-Infected Mice  

Digital Repository Infrastructure Vision for European Research (DRIVER)

To control tuberculosis worldwide, the burden of adult pulmonary disease must be reduced. Although widely used, Mycobacterium bovis BCG vaccination given at birth does not protect against adult pulmonary disease. Therefore, postexposure vaccination of adults with mycobacterial antigens is being considered. We examined the effect of various mycobacterial antigens on mice with prior M. tuberculosis infection. Subcutaneous administration of live or heat-treated BCG with or without lipid adjuvant...

Moreira, Andre L.; Tsenova, Liana; Haile Aman, Melles; Bekker, Linda-gail; Freeman, Sherry; Mangaliso, Bande; Schro?der, Ulf; Jagirdar, Jaishree; Rom, William N.; Tovey, Michael G.; Freedman, Victoria H.; Kaplan, Gilla

2002-01-01

113

Modulation of adjuvant arthritis in Lewis rats by recombinant vaccinia virus expressing the human 60-kilodalton heat shock protein.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The immune response to the mycobacterial 65-kDa heat shock protein (hsp65) is considered an important event in the induction of adjuvant arthritis (AA) in rats; this induction probably occurs through a molecular mimicry mechanism involving cross-reactivity against the rat homolog hsp60. To analyze the role of mammalian molecule hsp60 in arthritis, we generated a recombinant vaccinia virus (hsp60-VV) carrying the human hsp60 gene inserted into the thymidine kinase locus under the control of th...

Lo?pez-guerrero, J. A.; Lo?pez-bote, J. P.; Ortiz, M. A.; Gupta, R. S.; Pa?ez, E.; Bernabeu, C.

1993-01-01

114

Molecular-based mycobacterial identification in a clinical laboratory setting: a comparison of two methods.  

LENUS (Irish Health Repository)

Many mycobacterial species are pathogenic to humans, with infection occurring worldwide. Infection with Mycobacterium tuberculosis is a well-described global phenomenon, but other mycobacterial species are increasingly shown to be the cause of both pulmonary and extrapulmonary infection and are managed differently from M. tuberculosis infection. Rapid and accurate differentiation of mycobacterial species is, therefore, critical to guide timely and appropriate therapeutic and public health management. This study evaluates two commercially available DNA strip assays, the Genotype Common Mycobacteria (CM) assay (Hain Lifescience, Nehren, Germany) and the Speed-oligo Mycobacteria assay (Vircell, Spain) for their usefulness in a clinical laboratory setting. Both assays were evaluated on 71 clinical mycobacterial isolates, previously identified using Gen-Probe AccuProbe and through a UK mycobacteriology reference laboratory, as well as 29 non-mycobacterial isolates. Concordant results were obtained for 98% of isolates using both assays. The sensitivity was 97% (95% confidence interval [CI]: 93.3-100%) for the CM assay and 98.6% (95% CI: 95.9-100%) for the Speed-oligo assay. Overall, both assays proved to be useful tools for rapid and sensitive mycobacterial species identification, although interpretation of results was easier with the CM assay. Finally, results were available within one day, compared to current identification times which range between seven days and four weeks.

O'Donnell, N

2012-01-01

115

Mycobacterium tuberculosis and nontuberculous mycobacterial isolates among patients with recent HIV infection in Mozambique / Doença pulmonar por Mycobacterium tuberculosis e micobactérias não-tuberculosas entre pacientes recém-diagnosticados como HIV positivos em Moçambique, África  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: Portuguese Abstract in portuguese OBJETIVO: A micobacteriose é frequentemente diagnosticada entre pacientes infectados pelo HIV. Em Moçambique, onde apenas um pequeno número de pacientes encontra-se sob tratamento anti-retroviral, e a tuberculose tem alta prevalência, existe a necessidade de melhor caracterização destes agentes bact [...] erianos, em nível de espécie, bem como de se caracterizar os padrões de resistência às drogas antituberculosas. MÉTODOS: Em uma coorte de 503 indivíduos HIV positivos suspeitos de tuberculose pulmonar, 320 apresentaram positividade para baciloscopia ou cultura no escarro e no lavado brônquico. RESULTADOS: Bacilos álcool-ácido resistentes foram detectados no escarro em 73% dos casos com cultura positiva. De 277 isolados em cultura, apenas 3 mostraram-se tratar de micobactérias não-tuberculosas: 2 Mycobacterium avium e uma M. simiae. Todos os isolados de M. tuberculosis inicialmente caracterizados através de reação em cadeia de polimerase (RCP) do gene hsp65 foram posteriormente caracterizados como tal através de RCP do gene gyrB. Resistência à isoniazida foi encontrada em 14% dos casos; à rifampicina em 6%; e multirresistência em 5%. Pacientes previamente tratados para tuberculose mostraram tendência a taxas maiores de resistência às drogas de primeira linha. O padrão radiológico mais freqüente encontrado foi o infiltrado intersticial (67%), seguido da presença de linfonodos mediastinais (30%), bronquiectasias (28%), padrão miliar (18%) e cavidades (12%). Os pacientes infectados por micobactérias não-tuberculosas não apresentaram manifestações clínicas distintas das apresentadas pelos outros pacientes. A mediana de linfócitos CD4 entre todos os pacientes foi de 134 células/mm³. CONCLUSÕES: Tuberculose e AIDS em Moçambique estão fortemente associadas, como é de se esperar em países com alta prevalência de tuberculose. Embora as taxas de resistência a drogas sejam altas, o esquema isoniazida-rifampicina continua sendo a escolha apropriada para o início do tratamento. Abstract in english OBJECTIVE: Mycobacteriosis is frequently diagnosed among HIV-infected patients. In Mozambique, where few patients are under antiretroviral therapy and the prevalence of tuberculosis is high, there is need for better characterization of mycobacteria at the species level, as well as for the identifica [...] tion of patterns of resistance to antituberculous drugs. METHODS: We studied a sample of 503 HIV-infected individuals suspected of having pulmonary tuberculosis. Of those 503, 320 tested positive for mycobacteria through sputum smear microscopy or culture of bronchoalveolar lavage fluid. RESULTS: Acid-fast bacilli were observed in the sputum of 73% of the individuals presenting positive cultures. Of 277 isolates tested, only 3 were nontuberculous mycobacteria: 2 were identified as Mycobacterium avium and one was identified as M. simiae. Strains initially characterized as M. tuberculosis complex through polymerase chain reaction restriction analysis (PRA) of the hsp65 gene were later confirmed as such through PRA of the gyrB gene. Among the M. tuberculosis isolates, resistance patterns were as follows: to isoniazid, 14%; to rifampin, 6%; and multidrug resistance, 5%. Previously treated cases showed significantly higher rates of resistance to first-line antituberculous drugs. The most common radiological pattern was interstitial infiltrate (in 67%), followed by mediastinal lymph node enlargement (in 30%), bronchiectasis (in 28%), miliary nodules (in 18%) and cavitation (in 12%). Patients infected with nontuberculous mycobacteria presented clinical profiles indistinguishable from those of other patients. The median CD4 lymphocyte count in this group was 134 cells/mm³. CONCLUSIONS: There is a strong association between tuberculosis and AIDS in Mozambique, as expected in a country with a high prevalence of tuberculosis. Although drug resistance rates are high, the isoniazid-rifampin regimen continues to be the appr

Nunes, Elizabete Abrantes; De Capitani, Eduardo Mello; Coelho, Elizabete; Panunto, Alessandra Costa; Joaquim, Orvalho Augusto; Ramos, Marcelo de Carvalho.

116

Doença pulmonar por Mycobacterium tuberculosis e micobactérias não-tuberculosas entre pacientes recém-diagnosticados como HIV positivos em Moçambique, África Mycobacterium tuberculosis and nontuberculous mycobacterial isolates among patients with recent HIV infection in Mozambique  

Directory of Open Access Journals (Sweden)

Full Text Available OBJETIVO: A micobacteriose é frequentemente diagnosticada entre pacientes infectados pelo HIV. Em Moçambique, onde apenas um pequeno número de pacientes encontra-se sob tratamento anti-retroviral, e a tuberculose tem alta prevalência, existe a necessidade de melhor caracterização destes agentes bacterianos, em nível de espécie, bem como de se caracterizar os padrões de resistência às drogas antituberculosas. MÉTODOS: Em uma coorte de 503 indivíduos HIV positivos suspeitos de tuberculose pulmonar, 320 apresentaram positividade para baciloscopia ou cultura no escarro e no lavado brônquico. RESULTADOS: Bacilos álcool-ácido resistentes foram detectados no escarro em 73% dos casos com cultura positiva. De 277 isolados em cultura, apenas 3 mostraram-se tratar de micobactérias não-tuberculosas: 2 Mycobacterium avium e uma M. simiae. Todos os isolados de M. tuberculosis inicialmente caracterizados através de reação em cadeia de polimerase (RCP do gene hsp65 foram posteriormente caracterizados como tal através de RCP do gene gyrB. Resistência à isoniazida foi encontrada em 14% dos casos; à rifampicina em 6%; e multirresistência em 5%. Pacientes previamente tratados para tuberculose mostraram tendência a taxas maiores de resistência às drogas de primeira linha. O padrão radiológico mais freqüente encontrado foi o infiltrado intersticial (67%, seguido da presença de linfonodos mediastinais (30%, bronquiectasias (28%, padrão miliar (18% e cavidades (12%. Os pacientes infectados por micobactérias não-tuberculosas não apresentaram manifestações clínicas distintas das apresentadas pelos outros pacientes. A mediana de linfócitos CD4 entre todos os pacientes foi de 134 células/mm³. CONCLUSÕES: Tuberculose e AIDS em Moçambique estão fortemente associadas, como é de se esperar em países com alta prevalência de tuberculose. Embora as taxas de resistência a drogas sejam altas, o esquema isoniazida-rifampicina continua sendo a escolha apropriada para o início do tratamento.OBJECTIVE: Mycobacteriosis is frequently diagnosed among HIV-infected patients. In Mozambique, where few patients are under antiretroviral therapy and the prevalence of tuberculosis is high, there is need for better characterization of mycobacteria at the species level, as well as for the identification of patterns of resistance to antituberculous drugs. METHODS: We studied a sample of 503 HIV-infected individuals suspected of having pulmonary tuberculosis. Of those 503, 320 tested positive for mycobacteria through sputum smear microscopy or culture of bronchoalveolar lavage fluid. RESULTS: Acid-fast bacilli were observed in the sputum of 73% of the individuals presenting positive cultures. Of 277 isolates tested, only 3 were nontuberculous mycobacteria: 2 were identified as Mycobacterium avium and one was identified as M. simiae. Strains initially characterized as M. tuberculosis complex through polymerase chain reaction restriction analysis (PRA of the hsp65 gene were later confirmed as such through PRA of the gyrB gene. Among the M. tuberculosis isolates, resistance patterns were as follows: to isoniazid, 14%; to rifampin, 6%; and multidrug resistance, 5%. Previously treated cases showed significantly higher rates of resistance to first-line antituberculous drugs. The most common radiological pattern was interstitial infiltrate (in 67%, followed by mediastinal lymph node enlargement (in 30%, bronchiectasis (in 28%, miliary nodules (in 18% and cavitation (in 12%. Patients infected with nontuberculous mycobacteria presented clinical profiles indistinguishable from those of other patients. The median CD4 lymphocyte count in this group was 134 cells/mm³. CONCLUSIONS: There is a strong association between tuberculosis and AIDS in Mozambique, as expected in a country with a high prevalence of tuberculosis. Although drug resistance rates are high, the isoniazid-rifampin regimen continues to be the appropriate choice for initial therapy.

Elizabete Abrantes Nunes

2008-10-01

117

Adequate Th2-Type Response Associates with Restricted Bacterial Growth in Latent Mycobacterial Infection of Zebrafish  

Science.gov (United States)

Tuberculosis is still a major health problem worldwide. Currently it is not known what kind of immune responses lead to successful control and clearance of Mycobacterium tuberculosis. This gap in knowledge is reflected by the inability to develop sufficient diagnostic and therapeutic tools to fight tuberculosis. We have used the Mycobacterium marinum infection model in the adult zebrafish and taken advantage of heterogeneity of zebrafish population to dissect the characteristics of adaptive immune responses, some of which are associated with well-controlled latency or bacterial clearance while others with progressive infection. Differences in T cell responses between subpopulations were measured at the transcriptional level. It was discovered that a high total T cell level was usually associated with lower bacterial loads alongside with a T helper 2 (Th2)-type gene expression signature. At late time points, spontaneous reactivation with apparent symptoms was characterized by a low Th2/Th1 marker ratio and a substantial induction of foxp3 reflecting the level of regulatory T cells. Characteristic gata3/tbx21 has potential as a biomarker for the status of mycobacterial disease.

Hammaren, Milka Marjut; Luukinen, Bruno Vincent; Pesu, Marko; Ramet, Mika; Parikka, Mataleena

2014-01-01

118

Nontuberculous mycobacterial (NTM) lung disease: the top ten essentials.  

Science.gov (United States)

This review will utilize essential questions about nontuberculous mycobacterial (NTM) lung disease to succinctly address important new developments in the pathogenesis, diagnosis and management of NTM lung disease with a focus on practical information and "bottom line" answers. 1) What do I tell my patients who ask, “where did I get this infection” and, “should I take showers”? 2) What is the connection between bronchiectasis and the acquisition of NTM lung infection? 3) What other factors are important in the pathogenesis of NTM lung disease? 4) Why does it seem that am I seeing more new NTM lung disease patients? 5) Why is the diagnosis of NTM lung disease so complicated and does the diagnosis of NTM lung infection obligate specific treatment? 6) Unlike traditional tuberculosis, what is behind the irrelevance of most in vitro susceptibility testing reports for NTM infections? 7) Is there anything new for the management of patients with Mycobacterium avium complex lung disease? How does the radiographic appearance influence treatment? 8) Is there anything new for the management of patients with Mycobacterium abscessus lung disease? 9) What about the management of other NTM respiratory pathogens? 10) Is there a role for the use of macrolide monotherapy for non-cystic fibrosis bronchiectasis? PMID:24484653

Aksamit, Timothy R; Philley, Julie V; Griffith, David E

2014-03-01

119

Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition  

Science.gov (United States)

ABSTRACT The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3 of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ?mycP3ms mutant, failed to export the native Esx-3 substrates EsxHms and EsxGms to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxHms and EsxGms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxGms and EsxHms secretion in the ?mycP3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems.

Siegrist, M. Sloan; Steigedal, Magnus; Ahmad, Rushdy; Mehra, Alka; Dragset, Marte S.; Schuster, Brian M.; Philips, Jennifer A.; Carr, Steven A.

2014-01-01

120

Immunocytochemical detection of mycobacterial antigen in extrapulmonary tuberculosis.  

Science.gov (United States)

The aim of the study is to determine whether immunostaining for mycobacterial antigen can contribute to the cytological diagnosis of extrapulmonary tuberculosis (EPTB). The study was carried out on aspirated material of lymph nodes, and other accessible sites, from 65 patients with clinical diagnosis of tuberculosis (TB). Twenty patients, diagnosed by fine-needle aspiration, with non-tuberculous granulomas served as controls. The diagnosis of TB was based on the demonstration of acid-fast bacilli (AFB), culture positivity for Mycobacterium tuberculosis (M. tuberculosis), or response to treatment with standard anti-tubercular therapy. Immunostaining was done using polyclonal antibody to mycobacteria. AFB positivity by Ziehl Neelsen (ZN) staining was 21%, 65.38%, and 68% respectively in Pattern 1 (granulomas alone), in Pattern 2 (granulomas with necrosis), and in Pattern 3 (necrosis alone). Overall AFB positivity was 56.92%. Twenty-eight of 65 cases were negative for AFB on direct smear. Culture was positive in 46% (13/28). Sensitivity and specificity of immunostaining were 96.92% (63/65) and 95%, respectively. Immunoreactivity was seen in 26 (92.8%) of 28 cases which were negative by ZN staining. Except in the case of leprosy, in which cross reactivity was seen, there was no immunoreactivity in the control group. Immunocytochemistry (ICC) had high sensitivity (96.2%) and specificity (95%) in the diagnosis of EPTB. ICC may be a useful adjunct to evaluation of cytomorphology and ZN staining. PMID:24166859

Prapanna, Pooja; Srivastava, Ruchi; Arora, Vinod Kumar; Singh, Navjeevan; Bhatia, Arati; Kaur, Iqbal R

2014-05-01

 
 
 
 
121

Non-major histocompatibility complex-restricted cytotoxic activity of blood mononuclear cells stimulated with secreted mycobacterial proteins and other mycobacterial antigens  

DEFF Research Database (Denmark)

Several observations indicate that non-major histocompatibility complex (MHC)-restricted cytotoxicity, mediated for example by natural killer cells and lymphokine-activated killer cells, may serve as an important antimicrobial defense mechanism. The purpose of the present study was to investigate the influences of different mycobacterial antigens on non-MHC-restricted cytotoxicity and further to investigate the ways by which various lymphocyte subpopulations contribute to the development of this cytotoxicity. Non-MHC-restricted cytotoxicity was induced following stimulation of mononuclear cells with tuberculin purified protein derivative, Mycobacterium bovis bacillus Calmette-Guérin (BCG), short- and long-term culture filtrates of virulent Mycobacterium tuberculosis H37Rv, and 30-31-kDa secreted mycobacterial protein. These antigens also induced proliferation and production of gamma interferon. The CD4+ cells proliferated and expressed interleukin-2 receptors following stimulation with mycobacterial antigens. Depletion studies after antigen stimulation showed that the cytotoxic effector cells were CD16+ CD56+ and CD4-; the CD4+ cells alone did not mediate non-MHC-restricted cytotoxicity. To evaluate the influence of CD4+ cells on the development of non-MHC-restricted cytotoxicity, blood mononuclear cells were depleted of CD4+ cells before antigen stimulation. When mononuclear cells were incubated with purified protein derivative or short-term culture filtrate in the absence of CD4+ cells, cytotoxic activity was reduced. This reduction was abolished by interleukin-2 but not by gamma interferon. We conclude that several mycobacterial antigens are able to induce non-MHC-restricted cytotoxicity. This study indicates that non-MHC-restricted cytotoxicity following stimulation with mycobacterial antigens is induced by cytokines released by antigen-specific activated CD4+ cells.

Ravn, P; Pedersen, B K

1994-01-01

122

Atypical mycobacterial cutaneous infections in Egyptians: a clinicopathological study.  

Science.gov (United States)

Atypical mycobacteria comprise an uncommon heterogenous non-tuberculous group of acid-fast bacteria that rarely involve skin. The pattern of atypical mycobacterial cutaneous infections (AMCI) has not been previously studied in Egypt. The aim of this study was to describe the clinical characteristics, pathological features and species profile of AMCI among Egyptian patients. A retrospective study included 46 cases, diagnosed with AMCI during the period 2002 to 2012. The study included 34 males (73.9%) and 12 females (26.9%). The average age of patients was 39 years while the average duration of lesions was 15 months. The lesions were mostly located on the extremities (91.3%) and there was predominance of single (65.2%) and nodular (41.4%) lesions. History of trauma was confirmed in 91.3%. Histologically, the granulomas were mostly superficial (67.4%) with predominance of nodular suppurative pattern (84.8%). Other significant histological findings included epidermal hypertrophy (100%), presence of large-sized multinucleated giant cells (87%) and intrafollicular neutrophilic abscesses (84.8%). The diagnosis was proved by direct smear in 6.5%, skin biopsy in 10.9%, tissue culture in 47.8% and polymerase chain reaction (PCR) in 34.8%. Isolated species included Mycobacterium marinum (84.8%), Mycobacterium fortuitum (10.9%) and Mycobacterium kansasii (4.3%). Although the results of this study recommend that the diagnosis of AMCI is based mainly on culture and PCR, other clinicopathological features such as history of trauma, acral location of the lesion and suppurative granulomatous reaction with intrafollicular abscesses could be helpful clues in suspecting AMCI. PMID:24533920

El-Khalawany, Mohamed A

2014-04-01

123

Facilitated Oligomerization of Mycobacterial GroEL: Evidence for Phosphorylation-Mediated Oligomerization? †  

Science.gov (United States)

The distinctive feature of the GroES-GroEL chaperonin system in mediating protein folding lies in its ability to exist in a tetradecameric state, form a central cavity, and encapsulate the substrate via the GroES lid. However, recombinant GroELs of Mycobacterium tuberculosis are unable to act as effective molecular chaperones when expressed in Escherichia coli. We demonstrate here that the inability of M. tuberculosis GroEL1 to act as a functional chaperone in E. coli can be alleviated by facilitated oligomerization. The results of directed evolution involving random DNA shuffling of the genes encoding M. tuberculosis GroEL homologues followed by selection for functional entities suggested that the loss of chaperoning ability of the recombinant mycobacterial GroEL1 and GroEL2 in E. coli might be due to their inability to form canonical tetradecamers. This was confirmed by the results of domain-swapping experiments that generated M. tuberculosis-E. coli chimeras bearing mutually exchanged equatorial domains, which revealed that E. coli GroEL loses its chaperonin activity due to alteration of its oligomerization capabilities and vice versa for M. tuberculosis GroEL1. Furthermore, studying the oligomerization status of native GroEL1 from cell lysates of M. tuberculosis revealed that it exists in multiple oligomeric forms, including single-ring and double-ring variants. Immunochemical and mass spectrometric studies of the native M. tuberculosis GroEL1 revealed that the tetradecameric form is phosphorylated on serine-393, while the heptameric form is not, indicating that the switch between the single- and double-ring variants is mediated by phosphorylation.

Kumar, C. M. Santosh; Khare, Garima; Srikanth, C. V.; Tyagi, Anil K.; Sardesai, Abhijit A.; Mande, Shekhar C.

2009-01-01

124

Cytodiagnosis of coexistent cryptococcal and mycobacterial lymphadenitis in a case of AIDS  

Directory of Open Access Journals (Sweden)

Full Text Available Multiple infections are a common feature of acquired immunodeficiency syndrome (AIDS, but coexistent infections at the same site are rare. In this report, we describe a 35-year-old human immunodeficiency virus infected male with coexistent cryptococcal and mycobacterial lymphadenitis. He presented with generalised lymphadenopathy. Fine needle aspiration cytology of enlarged cervical lymph node, aided by special stains, revealed coexistent cryptococcal and mycobacterial infection. Coexistent infections pose diagnostic problems in AIDS patients and are likely to be missed. Special stains are valuable for accurate diagnosis of coexistent infections.

Anvikar Arti

2011-01-01

125

Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells  

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A group of T cells recognizes glycolipids presented by molecules of the CD1 family. The CD1d-restricted natural killer T cells (NKT cells) are primarily considered to be self-reactive. By employing CD1d-binding and T cell assays, the following structural parameters for presentation by CD1d were defined for a number of mycobacterial and mammalian lipids: two acyl chains facilitated binding, and a polar head group was essential for T cell recognition. Of the mycobacterial lipids tested, only a ...

Fischer, Karsten; Scotet, Emmanuel; Niemeyer, Marcus; Koebernick, Heidrun; Zerrahn, Jens; Maillet, Sophie; Hurwitz, Robert; Kursar, Mischo; Bonneville, Marc; Kaufmann, Stefan H. E.; Schaible, Ulrich E.

2004-01-01

126

Raised serum IgG and IgA antibodies to mycobacterial antigens in rheumatoid arthritis.  

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Autoantigens cross reactive with mycobacteria are implicated in the pathogenesis of adjuvant arthritis in the rat, and there are reports of changes in the immune response to mycobacteria in human rheumatoid arthritis (RA). We have therefore examined the IgM, IgG, and IgA antibody levels to crude mycobacterial antigens and to two recombinant mycobacterial heat shock/stress proteins (65 kD and 71 kD) in sera from patients with RA, systemic lupus erythematosus (SLE), and Crohn's disease, and fro...

Tsoulfa, G.; Rook, G. A.; Van-embden, J. D.; Young, D. B.; Mehlert, A.; Isenberg, D. A.; Hay, F. C.; Lydyard, P. M.

1989-01-01

127

Molecular evidence of lateral gene transfer in rpoB gene of Mycobacterium yongonense strains via multilocus sequence analysis.  

Science.gov (United States)

Recently, a novel species, Mycobacterium yongonense (DSM 45126(T)), was introduced and while it is phylogenetically related to Mycobacterium intracellulare, it has a distinct RNA polymerase ?-subunit gene (rpoB) sequence that is identical to that of Mycobacterium parascrofulaceum, which is a distantly related scotochromogen, which suggests the acquisition of the rpoB gene via a potential lateral gene transfer (LGT) event. The aims of this study are to prove the presence of the LGT event in the rpoB gene of the M. yongonense strains via multilocus sequence analysis (MLSA). In order to determine the potential of an LGT event in the rpoB gene of the M. yongonense, the MLSA based on full rpoB sequences (3447 or 3450 bp) and on partial sequences of five other targets [16S rRNA (1383 or 1395 bp), hsp65 (603 bp), dnaJ (192 bp), recA (1053 bp), and sodA (501 bp)] were conducted. Incongruences between the phylogenetic analysis of the full rpoB and the five other genes in a total of three M. yongonense strains [two clinical strains (MOTT-12 and MOTT-27) and one type strain (DSM 45126(T))] were observed, suggesting that rpoB gene of three M. yongonense strains may have been acquired very recently via an LGT event from M. parascrofulaceum, which is a distantly related scotochromogen. PMID:23382812

Kim, Byoung-Jun; Hong, Seok-Hyun; Kook, Yoon-Hoh; Kim, Bum-Joon

2013-01-01

128

Mycobacterial Esx-3 requires multiple components for iron acquisition.  

Science.gov (United States)

ABSTRACT The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3 of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ?mycP3ms mutant, failed to export the native Esx-3 substrates EsxHms and EsxGms to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxHms and EsxGms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxGms and EsxHms secretion in the ?mycP3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems. IMPORTANCE Mycobacteria have several paralogous type VII secretion systems, Esx-1 through Esx-5. Whereas Esx-1 is required for pathogenic mycobacteria to grow within an infected host, Esx-3 is essential for growth in vitro. We and others have shown that Esx-3 is required for siderophore-mediated iron acquisition. In this work, we identify individual Esx-3 components that contribute to this process. As in the Esx-1 system, most mutations that abolish Esx-3 protein export also disrupt its function. Unexpectedly, however, ultrasensitive quantitation of Esx-3 secretion by multiple-reaction-monitoring mass spectrometry (MRM-MS) revealed that very low levels of export were sufficient for iron acquisition under similar conditions. Although protein export clearly contributes to type VII function, the relationship is not absolute. PMID:24803520

Siegrist, M Sloan; Steigedal, Magnus; Ahmad, Rushdy; Mehra, Alka; Dragset, Marte S; Schuster, Brian M; Philips, Jennifer A; Carr, Steven A; Rubin, Eric J

2014-01-01

129

Characterization of the heparin-binding site of the mycobacterial heparin-binding hemagglutinin adhesin.  

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The mycobacterial adhesin heparin-binding hemagglutinin (HBHA) contains several lysine-rich repeats at its carboxyl-terminal end. Using truncated recombinant HBHA forms and hybrid proteins containing HBHA repeats grafted onto the Escherichia coli maltose-binding protein (MBP), we found that these repeats are responsible for heparin binding. Immunofluorescence microscopy studies revealed that their deletion abrogates binding of HBHA to human pneumocytes. Conversely, when fused to MBP, the HBHA repeats confer pneumocyte adherence properties to the hybrid protein. Treatment of pneumocytes with glycosaminoglycan-degrading enzymes showed that HBHA binding depends on the presence of heparan sulfate chains on the cell surface. The epitope of a monoclonal antibody that inhibits mycobacterial adherence to epithelial cells was mapped within the lysine-rich repeats, confirming their involvement in mycobacterial adherence to epithelial cells. Surface plasmon resonance analyses showed that recombinant HBHA binds to immobilized heparin with fast association kinetics (k(a) = 5.62 (+/- 0.10) x 10(5) m(-1) s(-1)), whereas the dissociation kinetics were slower (k(d) = 0.015 (+/- 0.002) s(-1)), yielding a K(D) value of 26 nm. Similar analyses with grafted MBP indicated similar kinetic constants, indicating that the carboxyl-terminal repeats contain the entire heparin-binding site of HBHA. The molecular characterization of the interactions of HBHA with epithelial glycosaminoglycans should help to better understand mycobacterial adherence within the lungs and may ultimately lead to new approaches for therapy or immunoprophylaxis. PMID:10799506

Pethe, K; Aumercier, M; Fort, E; Gatot, C; Locht, C; Menozzi, F D

2000-05-12

130

Immunology of atherosclerosis. Demonstration of heat shock protein 60 expression and T lymphocytes bearing alpha/beta or gamma/delta receptor in human atherosclerotic lesions.  

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Our previous work revealed the presence of a great number of activated T lymphocytes in early human atherosclerotic lesions, and we were able to induce atherosclerosis in normocholesterolemic rabbits by immunization with Mycobacterium tuberculosis heat-shock protein (HSP) 65. We hypothesized this latter phenomenon to arise from cross-reactivity of mycobacterial HSP 65 with the endogenously expressed homologous 60-kd form of this stress protein. To study HSP 60 expression and the phenotype of ...

Kleindienst, R.; Xu, Q.; Willeit, J.; Waldenberger, F. R.; Weimann, S.; Wick, G.

1993-01-01

131

Potential of two nucleic acid amplification assays for quantifying mycobacterial load in respiratory and non-respiratory specimens: a prospective study.  

Science.gov (United States)

Quantification of bacillary load plays a critical prognostic role in tuberculosis patients. This study evaluated the potential of the Cepheid GeneXpert (GX) MTB/RIF and the BD ProbeTec system as quantitative assays. The time to positivity (TTP) measured by the Mycobacterial Growth Index Tube system was compared to the cycle threshold (Ct) of GX MTB/RIF and the Metric Other than Acceleration (MOTA) scores generated by the ProbeTec system. Out of 714 samples examined, 44 culture confirmed cases were identified. The Ct values in 21 respiratory samples showed a high linear fit with the TTP in liquid culture (Spearman's correlation coefficient r = 0.9, P value < 0.0001), which was not the case in 23 non-respiratory samples. In both types of specimens, the MOTA scores did not correlate with the TTP in liquid culture. This indicates the suitability of GX as a quantitative measurement of mycobacterial load in respiratory but not non-respiratory specimens. PMID:24369994

Alnimr, Amani Mansour; Hassan, Manal Ismail

2014-03-01

132

Study of the gyrB gene polymorphism as a tool to differentiate among Mycobacterium tuberculosis complex subspecies further underlines the older evolutionary age of 'Mycobacterium canettii'.  

Science.gov (United States)

The present investigation evaluated the PCR-restriction fragment length polymorphism (RFLP) analysis of hsp65 and gyrB targets for differentiation of the species within the Mycobacterium tuberculosis complex (MTC) both by including new restriction enzymes and previously unstudied species. The hsp65 restriction analysis using HhaI resulted in a characteristic 'Mycobacterium canettii' pattern. A study of the gyrB gene polymorphism using TaqIalpha and HinfI allowed the initial division of MTC into two major groups, one consisting of M. tuberculosis and 'M. canettii' as opposed to another single group with other species. Three different patterns were observed with RsaI, the first characteristic of Mycobacterium microti, the second with Mycobacterium bovis, M. bovis BCG and Mycobacterium caprae (M. caprae was easily separated from M. bovis, and M. bovis BCG by SacII digestion), and the third with M. tuberculosis, 'M. canettii', Mycobacterium africanum, Mycobacterium pinnipedii, and the dassie bacillus. Although further discrimination within the last group was not obtained using additional restriction enzymes, the HaeIII and RsaI digestions highlighted an important gyrB polymorphism among 'M. canettii' strains. A study of the single nucleotide polymorphisms (SNP) within the gyrB by sequence analysis not only confirmed the results of the restriction analysis, but showed further differences among 'M. canettii' isolates that were not picked up using the existing battery of restriction enzymes. As many as 11 different SNPs were identified in the collection of eight 'M. canettii' isolates studied. Considering that gyrB variability among MTC member species other than 'M. canettii' is as restricted as hsp65 variability among MTC, our data corroborate a recent proposition that the 'M. canettii' group is evolutionary much older than the other MTC members. In conclusion, gyrB PCR-RFLP is a simple and rapid low-cost method that combined with phenotypic characteristics, may be helpful to differentiate most of the subspecies within the MTC. PMID:16517119

Goh, Khye Seng; Fabre, Michel; Huard, Richard C; Schmid, Solveig; Sola, Christophe; Rastogi, Nalin

2006-01-01

133

Disruption of the Gene Homologous to Mammalian Nramp1 in Mycobacterium tuberculosis Does Not Affect Virulence in Mice  

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Natural-resistance-associated macrophage protein 1 (Nramp1) is a divalent cation transporter belonging to a family of transporter proteins highly conserved in eukaryotes and prokaryotes. Mammalian and bacterial transporters may compete for essential metal ions during mycobacterial infections. The mycobacterial Nramp homolog may therefore be involved in Mycobacterium tuberculosis virulence. Here, we investigated this possibility by inactivating the M. tuberculosis Nramp1 gene (Mramp) by alleli...

Boechat, Neio; Lagier-roger, Be?atrice; Petit, Ste?phanie; Bordat, Yann; Rauzier, Jean; Hance, Allan J.; Gicquel, Brigitte; Reyrat, Jean-marc

2002-01-01

134

Isolation of Acid-Inducible Genes of Mycobacterium tuberculosis with the Use of Recombinase-Based In Vivo Expression Technology  

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A better understanding of mycobacterial gene regulation under certain stress conditions (e.g., low pH) may provide insight into mechanisms of adaptation during infection. To identify mycobacterial promoters induced at low pH, we adapted the recombinase-based in vivo expression technology (RIVET) promoter trap system for use with mycobacteria. Our results show that the TnpR recombinase of transposon ?? is active in Mycobacterium smegmatis and Mycobacterium tuberculosis. We developed a method...

Saviola, Beatrice; Woolwine, Samuel C.; Bishai, William R.

2003-01-01

135

Definition and annotation of (myco)bacterial non-coding RNA.  

Science.gov (United States)

RNA in bacteria may be broadly classified into coding and non-coding types. The prior, also known as messenger RNA, encode proteins as their final product. The non-coding RNA include all RNAs that are not translated into a protein. Examples of extensively studied and therefore prominent non-coding RNAs include rRNA, tRNA, tmRNA, whose designations reflect the functions performed by these RNAs. Discoveries of non-coding RNAs in mycobacteria have been reported in the recent years. At this early stage of this discipline of mycobacterial research, there is an opportunity for the scientific community to establish a consistent, systematic and objective approach to annotation of these RNAs. We are providing recommendations for this systematic annotation that we hope will be adopted by the mycobacterial research community. These may also serve as templates for annotation of non-coding RNAs in other bacteria. PMID:23291152

Lamichhane, Gyanu; Arnvig, Kristine B; McDonough, Kathleen A

2013-01-01

136

Mycobacterial phenolic glycolipid inhibits phagosome maturation and subverts the pro-inflammatory cytokine response.  

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Inhibition of phagosome maturation is an important hallmark of mycobacterial pathogenesis. A variety of genomic, transcriptomic and proteomic approaches have been used to pin down the molecule responsible for this pathogenic principle. We in this study characterize a glycolipid of Mycobacterium marinum identified through a screen of mutants disabled in inhibiting phagosome maturation to be phenolphthiocerol diester (phenolic glycolipid, PGL). This molecule is sufficient to impart its ability to inhibit phagosome maturation onto other microbial cells and even inert beads that are used as model pathogens. In addition, it abrogates pro-inflammatory cytokine secretion induced by strong inducers such as heat-killed Mycobacterium bovis bacille Calmette-Guérin. This strong dual agonistic effect of PGL overrides pro-inflammatory and pro-lysosomal delivery impulses set not only by mycobacteria but also by other pathogens and thus provides convincing evidence that this molecule is a vital mycobacterial virulence factor. PMID:18764820

Robinson, Nirmal; Kolter, Thomas; Wolke, Martina; Rybniker, Jan; Hartmann, Pia; Plum, Georg

2008-11-01

137

Synthetic UDP-furanoses as potent inhibitors of mycobacterial galactan biogenesis.  

Science.gov (United States)

UDP-galactofuranose (UDP-Galf) is a substrate for two types of enzymes, UDP-galactopyranose mutase and galactofuranosyltransferases, which are present in many pathogenic organisms but absent from mammals. In particular, these enzymes are involved in the biosynthesis of cell wall galactan, a polymer essential for the survival of the causative agent of tuberculosis, Mycobacterium tuberculosis. We describe here the synthesis of derivatives of UDP-Galf modified at C-5 and C-6 using a chemoenzymatic route. In cell-free assays, these compounds prevented the formation of mycobacterial galactan, via the production of short "dead-end" intermediates resulting from their incorporation into the growing oligosaccharide chain. Modified UDP-furanoses thus constitute novel probes for the study of the two classes of enzymes involved in mycobacterial galactan assembly, and studies with these compounds may ultimately facilitate the future development of new therapeutic agents against tuberculosis. PMID:21168771

Peltier, Pauline; Belá?ová, Martina; Dianišková, Petronela; Zhou, Ruokun; Zheng, Ruixiang Blake; Pearcey, Jean A; Joe, Maju; Brennan, Patrick J; Nugier-Chauvin, Caroline; Ferrières, Vincent; Lowary, Todd L; Daniellou, Richard; Mikušová, Katarína

2010-12-22

138

Organelle membrane proteomics reveals differential influence of mycobacterial lipoglycans on macrophage phagosome maturation and autophagosome accumulation.  

Science.gov (United States)

The mycobacterial cell wall component lipoarabinomannan (LAM) has been described as one of the key virulence factors of Mycobacterium tuberculosis. Modification of the terminal arabinan residues of this lipoglycan with mannose caps in M. tuberculosis or with phosphoinositol caps in Mycobacterium smegmatis results in distinct host immune responses. Given that M. tuberculosis typically persists in the phagosomal vacuole after being phagocytosed by macrophages, we performed a proteomic analysis of that organelle after treatment of macrophages with LAMs purified from the two mycobacterial species. The quantitative changes in phagosomal proteins suggested a distinct role for mannose-capped LAM in modulating protein trafficking pathways that contribute to the arrest of phagosome maturation. Enlightened by our proteomic data, we performed further experiments to show that only the LAM from M. tuberculosis inhibits accumulation of autophagic vacuoles in the macrophage, suggesting a new function for this virulence-associated lipid. PMID:21105745

Shui, Wenqing; Petzold, Christopher J; Redding, Alyssa; Liu, Jun; Pitcher, Austin; Sheu, Leslie; Hsieh, Tsung-Yen; Keasling, Jay D; Bertozzi, Carolyn R

2011-01-01

139

A study of mycobacterial transcriptional apparatus: identification of novel features in promoter elements.  

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Our earlier studies on transcriptional signals of mycobacteria had revealed that (i) strong promoters occur less frequently in the slowly growing pathogen Mycobacterium tuberculosis H37Rv than in the fast-growing saprophyte M. smegmatis and (ii) mycobacterial promoters function poorly in Escherichia coli. We now present evidence that RNA polymerases of M. smegmatis, M. tuberculosis, and M. bovis BCG recognize promoter elements with comparable efficiencies. Analysis of these randomly isolated ...

Bashyam, M. D.; Kaushal, D.; Dasgupta, S. K.; Tyagi, A. K.

1996-01-01

140

In Vitro Model of Mycobacterial Growth Arrest Using Nitric Oxide with Limited Air?  

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An in vitro model of mycobacterial growth arrest was developed using Mycobacterium bovis BCG. When an exponentially growing culture was transferred to an evacuated tube, growth continued; treatment with a source of nitric oxide (diethylenetriamine-nitric oxide adduct [DETA-NO] at 50 ?M) halted growth immediately, and aeration restored growth. When the period of growth arrest exceeded 4 h, a time lag occurred before aeration could restore growth. The lag time was maximal (24 h) after 16 h of ...

Hussain, Syed; Malik, Muhammad; Shi, Lanbo; Gennaro, Maria Laura; Drlica, Karl

2009-01-01

 
 
 
 
141

Esthetic outcome of surgical excision versus antibiotic therapy for nontuberculous mycobacterial cervicofacial lymphadenitis in children.  

Science.gov (United States)

One hundred children with microbiologically proven nontuberculous mycobacterial cervicofacial lymphadenitis were randomly assigned to excision of the involved lymph nodes, or antibiotic therapy consisting of clarithromycin and rifabutin. The esthetic outcome was rated using a revised and weighted Observer Scar Assessment Scale. The median weighted esthetic outcome in surgical patients was significantly better (30.6) than that for patients treated with antibiotics (42.2). PMID:19773678

Lindeboom, Jerome A; Lindeboom, Robert; Bruijnesteijn van Coppenraet, Elisabeth S; Kuijper, Ed J; Tuk, Jacco; Prins, Jan M

2009-11-01

142

Mycobacterial growth and sensitivity to H2O2 killing in human monocytes in vitro.  

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The intracellular growth and susceptibilities to killing by H2O2 in cultured human monocytes of a number of mycobacterial species including laboratory strains and clinical isolates of Mycobacterium tuberculosis, and Mycobacterium bovis bacillus Calmette-Guerin (BCG) and a clinical isolate of Mycobacterium avium-M. intracellulare were examined. The clinical isolate of M. avium-M. intracellulare did not replicate in freshly explanted monocytes (generation time of >400 h); BCG replicated with a ...

Laochumroonvorapong, P.; Paul, S.; Manca, C.; Freedman, V. H.; Kaplan, G.

1997-01-01

143

Differential Effects of Control and Antigen-Specific T Cells on Intracellular Mycobacterial Growth  

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We investigated the effects of peripheral blood mononuclear cells expanded with irrelevant control and mycobacterial antigens on the intracellular growth of Mycobacterium bovis bacillus Calmette-Guérin (BCG) in human macrophages. More than 90% of the cells present after 1 week of in vitro expansion were CD3+. T cells were expanded from purified protein derivative-negative controls, persons with latent tuberculosis, and BCG-vaccinated individuals. T cells expanded with nonmycobacterial antige...

Worku, S.; Hoft, D. F.

2003-01-01

144

Improving the characteristics of a mycobacterial 16 kDa-specific chicken scFv  

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Recombinant antibodies can be engineered to improve their binding or other characteristics. A chicken single chain variable fragment (scFv) phage display library was panned against the mycobacterial 16 kDa antigen. Three fusion phages which bound specifically to the antigenwere selected, each of which produced low signals in ELISA when secreted as a soluble scFv. One scFv was therefore chosen to be modified in an attempt to improve its binding. Firstly, a mutant sublibrary was cre...

Sixholo, Joy; Wyngaardt, Wouter; Mashau, Cordelia; Frischmuth, Janine; Du Plessis, Dion H.; Fehrsen, Jeanni

2011-01-01

145

The effect of endotoxin and endotoxin tolerance on inflammation induced by mycobacterial adjuvant.  

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Peptidoglycan, the substance in mycobacteria thought to be responsible for inducing adjuvant arthritis, and endotoxin (lipopolysaccharide or LPS) share many inflammatory properties. Since repeated administration of LPS produces tolerance, i.e., resistance to the toxic and inflammatory effects of LPS, we tested whether LPS and/or LPS tolerance might influence inflammation due to mycobacterial adjuvant. Male Sprague-Dawley rats were injected with Escherichia coli LPS or saline intraperitoneally...

1983-01-01

146

Hypoxia inducible factor signaling modulates susceptibility to mycobacterial infection via a nitric oxide dependent mechanism.  

Science.gov (United States)

Tuberculosis is a current major world-health problem, exacerbated by the causative pathogen, Mycobacterium tuberculosis (Mtb), becoming increasingly resistant to conventional antibiotic treatment. Mtb is able to counteract the bactericidal mechanisms of leukocytes to survive intracellularly and develop a niche permissive for proliferation and dissemination. Understanding of the pathogenesis of mycobacterial infections such as tuberculosis (TB) remains limited, especially for early infection and for reactivation of latent infection. Signaling via hypoxia inducible factor ? (HIF-?) transcription factors has previously been implicated in leukocyte activation and host defence. We have previously shown that hypoxic signaling via stabilization of Hif-1? prolongs the functionality of leukocytes in the innate immune response to injury. We sought to manipulate Hif-? signaling in a well-established Mycobacterium marinum (Mm) zebrafish model of TB to investigate effects on the host's ability to combat mycobacterial infection. Stabilization of host Hif-1?, both pharmacologically and genetically, at early stages of Mm infection was able to reduce the bacterial burden of infected larvae. Increasing Hif-1? signaling enhanced levels of reactive nitrogen species (RNS) in neutrophils prior to infection and was able to reduce larval mycobacterial burden. Conversely, decreasing Hif-2? signaling enhanced RNS levels and reduced bacterial burden, demonstrating that Hif-1? and Hif-2? have opposing effects on host susceptibility to mycobacterial infection. The antimicrobial effect of Hif-1? stabilization, and Hif-2? reduction, were demonstrated to be dependent on inducible nitric oxide synthase (iNOS) signaling at early stages of infection. Our findings indicate that induction of leukocyte iNOS by stabilizing Hif-1?, or reducing Hif-2?, aids the host during early stages of Mm infection. Stabilization of Hif-1? therefore represents a potential target for therapeutic intervention against tuberculosis. PMID:24367256

Elks, Philip M; Brizee, Sabrina; van der Vaart, Michiel; Walmsley, Sarah R; van Eeden, Fredericus J; Renshaw, Stephen A; Meijer, Annemarie H

2013-01-01

147

The mycobacterial Mpa–proteasome unfolds and degrades pupylated substrates by engaging Pup's N-terminus  

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Mycobacterium tuberculosis, along with other actinobacteria, harbours proteasomes in addition to members of the general bacterial repertoire of degradation complexes. In analogy to ubiquitination in eukaryotes, substrates are tagged for proteasomal degradation with prokaryotic ubiquitin-like protein (Pup) that is recognized by the N-terminal coiled-coil domain of the ATPase Mpa (also called ARC). Here, we reconstitute the entire mycobacterial proteasome degradation system for pupylated substr...

Striebel, Frank; Hunkeler, Moritz; Summer, Heike; Weber-ban, Eilika

2010-01-01

148

Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp  

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Abstract Background The histone-like Hlp protein is emerging as a key component in mycobacterial pathogenesis, being involved in the initial events of host colonization by interacting with laminin and glycosaminoglycans (GAGs). In the present study, nuclear magnetic resonance (NMR) was used to map the binding site(s) of Hlp to heparan sulfate and identify the nature of the amino acid residues directly involved in this interaction. Results The capacity of a panel...

Portugal Michelle I; Todeschini Adriane R; de Lima Cristiana S; Am, Silva Carlos; Mohana-Borges Ronaldo; Hm, Ottenhoff Tom; Mendonça-Previato Lucia; Previato Jose O; Cv, Pessolani Maria

2008-01-01

149

Osteopontin Expression Correlates with Clinical Outcome in Patients with Mycobacterial Infection  

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Osteopontin (OPN) is a protein that is expressed in chronic inflammatory diseases including tuberculosis, and its deficiency predisposes to more severe mycobacterial infections in mice. However, no reports have identified altered OPN expression in, or correlated these alterations to, infections in humans. The data presented herein identify alterations in the tissue expression of OPN protein and describe an inverse correlation between these levels and disease progression after inoculation of M...

2000-01-01

150

Single-Molecule Force Spectroscopy of Mycobacterial Adhesin-Adhesin Interactions?  

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The heparin-binding hemagglutinin (HBHA) is one of the few virulence factors identified for Mycobacterium tuberculosis. It is a surface-associated adhesin that expresses a number of different activities, including mycobacterial adhesion to nonphagocytic cells and microbial aggregation. Previous evidence indicated that HBHA is likely to form homodimers or homopolymers via a predicted coiled-coil region located within the N-terminal portion of the molecule. Here, we used single-molecule atomic-...

Verbelen, Claire; Raze, Dominique; Dewitte, Fre?de?rique; Locht, Camille; Dufre?ne, Yves F.

2007-01-01

151

Single-molecule force spectroscopy of mycobacterial adhesin-adhesin interactions.  

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The heparin-binding hemagglutinin (HBHA) is one of the few virulence factors identified for Mycobacterium tuberculosis. It is a surface-associated adhesin that expresses a number of different activities, including mycobacterial adhesion to nonphagocytic cells and microbial aggregation. Previous evidence indicated that HBHA is likely to form homodimers or homopolymers via a predicted coiled-coil region located within the N-terminal portion of the molecule. Here, we used single-molecule atomic-...

Verbelen, Claire; Raze, Dominique; Dewitte, Fre?de?rique; Locht, Camille; Dufre?ne, Yves

2007-01-01

152

Relationships between Mycobacterium Isolates from Patients with Pulmonary Mycobacterial Infection and Potting Soils? †  

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High numbers of mycobacteria, including known pathogenic species such as Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium chelonae, were recovered from aerosols produced by pouring commercial potting soil products and potting soil samples provided by patients with pulmonary mycobacterial infections. The dominant mycobacteria in the soil samples corresponded to the dominant species implicated clinically. Profiles of large restriction fragments obtained by pulsed-field gel e...

Groote, Mary Ann; Pace, Norman R.; Fulton, Kayte; Falkinham, Joseph O.

2006-01-01

153

Non-tuberculous and tuberculous mycobacterial peritonitis in peritoneal dialysis patients.  

Science.gov (United States)

Abstract Introduction: Peritoneal dialysis (pd)-associated mycobacterium peritonitis is an important clinical entity in patients with end stage renal disease. They present a significant diagnostic and therapeutic challenge for clinicians because clinical findings and laboratory investigations can not be differentiated from symptoms caused by non-tuberculous mycobacterium (ntm), Mycobacterium tuberculosis (tb) or other bacteria. The aim of the present article is to know the differences between the clinical manifestations and laboratory investigations, the appropriate diagnosis, treatment strategies and prognosis for tb and ntm disease in patients with pd-associated mycobacterial infections. Methods: This was a retrospective observational study conducted over a period of 25 years. Out of 1737 patients, only 7 were diagnosed with mycobacterial peritonitis. Result: Evaluable data showed that there were three patients diagnosed with ntm peritonitis and four patients with tuberculous peritonitis. The mean age of the patients was 53.9?±?11.8 years. Although all patients developed abdominal pain and cloudy dialysate, only four patients (57.1%) had fever. Two patients (28.6%) suffered severe sepsis and septic shock. Therefore, the patient survival rates for ntm and tuberculous peritonitis were 100.0% and 75.0%, respectively. Two patients were shifted to long-term hemodialysis; therefore, the technical survival rates for ntm and tuberculous peritonitis were 66.7% and 50.0%, respectively. Notably, recurrence of mycobacterial infection was found in one patient with both pulmonary tuberculosis and tuberculous peritonitis. Conclusion: The diagnosis of mycobacterial peritonitis remains a challenge to medical staffs because of its insidious nature, the variability of its presentation and the limitations of available diagnostic test. PMID:24827383

Lin, Jui-Hsiang; Wang, Wei-Jie; Yang, Huang-Yu; Cheng, Mei-Hua; Huang, Wen-Hung; Lin, Chih-Yuan; Lee, Shen-Yang; Yen, Tzung-Hai

2014-08-01

154

Atypical mycobacterial lymphadenitis in childhood--a clinicopathological study of 17 cases.  

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AIMS: To assess the clinical and pathological features of atypical mycobacterial lymphadenitis in childhood to define the salient clinical and histological features. METHODS: 17 cases were included on the basis of positive culture or demonstration of bacilli of appropriate morphology and staining characteristics. RESULTS: The mean age at diagnosis was 4.86 years. All children were systemically well, with clear chest x rays. Unilateral cervical lymphadenopathy was the commonest mode of present...

Evans, M. J.; Smith, N. M.; Thornton, C. M.; Youngson, G. G.; Gray, E. S.

1998-01-01

155

Recognition of multiple effects of ethambutol on metabolism of mycobacterial cell envelope.  

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Ethambutol is known to rapidly inhibit biosynthesis of the arabinan component of the mycobacterial cell wall core polymer, arabinogalactan (K. Takayama and J. O. Kilburn, Antimicrob. Agents Chemother. 33:1493-1499, 1989). This effect was confirmed, and it was also shown that ethambutol inhibits biosynthesis of the arabinan of lipoarabinomannan, a lipopolysaccharide noncovalently associated with the cell wall core. In contrast to cell wall core arabinan, which is completely inhibited by ethamb...

Deng, L.; Mikusova?, K.; Robuck, K. G.; Scherman, M.; Brennan, P. J.; Mcneil, M. R.

1995-01-01

156

Nonprocessive [2 + 2]e- off-loading reductase domains from mycobacterial nonribosomal peptide synthetases  

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In mycobacteria, polyketide synthases and nonribosomal peptide synthetases (NRPSs) produce complex lipidic metabolites by using a thio-template mechanism of catalysis. In this study, we demonstrate that off-loading reductase (R) domain of mycobacterial NRPSs performs two consecutive [2 + 2]e- reductions to release thioester-bound lipopeptides as corresponding alcohols, using a nonprocessive mechanism of catalysis. The first crystal structure of an R domain from Mycobacterium tuberculosis NR...

Chhabra, Arush; Haque, Asfarul S.; Pal, Ravi Kant; Goyal, Aneesh; Rai, Rajkishore; Joshi, Seema; Panjikar, Santosh; Pasha, Santosh; Sankaranarayanan, Rajan; Gokhale, Rajesh S.

2012-01-01

157

Mutually dependent secretion of proteins required for mycobacterial virulence  

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The ESX-1 locus is a region critical for full virulence in Mycobacterium tuberculosis, which encodes two secreted proteins as well as other genes involved in their secretion. The mechanism of secretion of the two proteins, ESAT-6 and CFP-10, and their function remain unknown. Using proteomic methods to search for additional proteins secreted by the ESX-1 locus, we discovered that a protein encoded by a chromosomally unlinked gene, espA, is also secreted by strains that contain the ESX-1 locus...

Fortune, S. M.; Jaeger, A.; Sarracino, D. A.; Chase, M. R.; Sassetti, C. M.; Sherman, D. R.; Bloom, B. R.; Rubin, E. J.

2005-01-01

158

Diagnostic potential of the pulsed discharged helium ionization detector (PDHID) for pathogenic Mycobacterial volatile biomarkers.  

Science.gov (United States)

Pathogenic Mycobacteria cause diseases in animals and humans with significant economic and societal consequences. Current methods for Mycobacterial detection relies upon time- and labor-intensive techniques such as culturing or DNA analysis. Using gas chromatography and mass spectrometry, four volatile compounds (methyl phenylacetate, methyl p-anisate, methyl nicotinate and o-phenyl anisole) were recently proposed as potential biomarkers for Mycobacteria. We demonstrate for the first time the capabilities of a field-deployable, pulsed discharge helium ionization detector (PDHID) for sensing these volatiles. We determined the analytical performance of the PDHID toward these Mycobacterial volatiles. Detector performance was moderately affected over the temperature range of 150 to 350 °C. The linear dynamic range for all four analytes exceeded three orders of magnitude. The limits of detection (LOD) and quantitation (LOQ) were calculated as 150 and 450 pg respectively, for all compounds, except methyl phenylacetate (LOD and LOQ, 90 and 270 pg, respectively). Control charts revealed that the PDHID detection system was generally stable, and deviations could be traced to common causes and excluded special causes. Grob tests and ionization potential data suggest that the PDHID is capable of detecting Mycobacterial volatiles in a complex milieu such as culture headspace or breath samples from tuberculosis patients. The diagnostic potential of the PDHID is critical to our goal of a handheld, field-deployable 'sniffer' system for biological pathogens and chemical warfare agents. PMID:23867723

Manginell, Ronald P; Pimentel, Adam S; Mowry, Curtis D; Mangan, Michael A; Moorman, Matthew W; Allen, Amy; Schares, Elizabeth S; Achyuthan, Komandoor E

2013-09-01

159

Mycobacterial laminin-binding histone-like protein mediates collagen-dependent cytoadherence  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular mat [...] rices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp), a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.

Dias, André Alves; Raze, Dominique; Lima, Cristiana Soares de; Marques, Maria Angela de Melo; Drobecq, Hervé; Debrie, Anne-Sophie; Ribeiro-Guimarães, Michelle Lopes; Biet, Franck; Pessolani, Maria Cristina Vidal.

160

Mycobacterium arosiense sp. nov., a slowly growing, scotochromogenic species causing osteomyelitis in an immunocompromised child  

DEFF Research Database (Denmark)

A yellow-pigmented, scotochromogenic, slowly growing mycobacterial strain, designated T1921(T), was isolated from the disseminated osteomyelitic lesions of a 7-year-old child with an underlying partial gamma interferon receptor alpha-1 deficiency. Hybridization by the line probe assay indicated the presence of a Mycobacterium species. Sequencing of the 16S rRNA gene, the internally transcribed spacer (ITS) region and the hsp65 and rpoB genes revealed that strain T1921(T) could be differentiated from all recognized species of the genus Mycobacterium. Phylogenetic analysis based on the 16S rRNA gene indicated that strain T1921(T) was related most closely to Mycobacterium intracellulare, whereas analysis based on the ITS and hsp65 and rpoB genes indicated that it was most closely related to Mycobacterium avium. Phenotypic tests were not able to differentiate strain T1921(T) from similar slowly growing mycobacteria. Strain T1921(T) is considered to represent a novel species of the genus Mycobacterium, for which the name Mycobacterium arosiense sp. nov. is proposed. The type strain is T1921(T) (=DSM 45069(T) =ATCC BAA-1401(T)) Udgivelsesdato: 2008/10

Bang, D.; Herlin, T.

2008-01-01

 
 
 
 
161

Pulmonary non-tuberculous mycobacterial infection in congenital contractural arachnodactyly.  

Science.gov (United States)

Congenital contractural arachnodactyly (CCA) is caused by mutations within the fibrillin-2 gene (FBN2), which is crucial for microfibril structure. Affected individuals may have contractures, chest wall deformities, scoliosis, abnormal ear folding and elongated limbs. We describe a novel FBN2 mutation in a woman with CCA who also had pulmonary non-tuberculous mycobacteria (NTM) infection. The population with pulmonary NTM infections shares phenotypic features with CCA, such as elongated body habitus, scoliosis and pectus deformities. While it is unlikely that FBN2 defects account for susceptibility to NTM infection in the majority of cases, the overlap between these two diseases suggests some shared pathophysiology. PMID:22325249

Paulson, M L; Olivier, K N; Holland, S M

2012-04-01

162

Purification and characterization of the acyltransferase involved in biosynthesis of the major mycobacterial cell envelope glycolipid - Monoacylated phosphatidylinositol dimannoside.  

Science.gov (United States)

Phosphatidylinositol mannosides are essential structural components of the mycobacterial cell envelope. They are implicated in host-pathogen interactions during infection and serve as a basis for biosynthesis of other unique molecules with immunomodulatory properties - mycobacterial lipopolysaccharides lipoarabinomannan and lipomannan. Acyltransferase Rv2611 is involved in one of the initial steps in the assembly of these molecules in Mycobacterium tuberculosis - the attachment of an acyl group to position-6 of the 2-linked mannosyl residue of the phosphatidylinositol mannoside anchor. Although the function of this enzyme was annotated 10years ago, it has never been completely biochemically characterized due to lack of the pure protein. We have successfully overexpressed and purified MSMEG_2934, the ortholog of Rv2611c from the non-pathogenic model organism Mycobacteriumsmegmatis mc(2)155 using mycobacterial pJAM2 expression system, which allowed confirmation of its in vitro acyltransferase activity, and establishment of its substrate specificity. PMID:24810911

Svetlíková, Zuzana; Baráth, Peter; Jackson, Mary; Korduláková, Jana; Mikušová, Katarína

2014-08-01

163

Rapid detection and differentiation of mycobacterial species using a multiplex PCR system  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Introduction The early diagnosis of mycobacterial infections is a critical step for initiating treatment and curing the patient. Molecular analytical methods have led to considerable improvements in the speed and accuracy of mycobacteria detection. Methods [...] The purpose of this study was to evaluate a multiplex polymerase chain reaction system using mycobacterial strains as an auxiliary tool in the differential diagnosis of tuberculosis and diseases caused by nontuberculous mycobacteria (NTM) Results Forty mycobacterial strains isolated from pulmonary and extrapulmonary origin specimens from 37 patients diagnosed with tuberculosis were processed. Using phenotypic and biochemical characteristics of the 40 mycobacteria isolated in LJ medium, 57.5% (n=23) were characterized as the Mycobacterium tuberculosis complex (MTBC) and 20% (n=8) as nontuberculous mycobacteria (NTM), with 22.5% (n=9) of the results being inconclusive. When the results of the phenotypic and biochemical tests in 30 strains of mycobacteria were compared with the results of the multiplex PCR, there was 100% concordance in the identification of the MTBC and NTM species, respectively. A total of 32.5% (n=13) of the samples in multiplex PCR exhibited a molecular pattern consistent with NTM, thus disagreeing with the final diagnosis from the attending physician. Conclusions Multiplex PCR can be used as a differential method for determining TB infections caused by NTM a valuable tool in reducing the time necessary to make clinical diagnoses and begin treatment. It is also useful for identifying species that were previously not identifiable using conventional biochemical and phenotypic techniques.

Andrea Santos, Lima; Rafael Silva, Duarte; Lilian Maria Lapa, Montenegro; Haiana Charifker, Schindler.

164

A chemically synthesized peptide which elicits humoral and cellular immune responses to mycobacterial antigens.  

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Monoclonal antibodies directed to Mycobacterium bovis BCG (BCG) and to M. tuberculosis H37Rv (H37Rv) were used in conjunction with affinity chromatography to prepare a mycobacterial component which was designated BCG-a. A synthetic peptide antigen was prepared based on the amino acid sequence of BCG-a and was designated BCG-a-P. Significant immunological similarities were found between BCG-a-P and antigens in extracts of BCG and H37Rv but not between BCG-a-P and antigens of nontuberculous myc...

1986-01-01

165

Isolation by genetic labeling of a new mycobacterial plasmid, pJAZ38, from Mycobacterium fortuitum.  

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In a two-step mating experiment with recipient strains of Mycobacterium smegmatis, the Mycobacterium fortuitum cryptic plasmid pJAZ38 was isolated. Plasmid pJAZ38 was genetically labeled by cointegration formation mediated by the kanamycin-resistant mycobacterial transposon Tn611. The region responsible for replication of pJAZ38 was located and sequenced. This region showed homology with the Mycobacterium avium plasmid pLR7 and the Mycobacterium scrofulaceum plasmid pMSC262, a family of plasm...

1997-01-01

166

Alteraciones en el reclutamiento y activación de proteínas Rab durante la infección micobacteriana Alterations in recruitment and activation of Rab proteins during mycobacterial infection  

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Full Text Available En el fagosoma, Mycobacterium spp. altera la activación y reclutamiento de diferentes proteínas "del gen Ras de cerebro de rata", comúnmente conocidas como Rab. En este manuscrito se revisa una serie de reportes que han demostrado que los fagosomas que contienen micobacterias tienen una expresión mayor y sostenida de Rab5, Rab11, Rab14 y Rab22a, y menor o ninguna expresión de Rab7, Rab9 y Rab6. Esto se correlaciona con aumento de la fusión de estos fagosomas con endosomas tempranos y de reciclaje, lo que les permite mantener ciertas características de compartimentos tempranos, permite que las bacterias obtengan acceso a nutrientes y previene la activación de mecanismos contra la micobacteria.
La expresión de mutantes constitutivamente activos de las Rab de endosomas tempranos impide la maduración de fagosomas que contienen esferas de látex o micobacterias inactivadas por calor. Mientras que su silenciamiento, mediante ARN de interferencia o mediante dominantes negativos, induce la maduración de fagosomas micobacterianos. Los mecanismos exactos por los que las micobacterias alteran la dinámica de expresión de estas GTPasas, afectando la maduración fagolisosómica, no se han establecido. El problema podría explicarse por defectos en el reclutamiento de las proteínas que interactúan con Rab, como la cinasa-3 del fosfatidilinositol y el antígeno endosómico temprano 1. La identificación de los mecanismos empleados por Mycobacterium spp. para interrumpir el ciclo de activación de las Rab, será esencial para comprender la fisiopatología de la infección micobacteriana y útil como posibles blancos farmacológicos.At the phagosome level, Mycobacterium spp. alters activation and recruitment of several "Ras gene from rat brain" proteins, commonly known as Rab. Mycobacterial phagosomes have a greater and sustained expression of Rab5, Rab11, Rab14 and Rab22a, and lowered or no expression of Rab7, Rab9 and Rab6. This correlates with increased fusion of the phagosomes with early and recycling endosomes acquiring some features of early phogosomes, allowing the bacteria to gain access to nutrients and preventing the activation of anti-mycobacterial mechanisms.
The expression of constitutively active mutants of Rab from the early stage endosomes prevents the maturation of phagosomes containing latex beads or heat-inactivated mycobacteria. Silencing of these mutants by interference RNA or dominant negative forms induces the maturation of mycobacterial phagosomes. The mechanisms have not been established by which mycobacteria alter the expression of these GTPases and thereby shift the phagolysosomal maturation. The problem can be explained by alterations in the recruitment of proteins that interact with Rab, such as phosphoinositide 3-kinases and early endosomal antigen 1. Identifying the mechanisms used by Mycobacterium spp. to disrupt the cycle of Rab activation will be essential to understand the pathophysiology of mycobacterial infections and usefully to potential drug targets.

Diana Castaño

2010-08-01

167

Non Mycobacterial Virulence Genes in the Genome of the Emerging Pathogen Mycobacterium abscessus  

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Mycobacterium abscessus is an emerging rapidly growing mycobacterium (RGM) causing a pseudotuberculous lung disease to which patients with cystic fibrosis (CF) are particularly susceptible. We report here its complete genome sequence. The genome of M. abscessus (CIP 104536T) consists of a 5,067,172-bp circular chromosome including 4920 predicted coding sequences (CDS), an 81-kb full-length prophage and 5 IS elements, and a 23-kb mercury resistance plasmid almost identical to pMM23 from Mycoba...

2009-01-01

168

Anti-mycobacterial activities of synthetic cationic ?-helical peptides and their synergism with rifampicin.  

Science.gov (United States)

The rapid emergence of multi-drug resistant tuberculosis (TB) and the lack of effective therapies have prompted the development of compounds with novel mechanisms of action to tackle this growing public health concern. In this study, a series of synthetic cationic ?-helical antimicrobial peptides (AMPs) modified with different hydrophobic amino acids was investigated for their anti-mycobacterial activity, both alone and in synergistic combinations with the frontline anti-tuberculosis drug rifampicin. The addition of thiol groups by incorporating cysteine residues in the AMPs did not improve anti-mycobacterial activity against drug-susceptible and drug-resistant Mycobacterium tuberculosis, while the enhancement of peptide hydrophobicity by adding methionine residues increased the efficacy of the primary peptide against all strains tested, including clinically isolated multidrug-resistant mycobacteria. The peptide with the optimal composition M(LLKK)2M was bactericidal, and eradicated mycobacteria via a membrane-lytic mechanism as demonstrated by confocal microscopic studies. Mycobacteria did not develop resistance after multiple exposures to sub-lethal doses of the peptide. In addition, the peptide displayed synergism with rifampicin against both Mycobacterium smegmatis and Mycobacterium bovis BCG and additivity against M. tuberculosis. Moreover, such combination therapy is effective in delaying the emergence of rifampicin resistance. The ability to potentiate anti-TB drug activity, kill drug-resistant bacteria and prevent drug resistance highlights the potential utility of the peptide in combating multidrug-resistant TB. PMID:24314557

Khara, Jasmeet S; Wang, Ying; Ke, Xi-Yu; Liu, Shaoqiong; Newton, Sandra M; Langford, Paul R; Yang, Yi Yan; Ee, Pui Lai Rachel

2014-02-01

169

Rapid susceptibility testing of Mycobacterium tuberculosis by bioluminescence assay of mycobacterial ATP  

International Nuclear Information System (INIS)

Mycobacterial growth was monitored by bioluminescence assay of mycobacterial ATP. Cultures of Mycobacterium tuberculosis H37Rv and of 25 clinical isolates of the same species were exposed to serial dilutions of ethambutol, isoniazid, rifampin, and streptomycin. A suppression of ATP, indicating growth inhibition, occurred for susceptible but not resistant strains within 5 to 7 days of incubation. Breakpoint concentrations between susceptibility and resistance were determined by comparing these results with those obtained by reference techniques. Full agreement was found in 99% of the assays with the resistance ratio method on Lowenstein-Jensen medium, and 98% of the assays were in full agreement with the radiometric system (BACTEC). A main advantage of the bioluminescence method is its rapidity, with results available as fast as with the radiometric system but at a lower cost and without the need for radioactive culture medium. The method provides kinetic data concerning drug effects within available in vivo drug concentrations and has great potential for both rapid routine susceptibility testing and research applications in studies of drug effects on mycobacteria

1988-01-01

170

Mycobacterial Induction of Autophagy Varies by Species and Occurs Independently of Mammalian Target of Rapamycin Inhibition*  

Science.gov (United States)

The interaction of host cells with mycobacteria is complex and can lead to multiple outcomes ranging from bacterial clearance to latent infection. Although many factors are involved, the mammalian autophagy pathway is recognized as a determinant that can influence the course of infection. Intervention aimed at utilizing autophagy to clear infection requires an examination of the autophagy and signal transduction induced by mycobacteria under native conditions. With both pathogenic and non-pathogenic mycobacteria, we show that infection correlates with an increase in the mammalian target of rapamycin (mTOR) activity indicating that autophagy induction by mycobacteria occurs in an mTOR-independent manner. Analysis of Mycobacterium smegmatis and Mycobacterium bovis bacille Calmette-Guérin (BCG), which respectively induce high and low autophagy responses, indicates that lipid material is capable of inducing both autophagy and mTOR signaling. Although mycobacterial infection potently induces mTOR activity, we confirm that bacterial viability can be reduced by rapamycin treatment. In addition, our work demonstrates that BCG can reduce autophagy responses to M. smegmatis suggesting that specific mechanisms are used by BCG to minimize host cell autophagy. We conclude that autophagy induction and mTOR signaling take place concurrently during mycobacterial infection and that host autophagy responses to any given mycobacterium stem from multiple factors, including the presence of activating macromolecules and inhibitory mechanisms.

Zullo, Alfred J.; Lee, Sunhee

2012-01-01

171

Mycobacterial spindle cell pseudotumor of the appendix vermiformis in a patient with AIDS  

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Full Text Available Mycobacterial pseudotumor (MP is a rare pathologic presentation of both Mycobacterium tuberculosis and non-tuberculous mycobacterial disease, hitherto reported to occur only in immunosuppressed patients with or without human immunodeficiency virus infection. This lesion shares close pathologic resemblance to certain mesenchymal neoplasms, particularly Kaposi's sarcoma (KS, from which it must be properly differentiated due to distinct prognosis and therapy. We report a case of MP obliterating the lumen of the appendix vermiformis in a 34-year-old patient who died of complications of AIDS at our hospital in Rio de Janeiro. A total of 24 cases of MP (including our patient have been described in the literature. MP has been found especially in lymph nodes, but extranodal lesions have been described in the skin, spleen, lung, bone marrow, brain and, in our patient, the appendix vermiformis. We offer a review of the other 23 published case reports of MP in both HIV-infected and uninfected patients and discuss the pathologic features that differentiate MP from KS.

Carlos Alberto Basílio-de-Oliveira

2001-04-01

172

Mycobacterial spindle cell pseudotumor of the appendix vermiformis in a patient with AIDS  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Mycobacterial pseudotumor (MP) is a rare pathologic presentation of both Mycobacterium tuberculosis and non-tuberculous mycobacterial disease, hitherto reported to occur only in immunosuppressed patients with or without human immunodeficiency virus infection. This lesion shares close pathologic rese [...] mblance to certain mesenchymal neoplasms, particularly Kaposi's sarcoma (KS), from which it must be properly differentiated due to distinct prognosis and therapy. We report a case of MP obliterating the lumen of the appendix vermiformis in a 34-year-old patient who died of complications of AIDS at our hospital in Rio de Janeiro. A total of 24 cases of MP (including our patient) have been described in the literature. MP has been found especially in lymph nodes, but extranodal lesions have been described in the skin, spleen, lung, bone marrow, brain and, in our patient, the appendix vermiformis. We offer a review of the other 23 published case reports of MP in both HIV-infected and uninfected patients and discuss the pathologic features that differentiate MP from KS.

Basílio-de-Oliveira, Carlos Alberto; Eyer-Silva, Walter A.; Valle, Heliomar de Azevedo; Rodrigues, Ana Lúcia; Pimentel, Ana Luisa Pinheiro; Morais-de-Sá, Carlos Alberto.

173

Single-molecule force spectroscopy of mycobacterial adhesin-adhesin interactions.  

Science.gov (United States)

The heparin-binding hemagglutinin (HBHA) is one of the few virulence factors identified for Mycobacterium tuberculosis. It is a surface-associated adhesin that expresses a number of different activities, including mycobacterial adhesion to nonphagocytic cells and microbial aggregation. Previous evidence indicated that HBHA is likely to form homodimers or homopolymers via a predicted coiled-coil region located within the N-terminal portion of the molecule. Here, we used single-molecule atomic-force microscopy to measure individual homophilic HBHA-HBHA interaction forces. Force curves recorded between tips and supports derivatized with HBHA proteins exposing their N-terminal domains showed a bimodal distribution of binding forces reflecting the formation of dimers or multimers. Moreover, the binding peaks showed elongation forces that were consistent with the unfolding of alpha-helical coiled-coil structures. By contrast, force curves obtained for proteins exposing their lysine-rich C-terminal domains showed a broader distribution of binding events, suggesting that they originate primarily from intermolecular electrostatic bridges between cationic and anionic residues rather than from specific coiled-coil interactions. Notably, similar homophilic HBHA-HBHA interactions were demonstrated on live mycobacteria producing HBHA, while they were not observed on an HBHA-deficient mutant. Together with the fact that HBHA mediates bacterial aggregation, these observations suggest that the single homophilic HBHA interactions measured here reflect the formation of multimers that may promote mycobacterial aggregation. PMID:17933894

Verbelen, Claire; Raze, Dominique; Dewitte, Frédérique; Locht, Camille; Dufrêne, Yves F

2007-12-01

174

Carboxyl terminal domain basic amino acids of mycobacterial topoisomerase I bind DNA to promote strand passage.  

Science.gov (United States)

Bacterial DNA topoisomerase I (topoI) carries out relaxation of negatively supercoiled DNA through a series of orchestrated steps, DNA binding, cleavage, strand passage and religation. The N-terminal domain (NTD) of the type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn(2+) finger motifs in the CTD. The Zn(2+) finger motifs were found to be essential in Escherichia coli topoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial topoI lacks Zn(2+) fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. We also show that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the catalytic step. Although the NTD binds to DNA in a site-specific fashion to carry out DNA cleavage and religation, the basic residues in CTD bind to non-scissile DNA in a sequence-independent manner to promote the crucial strand passage step during DNA relaxation. The loss of Zn(2+) fingers from the mycobacterial topoI could be associated with Zn(2+) export and homeostasis. PMID:23771144

Ahmed, Wareed; Bhat, Anuradha Gopal; Leelaram, Majety Naga; Menon, Shruti; Nagaraja, Valakunja

2013-08-01

175

Rapid susceptibility testing of Mycobacterium tuberculosis by bioluminescence assay of mycobacterial ATP  

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Mycobacterial growth was monitored by bioluminescence assay of mycobacterial ATP. Cultures of Mycobacterium tuberculosis H37Rv and of 25 clinical isolates of the same species were exposed to serial dilutions of ethambutol, isoniazid, rifampin, and streptomycin. A suppression of ATP, indicating growth inhibition, occurred for susceptible but not resistant strains within 5 to 7 days of incubation. Breakpoint concentrations between susceptibility and resistance were determined by comparing these results with those obtained by reference techniques. Full agreement was found in 99% of the assays with the resistance ratio method on Lowenstein-Jensen medium, and 98% of the assays were in full agreement with the radiometric system (BACTEC). A main advantage of the bioluminescence method is its rapidity, with results available as fast as with the radiometric system but at a lower cost and without the need for radioactive culture medium. The method provides kinetic data concerning drug effects within available in vivo drug concentrations and has great potential for both rapid routine susceptibility testing and research applications in studies of drug effects on mycobacteria.

Nilsson, L.E.; Hoffner, S.E.; Ansehn, S.

1988-08-01

176

Expression of tumor necrosis factor in vitro by human mononuclear phagocytes stimulated with whole Mycobacterium bovis BCG and mycobacterial antigens.  

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Four mycobacterial preparations directly stimulated human blood monocytes and alveolar macrophages to produce tumor necrosis factor (TNF). Monoclonal antibody against TNF blocked (99%) TNF activity. Lipopolysaccharide was not responsible for the TNF activity generated; activity was unaffected in the presence of polymyxin B.

Valone, S. E.; Rich, E. A.; Wallis, R. S.; Ellner, J. J.

1988-01-01

177

The DNA Damage-Regulated Autophagy Modulator DRAM1 Links Mycobacterial Recognition via TLP-MYD88 to Authophagic Defense.  

Science.gov (United States)

Autophagy is an important defense mechanism against mycobacteria, the causative agents of tuberculosis. The molecular mechanisms that link mycobacterial recognition to autophagy remain unclear. Our analysis in zebrafish and human macrophage models of mycobacterial infection reveals that the DNA damage-regulated autophagy modulator DRAM1 functions downstream of pathogen recognition by the Toll-like receptor (TLR)/interleukin-1 receptor (IL1R)-MYD88-NF-?B innate immune sensing pathway to activate selective autophagy. Mycobacterial infection of human macrophages and zebrafish embryos induced DRAM1 expression in a MYD88 and NF-?B-dependent manner. DRAM1 knockdown increased mycobacterial infection, whereas overexpression lowered infection by hyperactivating autophagy. DRAM1-mediated selective autophagic defenses require the cytosolic DNA sensor STING and the selective autophagy receptor p62/SQSTM1. Contrary to its known role in autophagy-mediated cell death and cancer, this DRAM1 function is p53 independent. We propose that DRAM1 mediates autophagic defense against a broader range of intracellular pathogens, since DRAM1 expression was also induced by the common bacterial endotoxin lipopolysaccharide. PMID:24922577

van der Vaart, Michiel; Korbee, Cornelis J; Lamers, Gerda E M; Tengeler, Anouk C; Hosseini, Rohola; Haks, Mariëlle C; Ottenhoff, Tom H M; Spaink, Herman P; Meijer, Annemarie H

2014-06-11

178

Sensitivity and Specificity of Immunocytochemical Staining of Mycobacterial Antigens in the Cytoplasm of Cerebrospinal Fluid Macrophages for Diagnosing Tuberculous Meningitis ?  

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The sensitivity and specificity of immunocytochemical staining of mycobacterial antigens in the cytoplasm of cerebrospinal fluid (CSF) macrophages for diagnosis of tuberculous meningitis (TBM) was prospectively compared with Ahuja criteria from 393 consecutive CSF specimens. The assay can play an important role for the diagnosis of TBM, with sensitivity of 73.5% and specificity of 90.7%.

Shao, Yuquan; Xia, Ping; Zhu, Tao; Zhou, Jiong; Yuan, Yuan; Zhang, Hao; Chen, Jianjun; Hu, Xingyue

2011-01-01

179

Immune reconstitution inflammatory syndrome in HIV-infected patients with mycobacterial infections starting highly active anti-retroviral therapy  

International Nuclear Information System (INIS)

AIM: To describe the radiological appearances of immune reconstitution inflammatory syndrome (IRIS) in human immunodeficiency virus (HIV)-infected patients with mycobacterial infections starting highly active anti-retroviral therapy (HAART). MATERIALS AND METHODS: Five consecutive HIV infected patients with IRIS due to mycobacterial infection were studied. Intercurrent infection and poor drug compliance were excluded as causes of presentation. The chest radiological appearances at the time of starting HAART and at the time of diagnosis of IRIS were compared. RESULTS: In these five patients there was clinical and radiological deterioration, occurring between 10 days and 7 months after starting HAART, leading to unmasking of previously undiagnosed mycobacterial infection or to worsening of mycobacterial disease. All five patients had HAART-induced increases in CD4+ T lymphocyte counts and reductions in peripheral blood HIV 'viral load'. Chest radiographic abnormalities due to IRIS included marked mediastinal lymphadenopathy in three patients--severe enough to produce tracheal compression in two patients (one of whom had stridor)--and was associated with new pulmonary infiltrates in two patients. The other two patients had new infiltrates, which in one patient was associated with a pleural effusion. CONCLUSION: These cases illustrate the diverse chest radiographic appearances of IRIS occurring after HAART in patients with mycobacterial and HIV co-infection. Marked mediastinal lymphadenopathy occurred in three of these five patients (with associated tracheal narrowing in two patients); four patients developed pulmonary infiltrates and one had an effusion. The cases further highlight that the onset of IRIS may be delayed for several months after HAART is started

2004-06-01

180

Immune reconstitution inflammatory syndrome in HIV-infected patients with mycobacterial infections starting highly active anti-retroviral therapy  

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AIM: To describe the radiological appearances of immune reconstitution inflammatory syndrome (IRIS) in human immunodeficiency virus (HIV)-infected patients with mycobacterial infections starting highly active anti-retroviral therapy (HAART). MATERIALS AND METHODS: Five consecutive HIV infected patients with IRIS due to mycobacterial infection were studied. Intercurrent infection and poor drug compliance were excluded as causes of presentation. The chest radiological appearances at the time of starting HAART and at the time of diagnosis of IRIS were compared. RESULTS: In these five patients there was clinical and radiological deterioration, occurring between 10 days and 7 months after starting HAART, leading to unmasking of previously undiagnosed mycobacterial infection or to worsening of mycobacterial disease. All five patients had HAART-induced increases in CD4+ T lymphocyte counts and reductions in peripheral blood HIV 'viral load'. Chest radiographic abnormalities due to IRIS included marked mediastinal lymphadenopathy in three patients--severe enough to produce tracheal compression in two patients (one of whom had stridor)--and was associated with new pulmonary infiltrates in two patients. The other two patients had new infiltrates, which in one patient was associated with a pleural effusion. CONCLUSION: These cases illustrate the diverse chest radiographic appearances of IRIS occurring after HAART in patients with mycobacterial and HIV co-infection. Marked mediastinal lymphadenopathy occurred in three of these five patients (with associated tracheal narrowing in two patients); four patients developed pulmonary infiltrates and one had an effusion. The cases further highlight that the onset of IRIS may be delayed for several months after HAART is started.

Buckingham, S.J.; Haddow, L.J.; Shaw, P.J.; Miller, R.F. E-mail: rmiller@gum.ucl.ac.uk

2004-06-01

 
 
 
 
181

CT features of pulmonary nontuberculous mycobacterial infection. Effects of underlying pulmonary disease  

International Nuclear Information System (INIS)

The CT findings of 50 cases of pulmonary nontuberculous mycobacterial infection (NMI) were evaluated by dividing patients into two clinical groups: group 1, with no history of pulmonary disease (n=34), and group 2, with underlying pulmonary disease (n=16), and observing serial CT images. Bronchiectasis, irregular opacity, and small nodule were common findings and were concomitantly seen in 80% of cases in both groups. In group 1, small nodule, peripheral bronchiectasis, and irregular opacity with bronchiectasis in the middle lobes were common findings. On the other hand, irregular opacity with cavity in the upper lobes was a common CT finding in group 2. Irregular opacity was a more frequent finding of NMI than previously reported and might be seen as the sole manifestation of NMI, especially in group 2 patients. Serial CT studies showed frequent changes in small nodule and irregular opacity. Irregular opacity with cavity tended to progress, whereas irregular opacity without bronchiectasis or cavity may or may not improve. (author)

2000-01-01

182

CT features of pulmonary nontuberculous mycobacterial infection. Effects of underlying pulmonary disease  

Energy Technology Data Exchange (ETDEWEB)

The CT findings of 50 cases of pulmonary nontuberculous mycobacterial infection (NMI) were evaluated by dividing patients into two clinical groups: group 1, with no history of pulmonary disease (n=34), and group 2, with underlying pulmonary disease (n=16), and observing serial CT images. Bronchiectasis, irregular opacity, and small nodule were common findings and were concomitantly seen in 80% of cases in both groups. In group 1, small nodule, peripheral bronchiectasis, and irregular opacity with bronchiectasis in the middle lobes were common findings. On the other hand, irregular opacity with cavity in the upper lobes was a common CT finding in group 2. Irregular opacity was a more frequent finding of NMI than previously reported and might be seen as the sole manifestation of NMI, especially in group 2 patients. Serial CT studies showed frequent changes in small nodule and irregular opacity. Irregular opacity with cavity tended to progress, whereas irregular opacity without bronchiectasis or cavity may or may not improve. (author)

Takada, Yukari; Sakai, Fumikazu; Suzuki, Keiko; Nagai, Atsushi [Tokyo Women' s Medical Coll. (Japan); Suzuki, Akira

2000-06-01

183

Mycobacterial bone marrow infections at a medical centre in Taiwan, 2001-2009.  

Science.gov (United States)

Mycobacterial bone marrow (BM) infection is the most common diagnosis established by BM examinations for fever of unknown origin. In this study, clinical features and outcomes of patients who fulfilled the criteria for BM infection due to Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) at a medical centre in Taiwan from 2001 to 2009 were investigated. The BM histopathological findings were also analysed. A total of 24 patients (16 men, eight women) with mycobacterial BM infections were found. Of these, nine (38%) were positive for human immunodeficiency virus (HIV) and six (25%) had no pre-existing immunocompromised conditions. MTB isolates were obtained from 11 (46%) patients and NTM species were isolated from 10 (42%) patients, including M. avium complex (MAC, n = 7) and M. kansasii (n = 3). Patients with MTB infections were significantly older than those with NTM infections (60·5 vs. 47·7 years, P = 0·043) and were less likely to have a positive BM culture (45% vs. 100%, P = 0·012). The 90-day survival rates for MTB and NTM BM infections were 68% and 60%, respectively (P = 0·61). In addition, the presence of BM granulomas was significantly more common in patients with MTB BM infections than in those with NTM infections (82% vs. 30%, P = 0·030). In Taiwan, the importance of NTM was not inferior to MTB and besides MAC, M. kansasii might be an important pathogen in non-HIV-infected patients. The presence of BM granulomas and caseation provides valuable information regarding early treatment pending culture results. PMID:24168831

Lin, S-H; Lai, C-C; Huang, S-H; Hung, C-C; Hsueh, P-R

2014-07-01

184

[Diagnosis and treatment of mycobacterial infections in patients with HIV/AIDS].  

Science.gov (United States)

Mycobacterial infection remains as a frequent complication associated to HIV infection. Although the widespread use of HAART has intensely decreased incidence of disseminated Mycobacterium avium (MAC) infection, it does not seem that it has affected tuberculosis occurrence so intensely. In spite of the intense search of new methods of rapid diagnosis, in the clinical practice the diagnosis of the mycobacterial illnesses continues based on culture, although the appearance of new media has allowed to shorten the time of growth. The combination of isoniazid (INH), rifampin (RIF) and pirazinamide (PZ) (with ethambutol [ETB] when primary resistance to INH is higher than 4%), remains as the elective treatment for tuberculosis in HIV infected patients. Due to the interaction between RIF and some antiretovirals drugs, such as proteasa inhibitors, a change in the usual regimens could be necessary. Combinations without RIF or antiretroviral therapy with drugs not interacting with RIF (nucleosides, ritonavir or nevirapin) have been suggested. The emergence of strains of Mycobacterium tuberculosis resistant to the antituberculosis drugs, the lack of adherence to treatment, and the frequency of adverse events hinders even more the control of the tuberculosis and they demand a narrow follow up of these patients. The treatment of the disseminated infection by MAC has improved in the last years with the generalization of the combinations including macrolides as claritromicin or azitromicin with ETB. The doubt persists about what combination is more effective, although like in other opportunists infections associated with a severe immunodeficiency, using antiretrovirals combinations that enhance the immune system could be a fundamental therapeutic approach. PMID:9859616

Pulido, F; Iribarren, J A; Kindelan, J M; Moreno, S

1998-01-01

185

Rapid radiometric methods to detect and differentiate Mycobacterium tuberculosis/M. bovis from other mycobacterial species  

Energy Technology Data Exchange (ETDEWEB)

Rapid methods for the differentiation of Mycobacterium tuberculosis/M. bovis (TB complex) from other mycobacteria (MOTT bacilli) were developed and evaluated in a three-phase study. In the first phase, techniques for identification of Mycobacterium species were developed by using radiometric technology and BACTEC Middlebrook 7H12 liquid medium. Based on /sup 14/CO/sub 2/ evolution, characteristic growth patterns were established for 13 commonly encountered mycobacterial species. Mycobacteria belonging to the TB complex were differentiated from other mycobacteria by cellular morphology and rate of /sup 14/CO/sub 2/ evolution. For further differentiation, radiometric tests for niacin production and inhibition by Q-nitro-alpha-acetyl amino-beta-hydroxy-propiophenone (NAP) were developed. In the second phase, 100 coded specimens on Lowenstein-Jensen medium were identified as members of the TB complex, MOTT bacilli, bacteria other than mycobacteria, or ''no viable organisms'' within 3 to 12 (average 6.4) days of receipt from the Centers for Disease Control. Isolation and identification of mycobacteria from 20 simulated sputum specimens were carried out in phase III. Out of 20 sputum specimens, 16 contained culturable mycobacteria, and all of the positives were detected by the BACTEC method in an average of 7.3 days. The positive mycobacterial cultures were isolated and identified as TB complex or MOTT bacilli in an average of 12.8 days. The radiometric NAP test was found to be highly sensitive and specific for a rapid identification of TB complex, whereas the radiometric niacin test was found to have some inherent problems. Radiometric BACTEC and conventional methodologies were in complete agreement in Phase II as well as in Phase III.

Siddiqi, S.H.; Hwangbo, C.C.; Silcox, V.; Good, R.C.; Snider, D.E. Jr.; Middlebrook, G.

1984-10-01

186

Rapid radiometric methods to detect and differentiate Mycobacterium tuberculosis/M. bovis from other mycobacterial species  

International Nuclear Information System (INIS)

Rapid methods for the differentiation of Mycobacterium tuberculosis/M. bovis (TB complex) from other mycobacteria (MOTT bacilli) were developed and evaluated in a three-phase study. In the first phase, techniques for identification of Mycobacterium species were developed by using radiometric technology and BACTEC Middlebrook 7H12 liquid medium. Based on 14CO2 evolution, characteristic growth patterns were established for 13 commonly encountered mycobacterial species. Mycobacteria belonging to the TB complex were differentiated from other mycobacteria by cellular morphology and rate of 14CO2 evolution. For further differentiation, radiometric tests for niacin production and inhibition by Q-nitro-alpha-acetyl amino-beta-hydroxy-propiophenone (NAP) were developed. In the second phase, 100 coded specimens on Lowenstein-Jensen medium were identified as members of the TB complex, MOTT bacilli, bacteria other than mycobacteria, or ''no viable organisms'' within 3 to 12 (average 6.4) days of receipt from the Centers for Disease Control. Isolation and identification of mycobacteria from 20 simulated sputum specimens were carried out in phase III. Out of 20 sputum specimens, 16 contained culturable mycobacteria, and all of the positives were detected by the BACTEC method in an average of 7.3 days. The positive mycobacterial cultures were isolated and identified as TB complex or MOTT bacilli in an average of 12.8 days. The radiometric NAP test was found to be highly sensitive and specific for a rapid identification of TB complex, whereas the radiometric niacin test was found to have some inherent problems. Radiometric BACTEC and conventional methodologies were in complete agreement in Phase II as well as in Phase III

1984-01-01

187

Rapid identification of strains belonging to the Mycobacterium abscessus group through erm(41) gene pyrosequencing.  

Science.gov (United States)

Mycobacterium abscessus and Mycobacterium massiliense lung infections have different clarithromycin susceptibilities, making proper identification important; however, standard multi-gene sequencing in clinical laboratories is laborious and time consuming. We developed a pyrosequencing-based method for rapid identification of strains belonging to the M. abscessus group by targeting erm(41). We examined 55 isolates from new pulmonary M. abscessus infections and identified 28 M. abscessus, 25 M. massiliense, and 2 Mycobacterium bolletii isolates. Multi-gene sequencing of 16S rRNA, hsp65, rpoB, and the 16S-23S ITS region was concordant with the results of erm(41) pyrosequencing; thus, the M. abscessus group can be identified by single-nucleotide polymorphisms in erm(41). The method also enables rapid identification of polymorphic, inducible clarithromycin-resistant sequevars (T28 or C28). Pyrosequencing of erm(41) is a rapid, reliable, high-throughput alternative method for identifying and characterizing M. abscessus species. Further testing of a diverse collection of isolates is necessary to demonstrate the discriminatory power of erm(41) sequencing to differentiating species with this highly divergent group. PMID:24809859

Yoshida, Shiomi; Tsuyuguchi, Kazunari; Suzuki, Katsuhiro; Tomita, Motohisa; Okada, Masaji; Shimada, Ryoko; Hayashi, Seiji

2014-07-01

188

Genes  

Science.gov (United States)

Illustration of the placement of genes in a chromosome. A gene can be defined as a region of DNA that controls a hereditary characteristic. It usually corresponds to a sequence used in the production of a specific protein or RNA. A gene carries biological information in a form that must be copied and transmitted from each cell to all its progeny. This includes the entire functional unit: coding DNA sequences, non-coding regulatory DNA sequences, and introns. Genes can be as short as 1000 base pairs or as long as several hundred thousand base pairs. It can even be carried by more than one chromosome. The estimate for the number of genes in humans has decreased as our knowledge has increased. As of 2001, humans are thought to have between 30,000 and 40,000 genes.

Excellence, Access

2005-03-12

189

Therapeutic DNA Vaccine Reduces Schistosoma mansoni-Induced Tissue Damage through Cytokine Balance and Decreased Migration of Myofibroblasts  

Science.gov (United States)

Helminths are known to elicit a wide range of immunomodulation characterized by dominant Th2-type immune responses. Our group previously showed that a DNA vaccine encoding the mycobacterial 65-kDa heat shock protein (DNA-hsp65) showed immunomodulatory properties. We also showed, using a helminth-tuberculosis (TB) co-infection model, that the DNA-hsp65 vaccine protected mice against TB. We next investigated the mechanistic role of the vaccine during helminth-TB co-infection. Clinically, helminth infection causes type 2 granulomas in the lung. Mice were immunized with DNA-hsp65 while they were submitted to the type 2 granuloma induction protocol by Schistosoma mansoni eggs infusion. In this work we investigated the effects of DNA-hsp65 on the pathology and immune response during the development of type 2 granuloma induced by S. mansoni eggs. Histologic analyses of lung parenchyma showed that the DNA-hsp65 vaccine protected mice against exacerbated fibrosis induced by Schistosoma eggs, and decreased the size of the granulomas. These changes were correlated with a reduction in the number of T cells specific for the egg antigens in the lung and also with modulation of Th2 cytokine expression. Taken together, our results showed that the adjuvant properties of the DNA-hsp65 vaccine regulated the immune response in this Th2 model, and resulted in a preserved lung parenchyma.

Frantz, Fabiani Gai; Ito, Toshihiro; Cavassani, Karen Angelica; Hogaboam, Cory M.; Lopes Silva, Celio; Kunkel, Steven L.; Faccioli, Lucia H.

2011-01-01

190

Attenuation of Mycobacterium tuberculosis by Disruption of a mas-Like Gene or a Chalcone Synthase-Like Gene, Which Causes Deficiency in Dimycocerosyl Phthiocerol Synthesis  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Tuberculosis is one of the leading preventable causes of death. Emergence of drug-resistant tuberculosis makes the discovery of new targets for antimycobacterial drugs critical. The unique mycobacterial cell wall lipids are known to play an important role in pathogenesis, and therefore the genes responsible for their biosynthesis offer potential new targets. To assess the possible role of some of the genes potentially involved in cell wall lipid synthesis, we disrupted a mas-like gene, msl7, ...

Sirakova, Tatiana D.; Dubey, Vinod S.; Cynamon, Michael H.; Kolattukudy, Pappachan E.

2003-01-01

191

Mycobacterial growth inhibition in murine splenocytes as a surrogate for protection against Mycobacterium tuberculosis (M. tb).  

Science.gov (United States)

Development of an improved vaccine against tuberculosis (TB) is hindered by the lack of a surrogate of protection. Efficacy of new TB vaccines in humans can only be evaluated by expensive and time consuming efficacy trials within TB endemic areas. It is critical that vaccines with the greatest potential to protect are selected for these trials. Mycobacterial growth inhibition assays (MGIAs) have been developed with the hope that these in-vitro functional assays will correlate with protection, which could aid in the selection of the best vaccine candidates. The present study describes the use of the BACTEC system to perform MGIAs in mice. We demonstrate reproducible mycobacterial growth inhibition in splenocytes from BCG immunised mice compared with unimmunised mice (P BACTEC MGIA can be used as a surrogate of protection in humans and preclinical animal models is now warranted. PMID:23726784

Marsay, Leanne; Matsumiya, Magali; Tanner, Rachel; Poyntz, Hazel; Griffiths, Kristin L; Stylianou, Elena; Marsh, Philip D; Williams, Ann; Sharpe, Sally; Fletcher, Helen; McShane, Helen

2013-09-01

192

[Comparative evaluation between MB/BacT system and Löwenstein-Jensen solid culture media for mycobacterial isolation].  

Science.gov (United States)

Tuberculosis is a public health issue in both developed and developing countries. Success of the treatment depend on the identification of patients with positive sputum smears, rapid confirmation of diagnosis in patients with negative microscopy and identification of mycobacterial strains with altered drug susceptibility. Data from the literature show that liquid culture media have a higher sensitivity for isolation mycobacteria than solid culture media as Löwenstein-Jensen. In our series inoculation on liquid media resulted in retrieval of a significant higher number of mycobacterial strains than on solid media (435 vs 250). The time needed to obtain a positive culture was also lower for liquid media (15.89 +/- 9 days, mean +/- standard deviation) than for solid media (27.77 +/- 10.13 days), p < 0.001. These differences were seen in both smear negative and smear positive cases. Culture in liquid media isolated more strains with altered drug susceptibility but this difference was not statistically significant. PMID:18019750

Grigoriu, Bogdan Drago?; Grigoriu, Carmen; Cojocaru, Cristian; Mih?escu, Traian; Diculencu, Daniela

2007-01-01

193

Fatty acid biosynthesis in Mycobacterium tuberculosis: Lateral gene transfer, adaptive evolution, and gene duplication  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Mycobacterium tuberculosis is a high GC Gram-positive member of the actinobacteria. The mycobacterial cell wall is composed of a complex assortment of lipids and is the interface between the bacterium and its environment. The biosynthesis of fatty acids plays an essential role in the formation of cell wall components, in particular mycolic acids, which have been targeted by many of the drugs used to treat M. tuberculosis infection. M. tuberculosis has ?250 genes invo...

Kinsella, Rhoda J.; Fitzpatrick, David A.; Creevey, Christopher J.; Mcinerney, James O.

2003-01-01

194

Conditional Depletion of KasA, a Key Enzyme of Mycolic Acid Biosynthesis, Leads to Mycobacterial Cell Lysis  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Inhibition or inactivation of InhA, a fatty acid synthase II (FASII) enzyme, leads to mycobacterial cell lysis. To determine whether inactivation of other enzymes of the mycolic acid-synthesizing FASII complex also leads to lysis, we characterized the essentiality of two ?-ketoacyl-acyl carrier protein synthases, KasA and KasB, in Mycobacterium smegmatis. Using specialized transduction for allelic exchange, null kasB mutants, but not kasA mutants, could be generated in Mycobacterium smegmati...

2005-01-01

195

Characterization of Mycobacterium tuberculosis Central Asian Strain1 using mycobacterial interspersed repetitive unit genotyping  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Abstract Background The Central Asian Strain1 (CAS1) genogroup of Mycobacterium tuberculosis (MTB) is the most prevalent in Pakistan, India and Bangladesh. Mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) typing is a reliable and reproducible method for differentiation of MTB isolates. However, information of its utility in determining the diversity of CAS1 strain is limited. We performed standard 12 loci based MIRU-VNTR typing on...

2007-01-01

196

CTLA-4 Blockade Enhances the Immune Response Induced by Mycobacterial Infection but Does Not Lead to Increased Protection  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The murine immune response to a pulmonary mycobacterial infection is slow to develop, allowing bacterial numbers to increase in the lung for several weeks after infection. We sought to enhance the protective immune response induced during Mycobacterium bovis BCG infection by administering an antibody that blocks the interaction of CTLA-4 with its ligands, CD80 and CD86. We found that injection of anti-CTLA-4 monoclonal antibody (MAb) greatly enhanced and accelerated the immune response, as me...

Kirman, Joanna; Mccoy, Kathy; Hook, Sarah; Prout, Melanie; Delahunt, Brett; Orme, Ian; Frank, Anthony; Le Gros, Graham

1999-01-01

197

Mechanisms involved in mycobacterial growth inhibition by gamma interferon-activated bone marrow macrophages: role of reactive nitrogen intermediates.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Murine bone marrow-derived macrophages are able to inhibit the growth of Mycobacterium bovis after stimulation with recombinant gamma interferon. This antimycobacterial activity was inhibited by NG-monomethyl-L-arginine, a specific inhibitor of nitrite and nitrate synthesis from L-arginine. Furthermore, there was a complete lack of mycobacterial growth inhibition in a medium deficient in L-arginine. Nitrite is generated by gamma interferon-activated bone marrow-derived macrophages after infec...

Flesch, I. E.; Kaufmann, S. H.

1991-01-01

198

Identification of Beijing Lineage Mycobacterium tuberculosis with Combined Mycobacterial Interspersed Repetitive Unit Loci 26, 31, and ETR-A?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A rapid method for identification of Beijing lineage Mycobacterium tuberculosis is still needed in regions of tuberculosis endemicity, especially if genotyping methods are not readily accessible. After analyzing 1,557 clinical isolates, a PCR method with combined mycobacterial interspersed repetitive unit loci 26, 31, and ETR-A for differentiation of Beijing lineage isolates was established, the sensitivity and specificity of which are 94.7% and 98.5%, respectively.

Chin, Pei-ju; Chiu, Chen-che; Jou, Ruwen

2007-01-01

199

Predicting results of mycobacterial culture on sputum smear reversion after anti-tuberculous treatment: a case control study  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Abstract Background Little is currently known regarding sputum smear reversion (acid-fast smear becomes positive again after negative conversion) during anti-tuberculous treatment. This study aimed to evaluate its occurrence in patients with pulmonary tuberculosis (TB) and identify factors predicting results of mycobacterial culture for smear-reversion of sputum samples. Methods The retrospective review was performed in a tertiary referral center and a local tea...

Shu Chin-Chung; Wang Jann-Tay; Lee Chih-Hsin; Wang Jann-Yuan; Lee Li-Na; Yu Chong-Jen

2010-01-01

200

Functional capacity of Mycobacterium tuberculosis-specific T cell responses in humans is associated with mycobacterial load1  

Digital Repository Infrastructure Vision for European Research (DRIVER)

High antigen load in chronic viral infections has been associated with impairment of antigen-specific T cell responses; however, the relationship between antigen load in chronic Mycobacterium tuberculosis (Mtb) infection and functional capacity of Mtb-specific T cells in humans is not clear. We compared Mtb-specific T cell-associated cytokine production and proliferative capacity in peripheral blood from adults with progressively higher mycobacterial loads, i.e., persons with latent Mtb infec...

Day, Cheryl L.; Abrahams, Deborah A.; Lerumo, Lesedi; Rensburg, Esme Janse; Stone, Lynnett; O’rie, Terrence; Pienaar, Bernadette; Kock, Marwou; Kaplan, Gilla; Mahomed, Hassan; Dheda, Keertan; Hanekom, Willem A.

2011-01-01

 
 
 
 
201

Detection of IgG and IgA against the mycobacterial antigen A60 in patients with extrapulmonary tuberculosis  

Digital Repository Infrastructure Vision for European Research (DRIVER)

BACKGROUND—Diagnosis of extrapulmonary tuberculosis is often difficult to establish using standard methods. Serological techniques based on detection of antibodies against mycobacterial antigen A60 have shown good sensitivity and specificity in pulmonary tuberculosis. The present study was undertaken to define the diagnostic accuracy of testing for IgG and IgA against A60 in extrapulmonary tuberculosis.?METHODS—One hundred and ninety eight subjects were studied:...

Alifano, M.; Pascalis, R.; Sofia, M.; Faraone, S.; Del Pezzo, M.; Covelli, I.

1998-01-01

202

Rapid Diagnosis of Tuberculous Meningitis by a Dot Immunobinding Assay To Detect Mycobacterial Antigen in Cerebrospinal Fluid Specimens  

Digital Repository Infrastructure Vision for European Research (DRIVER)

In the present prospective study, a dot immunobinding assay (Dot-Iba) was standardized to measure the circulating mycobacterial antigen in cerebrospinal fluid (CSF) specimens for the laboratory diagnosis of tuberculous meningitis (TBM). Immunoglobulin G antibody specific for Mycobacterium tuberculosis in a CSF specimen from a patient with culture-proven TBM was isolated and was coupled with activated cyanogen bromide-Sepharose 4B. By immunosorbent affinity chromatography, a 14-kDa antigen was...

Sumi, M. G.; Mathai, A.; Sarada, C.; Radhakrishnan, V. V.

1999-01-01

203

Rapid Identification of Mycobacterium tuberculosis Beijing Genotypes on the Basis of the Mycobacterial Interspersed Repetitive Unit Locus 26 Signature  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Mycobacterium tuberculosis Beijing strains are prevalent in many parts of the world and often give rise to large institutional outbreaks. Such highly transmissible strains, often associated with multidrug resistance, are likely underrepresented in outbreaks reported from developing countries, mainly due to nonavailability of fast detection methods suitable in epidemiological surveillance studies. We evaluated a PCR assay based on amplification of mycobacterial interspersed repetitive unit loc...

2006-01-01

204

Exploring the Structure and Function of the Mycobacterial KatG Protein Using trans-Dominant Mutants  

Digital Repository Infrastructure Vision for European Research (DRIVER)

In order to probe the structure and function of the mycobacterial catalase-peroxidase enzyme (KatG), we employed a genetic approach using dominant-negative analysis of katG merodiploids. Transformation of Mycobacterium bovis BCG with various katG point mutants (expressed from low-copy-number plasmids) resulted in reductions in peroxidase and catalase activities as measured in cell extracts. These reductions in enzymatic activity usually correlated with increased resistance to the antitubercul...

Devito, Joseph A.; Morris, Sheldon

2003-01-01

205

Mycobacterial lipoarabinomannans modulate cytokine production in human T helper cells by interfering with raft/microdomain signalling.  

Science.gov (United States)

Lipoarabinomannans (LAMs) are major lipoglycans of the mycobacterial envelope and constitute immunodominant epitopes of mycobacteria. In this paper, we show that mannose-capped (ManLAM) and non-mannose-capped (PILAM) mycobacterial lipoglycans insert into T helper cell rafts without apparent binding to known receptors. T helper cells modified by the insertion of PILAM responded to CD3 cross-linking by decreasing type 1 (IL-2 and IFN-gamma) and increasing type 2 (IL-4 and IL-5) cytokine production. Modification by the mannose-capped ManLAMs had similar, but more limited effects on T helper cell cytokine production. When incorporated into isolated rafts, PILAMs modulated membrane-associated kinases in a dose-dependent manner, inducing increased phosphorylation of Src kinases and Cbp/PAG in Th1 rafts, while decreasing phosphorylation of the same proteins in Th2 rafts. Mycobacterial lipoglycans thus modify the signalling machineries of rafts/microdomains in T helper cells, a modification of the membrane organization that eventually leads to an overall enhancement of type 2 and inhibition of type 1 cytokine production. PMID:15666089

Shabaana, A K; Kulangara, K; Semac, I; Parel, Y; Ilangumaran, S; Dharmalingam, K; Chizzolini, C; Hoessli, D C

2005-01-01

206

Detection of mycobacterial lipoarabinomannan with an antigen-capture ELISA in unprocessed urine of Tanzanian patients with suspected tuberculosis.  

Science.gov (United States)

A direct antigen-capture ELISA based on the detection of mycobacterial lipoarabinomannan (LAM) in unprocessed urine was evaluated for its usefulness in clinical practice. In Tanzania, 231 patients with suspected pulmonary tuberculosis (TB) and 103 healthy volunteers were screened with standard TB tests and with the new LAM-ELISA. Of 132 patients with confirmed pulmonary mycobacterial disease (positive sputum culture), 106 were positive using the LAM-ELISA (sensitivity 80.3%). In comparison, the sensitivity of acid-fast bacilli (AFB) sputum microscopy was 62.1% (82 of 132 confirmed cases). Of the 231 patients, 17 were both culture- and AFB-negative, but had typical radiographic signs of pulmonary mycobacterial infection and did not respond to antibiotic treatment. Of these 17 patients, 13 (76.5%) had positive LAM-ELISA test results. To define the specificity of the assay, urine samples from 103 healthy volunteers were also screened using LAM-ELISA. All but one had an optical density below the cut-off (specificity 99%). Of interest was a significant correlation between level of microscopic density of mycobacteria in sputum and LAM antigen concentration in urine (chi2=8.44). The LAM-ELISA is a field-adapted tool that can improve screening standards in countries with a high incidence of TB. PMID:16139316

Boehme, C; Molokova, E; Minja, F; Geis, S; Loscher, T; Maboko, L; Koulchin, V; Hoelscher, M

2005-12-01

207

Increased serum anti-mycobacterial antibody titers in rheumatoid arthritis patients: Is there any specific antigenic target?  

International Nuclear Information System (INIS)

Objective was to investigate the presence of immunoreactivity against mycobacterial antigens in the sera of patients with rheumatoid arthritis (Ra) and to detect the target of the immune reaction. This study was carried out on 60 patients with RA, and 25 patients with no joint diseases in the laboratory of Clinical Microbiology Department of Ankara University Medical Faculty, Ankara, Turkey between July 2003 to January 2004. Secreted and cellular antigens of Mycobacterium tuberculosis (M. tuberculosis) H37Rv and Mycobacterium bovis (M. bovis) were isolated and purified by high performance liquid chromatography to antigenic fractions. The immunoreactivity of patient and control sera against these antigens were determined by enzyme-linked immunosorbent assay (ELISA). Immunoreactivity against mycobacterial antigens in RA patients were significantly higher than controls. Significant difference between patients and controls has been determined with M. bovis Bacillus Calmette Guerin (BCG) culture fluid and sonicate antigens, but not with M. tuberculosis H37Rv. This suggests that the antigen triggering immune response in patients with RA may belong to or mainly expressed on M. bovis BCG. The ELISA results showed significant difference between RA patients and controls with all antigenic fractions. Presence of increased immunoreactivity against mycobacterial antigens in the sera of patients with RA was detected. When statistical analysis was considered, we cannot put forward any antigenic fraction alone as the one responsible for the increased reactivity. (author)

2007-01-01

208

Antimicrobial peptides and proteins in mycobacterial therapy: Current status and future prospects.  

Science.gov (United States)

Tuberculosis (TB), an infectious disease caused by the pathogen Mycobacterium tuberculosis (Mtb), kills about 1.5 million people every year worldwide. An increase in the prevalence of drug-resistant strains of Mtb in the last few decades now necessitates the development of novel drugs that combat infections by both drug-sensitive and resistant Mtb. Moreover, as Mtb can persist in host cells by modulating their immune responses, it is essential that anti-TB agents be able to penetrate macrophages and kill the pathogen intracellularly without harming the host cells. In this context, antimicrobial peptides (AMPs) and proteins are being harnessed as anti-infective agents for the treatment of various diseases. Due to their direct and rapid bactericidal activity it is unlikely that pathogens acquire resistance against AMPs. Several short and potent AMP derivatives have been prepared by peptide engineering, and several of them are currently evaluated in clinical trials. The present review summarizes the role of endogenously expressed AMPs and proteins in the treatment of tuberculosis infections. In addition, mechanisms of direct anti-mycobacterial activity, manipulation of host immune responses, and future prospects of AMPs as therapeutic agents are discussed. PMID:24813349

Padhi, Avinash; Sengupta, Mitali; Sengupta, Srabasti; Roehm, Klaus H; Sonawane, Avinash

2014-07-01

209

Antibodies against Mycobacterial Proteins as Biomarkers for HIV-Associated Smear-Negative Tuberculosis.  

Science.gov (United States)

Serology data are limited for patients with sputum smear-negative HIV-associated active tuberculosis (TB). We evaluated the serum antibody responses against the mycobacterial proteins MPT51, MS, and echA1 and the 38-kDa protein via enzyme-linked immunosorbent assay (ELISA) in South African (S.A.) HIV-positive (HIV(+)) smear-negative TB patients (n = 56), U.S. HIV(+) controls with a positive tuberculin skin test (TST(+); n = 21), and S.A. HIV-negative (HIV(-)) (n = 18) and HIV(+) (n = 24) controls. TB patients had positive antibody reactivity against MPT51 (73%), echA1 (59%), MS (36%), and the 38-kDa protein (11%). Little reactivity against MPT51 and echA1 was observed in control groups at low risk for TB, i.e., S.A. HIV(-) (0% and 6%, respectively), and at moderate risk for TB development, i.e., U.S. HIV(+) TST(+) controls (14% and 10%, respectively). By contrast, more reactivity was detected in the S.A. HIV(+) control group at higher risk for TB (25% and 45%, respectively). Our data hold promise that antibody detection against MPT51 and echA1 might have adjunctive value in the detection of HIV(+) smear-negative TB and might reflect increasing Mycobacterium tuberculosis infection activity in asymptomatic HIV(+) individuals. PMID:24671553

Siev, Michael; Wilson, Douglas; Kainth, Supreet; Kasprowicz, Victoria O; Feintuch, Catherine M; Jenny-Avital, Elizabeth R; Achkar, Jacqueline M

2014-06-01

210

X-ray and CT appearance of pulmonary nontuberculosis mycobacterial infection  

International Nuclear Information System (INIS)

Objective: To study the chest X-ray and CT appearance of pulmonary non-tuberculosis mycobacterial infection. Methods: The chest X-ray or CT findings of 42 cases with cultures positive for pulmonary non-tuberculosis mycobacterium were reviewed. All abnormalities and predominant lobe involvement were recorded. The findings of chest X-ray and CT were compared. Results: The chest X-ray showed that air space consolidation and cavities were most frequently seen, nodules (n=19) and linear disease (n=19) were observed too. The abnormalities involved bilateral multiple lobes, with upper lobe more often than lower ones. Multiple manifestations were often co-existed. On CT scans, bronchiectasis, 'tree in bud' sign, and mediastinal lymphadenopathy were seen on CT but not on chest X-ray. Conclusion: Space con- solidation, cavity, nodule, fibrosis, bronchiectasis, and 'tree in bud' sign were major abnormalities of pulmonary NTMB, which were indistinguishable from those of secondary pulmonary TB, and the CT findings were helpful in the diagnosis of pulmonary NTMB infection. (authors)

2007-08-01

211

HAMP domain-mediated signal transduction probed with a mycobacterial adenylyl cyclase as a reporter.  

Science.gov (United States)

HAMP domains, ?55 amino acid motifs first identified in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases, operate as signal mediators in two-component signal transduction proteins. A bioinformatics study identified a coevolving signal-accepting network of 10 amino acids in membrane-delimited HAMP proteins. To probe the functionality of this network we used a HAMP containing mycobacterial adenylyl cyclase, Rv3645, as a reporter enzyme in which the membrane anchor was substituted by the Escherichia coli chemotaxis receptor for serine (Tsr receptor) and the HAMP domain alternately with that from the protein Af1503 of the archaeon Archaeoglobus fulgidus or the Tsr receptor. In a construct with the Tsr-HAMP, cyclase activity was inhibited by serine, whereas in a construct with the HAMP domain from A. fulgidus, enzyme activity was not responsive to serine. Amino acids of the signal-accepting network were mutually swapped between both HAMP domains, and serine signaling was examined. The data biochemically tentatively established the functionality of the signal-accepting network. Based on a two-state gearbox model of rotation in HAMP domain-mediated signal propagation, we characterized the interaction between permanent and transient core residues in a coiled coil HAMP structure. The data are compatible with HAMP rotation in signal propagation but do not exclude alternative models for HAMP signaling. Finally, we present data indicating that the connector, which links the ?-helices of HAMP domains, plays an important structural role in HAMP function. PMID:22094466

Mondéjar, Laura García; Lupas, Andrei; Schultz, Anita; Schultz, Joachim E

2012-01-01

212

Cervical Nontuberculous Mycobacterial Lymphadenitis Mimicking a Thyroid Tumor and Infiltrating Deep into the Neck  

Directory of Open Access Journals (Sweden)

Full Text Available Aim: Nontuberculous Mycobacterial Lymphadenitis (NML, which occurs in 1.2 per 100,000 children, is very rare. And those which emerge at the anterior cervical portion and infiltrate deep into the neck are even more rare. Generally, this disorder is uncommon existed near the thyroid gland. We report here a case of NML mimicking a thyroid tumor and infiltrating into the deep part of the anterior neck. Case: A mass at the anterior portion of her neck was found at 5 years old. It was not mobile and palpated as an irregularly surfaced hard mass whose size was 3 cm at the anterior lower portion of her neck. Ultrasonography showed an oval mass which existed near the slightly inferior part of the right lobe of the thyroid gland. Enhanced computed tomography showed a mass near the slightly inferior part of the right lobe of the thyroid gland. The mass was resected with the platysma and the right sternohyoid muscle. In the HE staining, epithelioid cell and Langhans type giant cells surrounding coagulative necrosis lesions which seemed to be caseation necrosis existed, similarly to cervical NML. Discussion: No consensus exists for the treatment of NML, but many documents advise complete excision. When the lesion cannot be completely removed, excision as far as possible and additional antibiotics are recommended. The characteristics of imaging of NML around the thyroid gland and infiltrating deep into the anterior neck and mediastinum are discussed.

Kazuhiro Mino

2012-12-01

213

Nontuberculous Mycobacterial (NTM) Disease in Immunocompetent Patients: Expanding Image Findings on Chest CT  

Energy Technology Data Exchange (ETDEWEB)

The aim of this study was to evaluate the chest CT features of nontuberculous mycobacterial (NTM) disease regardless of the specific organisms. This study included 74 consecutive patients (35 men, 39 women; mean age, 63 years; age range, 25-89 years) who were diagnosed with NTM disease according to the American Thoracic Society Guidelines (1997 and 2007) between January 2005 and July 2007. Chest CT images were randomly reviewed by two radiologists with consensus. The most common organism associated with NTM disease is M. avium-intracellulare complex (87.8%), followed by M. abscesses, M. kansasii, and M. chelonae. The most common chest CT finding was a nodular bronchiectatic lesion (n = 35, 46.7%), followed by a cavitary lesion of the upper lobe (n = 21, 28.0%), combined lesions of two prior subtypes (n = 6, 8.0%), consolidative lesion (s) (n = 5, 6.7%), a bronchogenic spreading pulmonary tuberculosis-like lesion (n = 5, 6.7%), a cavitary mass lesion with small satellite nodules (n = 2, 2.7%), and a miliary nodular lesion (n = 1, 1.3%). More than 5 segments were involved in 60 cases (81.1%). The nodular bronchiectatic lesion or cavitary lesion of upper lobe presents with multi-segmental involvement and the occurrence of combined consolidation is indicative of NTM disease

Shin, Hyo Hyun; Seon, Hyun Ju; Kim, Mok Hee; Choi, Song; Song, Sang Gook; Shin, Sang Soo; Kim, Yun Hyeon; Park, Jin Gyoon [Chonnam National University Hospital, Gwangju (Korea, Republic of)

2010-04-15

214

Effective generation of reactive oxygen species in the mycobacterial phagosome requires K+ efflux from the bacterium.  

Science.gov (United States)

Efficient killing of mycobacteria by host macrophages depends on a number of mechanisms including production of reactive oxygen species (ROS) by the phagosomal NADPH oxidase, NOX2. Survival of pathogenic mycobacteria in the phagosome relies on the ability to control maturation of the phagosome such that it is biologically and chemically altered in comparison to phagosomes containing non-pathogenic bacteria. In this study we show that the action of NOX2 to produce ROS in the mycobacterial phagosome is paradoxically dependent on a bacterial potassium transporter. We show that a Mycobacterium bovis BCG mutant (BCGDeltakef), deficient in a Kef-type K+ transporter, exhibits an increased intracellular survival phenotype in resting and activated macrophages, yet retains the ability to inhibit phagosome acidification, and does not show increased resistance to acidic conditions or ROS. Addition of a ROS scavenger replicates this phenotype in macrophages infected with wild-type BCG, and the production of ROS by macrophages infected with BCGDeltakef is substantially decreased compared with those infected with wild-type BCG. Our results suggest that increased intracellular survival of BCGDeltakef is mediated by inducing a decreased macrophage oxidative burst, and are consistent with Kef acting to alter the ionic contents of the phagosome and promoting NOX2 production of ROS. PMID:20331644

Butler, Rachel E; Cihlarova, Vera; Stewart, Graham R

2010-08-01

215

Protective role for heat shock protein-reactive alpha beta T cells in murine yersiniosis.  

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To investigate the role of heat shock proteins (HSP) of Yersinia enterocolitica for the host immune response against this pathogen, we cloned and expressed a 60-kDa HSP of Y. enterocolitica serotype O8. A fragment of Y. enterocolitica O8 HSP60 encoded by amino acids 90 to 286 was sequenced and showed more than 90% homology with HSP60 of Y. enterocolitica O3 and GroEL of Escherichia coli and 59% homology with HSP65 of Mycobacterium bovis. The arthritogenic T-cell epitope of mycobacterial HSP65...

Noll, A.; Roggenkamp, A.; Heesemann, J.; Autenrieth, I. B.

1994-01-01

216

Expression and Immunogenicity of the Mycobacterial Ag85B/ESAT-6 Antigens Produced in Transgenic Plants by Elastin-Like Peptide Fusion Strategy  

Directory of Open Access Journals (Sweden)

Full Text Available This study explored a novel system combining plant-based production and the elastin-like peptide (ELP fusion strategy to produce vaccinal antigens against tuberculosis. Transgenic tobacco plants expressing the mycobacterial antigens Ag85B and ESAT-6 fused to ELP (TBAg-ELP were generated. Purified TBAg-ELP was obtained by the highly efficient, cost-effective, inverse transition cycling (ICT method and tested in mice. Furthermore, safety and immunogenicity of the crude tobacco leaf extracts were assessed in piglets. Antibodies recognizing mycobacterial antigens were produced in mice and piglets. A T-cell immune response able to recognize the native mycobacterial antigens was detected in mice. These findings showed that the native Ag85B and ESAT-6 mycobacterial B- and T-cell epitopes were conserved in the plant-expressed TBAg-ELP. This study presents the first results of an efficient plant-expression system, relying on the elastin-like peptide fusion strategy, to produce a safe and immunogenic mycobacterial Ag85B-ESAT-6 fusion protein as a potential vaccine candidate against tuberculosis.

Udo Conrad

2010-01-01

217

Genes  

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In order to describe a cell at molecular level, a notion of a “gene” is neither necessary nor helpful. It is sufficient to consider the molecules (i.e., chromosomes, transcripts, proteins) and their interactions to describe cellular processes. The downside of the resulting high resolution is that it becomes very tedious to address features on the organismal and phenotypic levels with a language based on molecular terms. Looking for the missing link between biological disciplines dealing w...

Prohaska, Sonja J.; Stadler, Peter F.

2008-01-01

218

CT findings of mycobacterial infection other than tuberculosis : comparison with tuberculosis  

Energy Technology Data Exchange (ETDEWEB)

To compare the CT findings of mycobacterial infection other than tuberculosis (MOTT) with those of tuberculosis (TB). The chest CT scans of 30 immunocompetent patients with culture-proven pulmonary MOTT (M:F =3D 11:19; mean age, 51.2 yrs.) and of 24 patients with active tuberculosis (M:F 12:12; mean age, 42.5 yrs.) were analyzed by two radiologists; decisions were reached by consensus. Common findings for both MOTT and TB included brochogenically-spread bronchogenic spread nodular lesion (93.3% for MOTT, 100% for TB), bronchiectasis (90%, 83.3%), bronchial wall thickening (66.7%, 54.2%), granuloma (63.3%, 75%), parenchymal scarring (53.3%, 54.2%), and mediastinal lymphadenopathy (50%, 37.5%). Less commonly observed findings were emphysema (46.7%, 29.7%), atelectasis (36.7%, 29.2%), narrowing of a major airway (23.3%, 25%), consolidation (23.3%, 29.2%)and pleural disease (16.7%, 29.2%). Except for cavity (30%, 53.3%; P less than 0.05), the frequencies of each finding were not different between the two groups. A lobe-matched frequency comparison showed that only bronchiectasis in the right middle lobe (40%, 16.7%), right lower lobe (63.3%, 33.3%) and lingula division (53.3%, 25%) was significantly more common in MOTT than in TB (p less than 0.05). The number of lobes in which bronchiectasis and bronchial wall thickening were involved was greater in MOTT (3.20) than in TB (2.04) (p=3D 0.011). Although the CT findings of MOTT and TB overlap considerably, cavities are more common in TB, while in MOTT, bronchiectasis in the lower lung zone is more common and bronchiectasis tends to be more extensive. (author)

Yoon, Chang Jin; Goo, Jin Mo; Seo, Joon Beom; Kim, Se Hyung; Im, Jung Gi [College of Medicine and the Institute of Radiation Medicine, Seoul National University, Seoul (Korea, Republic of)

2000-03-01

219

CT findings of mycobacterial infection other than tuberculosis : comparison with tuberculosis  

International Nuclear Information System (INIS)

To compare the CT findings of mycobacterial infection other than tuberculosis (MOTT) with those of tuberculosis (TB). The chest CT scans of 30 immunocompetent patients with culture-proven pulmonary MOTT (M:F =3D 11:19); mean age, 51.2 yrs.) and of 24 patients with active tuberculosis (M:F 12:12; mean age, 42.5 yrs.) were analyzed by two radiologists; decisions were reached by consensus. Common findings for both MOTT and TB included brochogenically-spread bronchogenic spread nodular lesion (93.3% for MOTT, 100% for TB), bronchiectasis (90%, 83.3%), bronchial wall thickening (66.7%, 54.2%), granuloma (63.3%, 75%), parenchymal scarring (53.3%, 54.2%), and mediastinal lymphadenopathy (50%, 37.5%). Less commonly observed findings were emphysema (46.7%, 29.7%), atelectasis (36.7%, 29.2%), narrowing of a major airway (23.3%, 25%), consolidation (23.3%, 29.2%)and pleural disease (16.7%, 29.2%). Except for cavity (30%, 53.3%; P less than 0.05), the frequencies of each finding were not different between the two groups. A lobe-matched frequency comparison showed that only bronchiectasis in the right middle lobe (40%, 16.7%), right lower lobe (63.3%, 33.3%) and lingula division (53.3%, 25%) was significantly more common in MOTT than in TB (p less than 0.05). The number of lobes in which bronchiectasis and bronchial wall thickening were involved was greater in MOTT (3.20) than in TB (2.04) (p=3D 0.011). Although the CT findings of MOTT and TB overlap considerably, cavities are more common in TB, while in MOTT, bronchiectasis in the lower lung zone is more common and bronchiectasis tends to be more extensive. (author)

2000-03-01

220

Nontuberculous Mycobacterial Infection Is Associated with Increased Respiratory Failure: A Nationwide Cohort Study  

Science.gov (United States)

Background and Purpose Population study on relationship between nontuberculous mycobacterial (NTM) infection and respiratory failure (RF) is limited. This study evaluated the RF risk, including acute respiratory failure (ARF), chronic respiratory failure (CRF) and ARF on CRF, in patients with NTM infection in Taiwan. Methods We used the National Health Insurance Research Database of Taiwan to identify 3864 newly diagnosed NTM patients (NTM cohort) from 1999 to 2009, and 15456 non-NTM patients (non-NTM cohort), frequency matched by demographic status for comparison. Incidence and hazard of developing RF were measured by the end of 2010. Results The incidence rate of RF was 4.31-fold higher in the NTM cohort than in the non-NTM cohort (44.0 vs.10.2 per 1000 person-years), with an adjusted hazard ratio (HR) of 3.11 (95% CI: 2.73–3.54). The cumulative proportional incidence of RF was 10% higher in the NTM cohort than in the non-NTM cohort (P<0.0001). The RF risk was much greater within 6 months after the diagnosis of NTM infection with a HR of 7.45 (95% CI?=?5.50–10.09). Age-specific comparison showed that the younger NTM patients had a higher HR of RF than the elderly NTM patients (HR: 4.42, 95% CI: 3.28–5.96 vs. HR: 2.52, 95% CI: 2.17–2.92). Comorbidity increased the risk of RF in both cohorts, particularly in those with chronic obstructive pulmonary disease. Conclusion Our study suggests patients with NTM infection are at a high risk of RF. The risk appears much greater soon after patients diagnosed with NTM infection.

Yeh, Jun-Jun; Wang, Yu-Chiao; Lin, Cheng-Li; Chou, Christine Yi-Ting; Yeh, Ting-Chun; Wu, Bing-Tsang

2014-01-01

 
 
 
 
221

Nontuberculous Mycobacterial Disease Mortality in the United States, 1999-2010: A Population-Based Comparative Study  

Science.gov (United States)

Background Environmental nontuberculous mycobacteria (NTM) are ubiquitous organisms with which humans commonly interact. The epidemiologic characteristics of NTM diseases including mortality rate and its associated factors remain largely unknown. In this study, we explored the geographical area of exposure and mortality and comorbid conditions of affected persons to determine environment, host, and host-pathogen interactive factors. Methods We analyzed mortality related to nontuberculous mycobacterial infections from 1999 through 2010 by examining multiple-cause-of-death data from the National Center for Health Statistics. Among those who died with these diseases, we analyzed age-adjusted mortality rates, trends, associations with demographic variables, and comorbid conditions and correlated this information with similar data for tuberculosis-related mortality during the same time. Measurements and Mean Results From 1999 through 2010, nontuberculous mycobacterial disease was reported as an immediate cause of death in 2,990 people in the United States with a combined overall mean age-adjusted mortality rate of 0.1 per 100,000 person-years. A significant increase in the number of NTM related deaths was seen from 1999 through 2010 (R2?=?0.72, p<0.0001), but it was not significant after adjustment for age. Persons aged 55 years and older, women, those living in Hawaii and Louisiana, and those of non-Hispanic, white ethnicity had higher mortality rates. Compared to tuberculosis-related mortality, chronic obstructive pulmonary disease, bronchiectasis, HIV, interstitial lung diseases, and tobacco use were significantly more common in persons with nontuberculous mycobacteria-related deaths. Conclusions Nontuberculous mycobacteria-related death numbers are rising and are unevenly distributed. The strong association of nontuberculous mycobacterial disease with age suggests that its prevalence will increase as the United States population ages.

Mirsaeidi, Mehdi; Machado, Roberto F.; Garcia, Joe G. N.; Schraufnagel, Dean E.

2014-01-01

222

Characterization of the Mycobacterial AdnAB DNA Motor Provides Insights into the Evolution of Bacterial Motor-Nuclease Machines*  

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Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (kcat 415 s?1), and the affinity of the motor for the ssDNA co...

Unciuleac, Mihaela-carmen; Shuman, Stewart

2010-01-01

223

Mycobacterial HSP60 and HSP70 prevents the severe form of acute DSS colitis in Balb/c mice.  

Czech Academy of Sciences Publication Activity Database

. Innsbruck : Elsevier, 2007, s. 49-49.[European Crohn´s and Colitis Organisation (ECCO) Congress. Innsbruck (AT), 01.03.2007-03.03.2007]Grant CEP: GA AV ?R IAA5020205; GA AV ?R 1QS500200572; GA ?R GD310/03/H147Grant ostatní: XE(XE) Broad Medical Research Program IBD-0159Výzkumný zám?r: CEZ:AV0Z50200510Zdroj financování: R - rámcový projekt EKKlí?ová slova: mycobacterial hsp60Kód oboru RIV: EE - Mikrobiologie, virologie

Kverka, Miloslav; Zákostelská, Zuzana; Tlaskalová, Helena; Van der Zee, R.; Van Eden, W.

224

Diffuse Pulmonary Uptake of Tc-99m Methylene Diphosphonate in a Patient with Non-tuberculosis Mycobacterial Infection  

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Extra-osseous uptake of bone-seeking radiopharmaceuticals has been reported at various sites and it is known to be induced by various causes. Diffuse pulmonary infection, such as tuberculosis, can be a cause of lung uptake of bone-scan agent. Here we report on a patient with non-tuberculosis mycobacterial infection (NTM) who demonstrated diffuse pulmonary uptake on Tc-99m MDP bone scan. After medical treatment for NTM, the patient's lung lesions improved. Estra skeletal lung Tc-99m MDP uptake on bone scan may suggest lung parenchymal damage associated with disease activity.

Kwon, Hyun Woo; Chung, June Key; Lee, Dong Soo [Seoul National University College of Medicine, Seoul (Korea, Republic of); Ab-Aziz, Aini [University Kebangsaan Malaysia Medical Centre, Kuala Lumpur, (Morocco)

2010-06-15

225

Diffuse Pulmonary Uptake of Tc-99m Methylene Diphosphonate in a Patient with Non-tuberculosis Mycobacterial Infection  

International Nuclear Information System (INIS)

Extra-osseous uptake of bone-seeking radiopharmaceuticals has been reported at various sites and it is known to be induced by various causes. Diffuse pulmonary infection, such as tuberculosis, can be a cause of lung uptake of bone-scan agent. Here we report on a patient with non-tuberculosis mycobacterial infection (NTM) who demonstrated diffuse pulmonary uptake on Tc-99m MDP bone scan. After medical treatment for NTM, the patient's lung lesions improved. Estra skeletal lung Tc-99m MDP uptake on bone scan may suggest lung parenchymal damage associated with disease activity.

2010-06-01

226

Conserved mycobacterial lipoglycoproteins activate TLR2 but also require glycosylation for MHC class II-restricted T cell activation1  

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CD4+ T cell clones derived from a leprosy lesion and patient blood were used to monitor the isolation and identification of an antigen associated with the self-limited form of the disease. Biochemical purification and genetic analysis identified the T cell antigen as a conserved mycobacterial lipoglycoprotein LprG. LprG-mediated activation of CD4+T cells required specific MHC class II restriction molecules and intracellular processing. Although LprG activated TLR2, this alone was not sufficie...

Sieling, Peter A.; Hill, Preston J.; Dobos, Karen M.; Brookman, Kerry; Kuhlman, Andrew M.; Fabri, Mario; Krutzik, Stephan R.; Rea, Thomas H.; Heaslip, Darragh G.; Belisle, John T.; Modlin, Robert L.

2008-01-01

227

Double Strand Break Unwinding and Resection by the Mycobacterial Helicase-Nuclease AdnAB in the Presence of Single Strand DNA-binding Protein (SSB)*  

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Mycobacterial AdnAB is a heterodimeric DNA helicase-nuclease and 3? to 5? DNA translocase implicated in the repair of double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Inclusion of mycobacterial single strand DNA-binding protein (SSB) in reactions containing linear plasmid dsDNA allowed us to study the AdnAB helicase under conditions in which the unwound single strands are coated by SSB and thereby pre...

Unciuleac, Mihaela-carmen; Shuman, Stewart

2010-01-01

228

Direct contacts between conserved motifs of different subunits provide major contribution to active site organization in human and mycobacterial dUTPases  

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dUTPases are essential for genome integrity. Recent results allowed characterization of the role of conserved residues. Here we analyzed the Asp/Asn mutation within conserved Motif I of human and mycobacterial dUTPases, wherein the Asp residue was previously implicated in Mg2+-coordination. Our results on transient/steady-state kinetics, ligand-binding and a 1.80 Å-resolution structure of the mutant mycobacterial enzyme, in comparison with wild type and C-terminally truncated structures, arg...

2010-01-01

229

Host immune responses to mycobacterial antigens and their implications for the development of a vaccine to control tuberculosis.  

Science.gov (United States)

Tuberculosis (TB) remains a worldwide health problem, causing around 2 million deaths per year. Despite the bacillus Calmette Guérin vaccine being available for more than 80 years, it has limited effectiveness in preventing TB, with inconsistent results in trials. This highlights the urgent need to develop an improved TB vaccine, based on a better understanding of host-pathogen interactions and immune responses during mycobacterial infection. Recent studies have revealed a potential role for autophagy, an intracellular homeostatic process, in vaccine development against TB, through enhanced immune activation. This review attempts to understand the host innate immune responses induced by a variety of protein antigens from Mycobacterium tuberculosis, and to identify future vaccine candidates against TB. We focus on recent advances in vaccine development strategies, through identification of new TB antigens using a variety of innovative tools. A new understanding of the host-pathogen relationship, and the usefulness of mycobacterial antigens as novel vaccine candidates, will contribute to the design of the next generation of vaccines, and to improving the host protective immune responses while limiting immunopathology during M. tuberculosis infection. PMID:25003089

Yuk, Jae-Min; Jo, Eun-Kyeong

2014-07-01

230

Tratamiento antirretroviral en pacientes con sida y micobacteriosis / Anti-retroviral treatment in patients with AIDS and mycobacterial diseases  

Scientific Electronic Library Online (English)

Full Text Available SciELO Argentina | Language: Spanish Abstract in spanish La tuberculosis y otras micobacteriosis constituyen asociaciones o coinfecciones frecuentes en pacientes con sida y se asocian con una elevada mortalidad. En esta revisión se actualizan los tratamientos de las principales enfermedades micobacterianas asociadas al sida (tuberculosis y micobacteriosis [...] por Mycobacterium avium), con especial énfasis en las interacciones farmacológicas entre antimicobacterianos, principalmente rifampicina y claritromicina, y fármacos antirretrovirales. Se analizan los esquemas de tratamiento, su duración, la quimioprofilaxis primaria y secundaria y el momento óptimo de iniciación del tratamiento antirretroviral. Finalmente se describe el síndrome inflamatorio de reconstitución inmune y su tratamiento. Abstract in english Tuberculosis and other mycobacterial diseases are frequent coinfections in AIDS patients with an increased related mortality. In this review we have updated the treatment of the main mycobacterial diseases (tuberculosis and Mycobacterium avium disease), under the scope of pharmacological interaction [...] s between antimycobacterial drugs, specially rifampicin and clarithromycin, and anti-retroviral drugs. Antimycobacterial treatment schemes, their duration, primary and secondary chemoprophylaxis and the optimal time to start the anti-retroviral therapy are analized. Finally, the immnune reconstitution inflammatory syndrome and its treatment are discussed.

Marcelo E., Corti; Domingo J., Palmero.

231

In vitro and in vivo studies of a rapid and selective breath test for tuberculosis based upon mycobacterial CO dehydrogenase.  

Science.gov (United States)

One of the major hurdles in treating tuberculosis (TB) is the time-consuming and difficult methodology for diagnosis. Stable-isotope breath tests hold great potential for rapidly diagnosing an infectious disease, monitoring therapy, and determining a bacterial phenotype in a rapid, point-of-care manner that does not require invasive sampling. Here we describe the preclinical development of a potentially highly selective TB diagnostic breath test based upon the organism's CO dehydrogenase activity. After development of the test in vitro, we were able to use the breath test to discriminate between infected and control rabbits, demonstrating that a diagnosis can potentially be made and also that a complex bacterial phenotype can be noninvasively and rapidly studied in the host. IMPORTANCE Tuberculosis (TB) remains a major infectious cause of disease and death worldwide, and effective diagnosis and then treatment are the tools with which we fight TB. The more quickly and more specific the diagnosis can be made, the better, and this is also true of diagnosis being as close to the patient (point of care) as possible. Here we report our preclinical development of breath tests based upon specific mycobacterial metabolism that could, with development, allow rapid point-of-care diagnosis through measuring the mycobacterial conversion of labeled CO to labeled CO2. PMID:24736224

Maiga, Mamoudou; Choi, Seong Won; Atudorei, Viorel; Maiga, Mariama C; Sharp, Zachary D; Bishai, William R; Timmins, Graham S

2014-01-01

232

Correlation Between Microscopic Examination and Culture for Detection and Differentiation of Mycobacterial Isolates from Cattle in the Sudan  

Directory of Open Access Journals (Sweden)

Full Text Available One hundred and sixty seven caseated lymph nodes and tuberculous lungs were collected from cattle slaughtered in various locations in Khartoum State (Sudan and examined bacteriologically. Microscopic examination using Zeilh-Neelsen stain and culture on Lowenstein-Jensen (L-J medium were carried out to detect and differentiate between the mycobacterial species in the specimens. Thirty-five, 35 (20.96% samples were found to harbor acid-fast bacteria when examined microscopically. Out of the 35 acid-fast bacteria, 22 (62.86% showed branching filaments and were identified as Mycobacterium farcinogenes. The remaining 13(37.14% were bacilli and identified as Mycobacterium bovis. The 35 specimens that proved to harbor acid fast bacteria were cultured on L-J medium. None of the 22 specimens with branching filaments (Mycobacterium farcinogenes grew on L-J medium when incubated aerobically at 37 ° C between four and eight weeks. 12 (92.31% of the 13 bacilli (Mycobacterium bovis showed visible growth using the above growth conditions. In conclusion, microscopic examination can only detect acid fast organisms in the clinical samples whereas culture on L-J medium can differentiate between acid-fast mycobacterial species.

S.H. Manal

2005-01-01

233

Deletion of the Mycobacterium tuberculosis pknH Gene Confers a Higher Bacillary Load during the Chronic Phase of Infection in BALB/c Mice‡  

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The role of the serine/threonine kinase PknH in the physiology and virulence of Mycobacterium tuberculosis was assessed by the construction of a pknH deletion mutant. Deletion of the pknH gene did not affect sensitivity to the antimycobacterial drug ethambutol, although it was previously thought to be involved in regulating expression of emb genes encoding arabinosyl transferases, the targets of ethambutol. Nevertheless, transcription analyses revealed that genes associated with mycobacterial...

Papavinasasundaram, K. G.; Chan, Bosco; Chung, Ji-hae; Colston, M. Joseph; Davis, Elaine O.; Av-gay, Yossef

2005-01-01

234

Trafficking of Superinfecting Mycobacterium into Established Granulomas Occurs in Mammals and is Independent of the Mycobacterial Erp and ESX-1 Virulence Loci  

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Whereas tuberculous granulomas, comprised of infected macrophages and other immune cells, were considered impermeable structures, recent studies showed that superinfecting Mycobacterium marinum traffic rapidly to established fish and frog granulomas by host-mediated and Mycobacterium-directed mechanisms. The present study shows that superinfecting Mycobacterium tuberculosis and Mycobacterium bovis BCG similarly home to established granulomas in mice. Furthermore, two prominent mycobacterial v...

Cosma, Christine L.; Humbert, Olivier; Sherman, David R.; Ramakrishnan, Lalita

2008-01-01

235

Use of Gene Dosage Effects for a Whole-Genome Screen To Identify Mycobacterium marinum Macrophage Infection Loci?  

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We recently identified two loci, mel1 and mel2, that affect macrophage infection by Mycobacterium marinum. The ability of these loci to confer enhanced infection in trans is presumably due to gene dosage effects since their presence on plasmids increases expression from five- to eightfold. Reasoning that this phenomenon would allow identification of other mycobacterial genes involved in macrophage infection, we conducted a screen of an M. marinum DNA library that provides 2.6-fold coverage of...

Park, Bonggoo; Subbian, Selvakumar; El-etr, Sahar H.; Cirillo, Suat L. G.; Cirillo, Jeffrey D.

2008-01-01

236

Regulation of the Mycobacterium tuberculosis PE/PPE genes.  

Science.gov (United States)

The genome of Mycobacterium tuberculosis encodes approximately 170 members of the unique mycobacterial PE and PPE gene families. Evidence suggests members of these families are surface-associated cell wall proteins that may provide a diverse antigenic profile and affect immunity. To determine if the expression patterns of PE/PPE genes are consistent with a role in antigenic variability, we analyzed microarray data from 132 experimental conditions for expression of PE/PPE genes. Whole genome expression patterns show that the PE/PPE genes are regulated in a variable and largely independent manner. Gene expression profiling of 15 unique conditions identified differential regulation of 128 of the 169 PE/PPE genes. Expression of the PE/PPE genes appears to be controlled by a variety of independent mechanisms. These data indicate that differential expression of the PE/PPE genes has the potential to provide a dynamic antigenic profile during the course of changing microenvironments within the host. PMID:15207495

Voskuil, M I; Schnappinger, D; Rutherford, R; Liu, Y; Schoolnik, G K

2004-01-01

237

Anti-mycobacterial treatment reduces high plasma levels of CXC-chemokines detected in active tuberculosis by cytometric bead array  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Chemokines recruit and activate leukocytes, assisting granuloma formation. Herein, we evaluated plasma chemokines in patients with active tuberculosis (ATB) and after completing treatment (TTB) and compared them to BCG-vaccinated healthy controls (HC). Levels of chemokines were measured by cytometri [...] c bead array. Levels of CXCL8, CXCL9 and CXCL10 were higher in ATB patients compared to HC, but they decreased in TTB. Levels of CCL2 and CCL5 in ATB patients were similar to those observed in HC. Thus, the high levels of CXC-chemokines detected during ATB, which can modulate the trafficking of immune cells from the periphery to the site of infection, were reversed by anti-mycobacterial treatment.

Almeida, Caroline de Souza; Abramo, Clarice; Alves, Caio César de Souza; Mazzoccoli, Luciano; Ferreira, Ana Paula; Teixeira, Henrique Couto.

238

Mycobacterial Phosphatidylinositol Mannoside 6 (PIM6) Up-Regulates TCR-Triggered HIV-1 Replication in CD4+ T Cells  

Science.gov (United States)

Tuberculosis (TB) is the leading cause of mortality among those infected with human immunodeficiency virus (HIV-1) worldwide. HIV-1 load and heterogeneity are increased both locally and systemically in active TB. Mycobacterium tuberculosis (MTB) infection supports HIV-1 replication through dysregulation of host cytokines, chemokines, and their receptors. However the possibility that mycobacterial molecules released from MTB infected macrophages directly interact with CD4+ T cells triggering HIV-1 replication has not been fully explored. We studied the direct effect of different MTB molecules on HIV-1 replication (R5-tropic strain Bal) in anti-CD3- stimulated CD4+ T cells from healthy donors in an antigen presenting cell (APC)-free system. PIM6, a major glycolipid of the mycobacterial cell wall, induced significant increases in the percent of HIV-1 infected T cells and the viral production in culture supernatants. In spite of structural relatedness, none of the other three major MTB cell wall glycolipids had significant impact on HIV-1 replication in T cells. Increased levels of IFN-? in culture supernatants from cells treated with PIM6 indicate that HIV-1 replication is likely dependent on enhanced T cell activation. In HEK293 cells transfected with TLR2, PIM6 was the strongest TLR2 agonist among the cell wall associated glycolipids tested. PIM6 increased the percentage of HIV infected cells and viral particles in the supernatant in a T-cell-based reporter cell line (JLTRg-R5) transfected with TLR1 and TLR2 but not in the cells transfected with the empty vector (which lack TLR2 expression) confirming that PIM6-induced HIV-1 replication depends at least partially on TLR2 signaling.

Rodriguez, Myriam E.; Loyd, Candace M.; Ding, Xuedong; Karim, Ahmad F.; McDonald, David J.; Canaday, David H.; Rojas, Roxana E.

2013-01-01

239

Direct contacts between conserved motifs of different subunits provide major contribution to active site organization in human and mycobacterial dUTPases.  

Science.gov (United States)

dUTP pyrophosphatases (dUTPases) are essential for genome integrity. Recent results allowed characterization of the role of conserved residues. Here we analyzed the Asp/Asn mutation within conserved Motif I of human and mycobacterial dUTPases, wherein the Asp residue was previously implicated in Mg(2+)-coordination. Our results on transient/steady-state kinetics, ligand binding and a 1.80 A resolution structure of the mutant mycobacterial enzyme, in comparison with wild type and C-terminally truncated structures, argue that this residue has a major role in providing intra- and intersubunit contacts, but is not essential for Mg(2+) accommodation. We conclude that in addition to the role of conserved motifs in substrate accommodation, direct subunit interaction between protein atoms of active site residues from different conserved motifs are crucial for enzyme function. PMID:20493855

Takács, Eniko; Nagy, Gergely; Leveles, Ibolya; Harmat, Veronika; Lopata, Anna; Tóth, Judit; Vértessy, Beáta G

2010-07-16

240

Rapid Identification of Mycobacterial Whole Cells in Solid and Liquid Culture Media by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry ?  

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Mycobacterial identification is based on several methods: conventional biochemical tests that require several weeks for accurate identification, and molecular tools that are now routinely used. However, these techniques are expensive and time-consuming. In this study, an alternative method was developed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). This approach allows a characteristic mass spectral fingerprint to be obtained from whole ina...

Lotz, Aure?lie; Ferroni, Agne?s; Beretti, Jean-luc; Dauphin, Brunhilde; Carbonnelle, Etienne; Guet-revillet, He?le?ne; Veziris, Nicolas; Heym, Be?ate; Jarlier, Vincent; Gaillard, Jean-louis; Pierre-audigier, Catherine; Frapy, Eric; Berche, Patrick; Nassif, Xavier; Bille, Emmanuelle

2010-01-01

 
 
 
 
241

Confirmation of the presence of Mycobacterium tuberculosis and other mycobacteria in mycobacterial growth indicator tubes (MGIT) by multiplex strand displacement amplification.  

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Multiplex strand displacement amplification (mSDA) is capable of amplifying three distinct DNA sequences simultaneously. These include sequences present in most genera of mycobacteria, a sequence specific for Mycobacterium tuberculosis, and an internal control. mSDA was used to detect the presence of these target sequences in 154 (72 positive, 76 negative, and 6 failed) clinical specimens cultured in the mycobacterial growth indicator tube (MGIT) system. A wide variety of specimen types were ...

Badak, F. Z.; Kiska, D. L.; O Connell, M.; Nycz, C. M.; Hartley, C.; Setterquist, S.; Hopfer, R. L.

1997-01-01

242

A recombinant 64 kilodalton protein of Mycobacterium bovis bacillus Calmette-Guerin specifically stimulates human T4 clones reactive to mycobacterial antigens  

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A recombinant 64 kD protein of Mycobacterium bovis bacillus Calmette- Guerin (BCG) (antigen A), which amounted to approximately 2% of an E. coli lysate, was tested for its capacity to stimulate human T4 clones reactive to mycobacterial proteins. Two out of four crossreactive clones, established from a patient with tuberculoid leprosy, which could be stimulated by protein preparations of M. leprae and M. tuberculosis, and by particulate M. bovis BCG were also reactive to antigen A without furt...

1986-01-01

243

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India.  

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The aim of the present study was to compare polymerase chain reaction (PCR)-based methods--spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing--with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotyp...

Varma-basil, Mandira; Kumar, Sujeet; Arora, Jyoti; Angrup, Archana; Zozio, Thierry; Banavaliker, Jayant Nagesh; Singh, Urvashi Balbir; Rastogi, Nalin; Bose, Mridula

2011-01-01

244

Rapid Mycobacterial Liquid Culture-Screening Method for Mycobacterium avium Complex Based on Secreted Antigen-Capture Enzyme-Linked Immunosorbent Assay?  

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Sensors in automated liquid culture systems for mycobacteria, such as MGIT, BacT/Alert 3D, and Trek ESP II, flag growth of any type of bacteria; a positive signal does not mean that the target mycobacteria are present. All signal-positive cultures thus require additional and often laborious testing. An immunoassay was developed to screen liquid mycobacterial cultures for evidence of Mycobacterium avium complex (MAC). The method, called the MAC-enzyme-linked immunosorbent assay (ELISA), relies...

2009-01-01

245

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods - spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing - with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligot...

2011-01-01

246

Association between caudal fold tuberculin test responses and results of an ELISA for Mycobacterium avium subsp paratuberculosis and mycobacterial culture of feces in tuberculosis-free dairy herds.  

Science.gov (United States)

OBJECTIVE--To evaluate associations between Mycobacterium avium subsp paratuberculosis (MAP) and caudal fold tuberculin (CFT) test results in cattle. DESIGN--Longitudinal and cross-sectional evaluations. ANIMALS--1 California (approx 3,600 cows) and 3 Colorado (approx 640, 1,190, and 1,480 cows) dairy herds considered free of Mycobacterium bovis infection. PROCEDURES--In the California herd, the association between CFT response and MAP status was determined with ELISA and mycobacterial culture of feces within 1 year before and after CFT testing. The association between CFT and MAP status in all herds was modeled with mixed-effects logistic regression. RESULTS--In the California herd, significantly higher odds of being classified as suspect by CFT were found for cows with results of MAP ELISA negative before and positive after CFT testing (OR, 5.6) and cows positive before and after CFT testing (OR, 8.1). Higher odds were found for cows positive for mycobacterial culture of feces before and negative for culture after CFT testing (OR, 4.6) and cows negative for mycobacterial culture of feces before and positive for culture after CFT testing (OR, 13.2). All herds had higher odds of being classified as suspect by CFT testing for cows with positive results for ELISA (OR, 2.9) or mycobacterial culture of feces (OR, 5.0), compared with cows with negative results of the same tests. CONCLUSIONS AND CLINICAL RELEVANCE--A strong association was found between positive MAP test results and being classified as a suspect by CFT testing. Within-herd MAP prevalence may affect specificity of CFT testing for tuberculosis in cattle. PMID:24548233

Brito, Barbara P; Aly, Sharif S; Anderson, Randall J; Fossler, Charles P; Garry, Franklyn B; Gardner, Ian A

2014-03-01

247

Can 15-Locus Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Analysis Provide Insight into the Evolution of Mycobacterium tuberculosis?  

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The phylogeny and evolution of the bacterium Mycobacterium tuberculosis is still poorly understood despite the application of a variety of molecular techniques. We analyzed 469 M. tuberculosis and 49 Mycobacterium bovis isolates to evaluate if the mycobacterial interspersed repetitive units-variable-number tandem repeats (MIRU-VNTR) commonly used for epidemiological studies can define the phylogeny of the M. tuberculosis complex. This population was characterized by previously identified sile...

Gibson, Andrea; Brown, Timothy; Baker, Lucy; Drobniewski, Francis

2005-01-01

248

Expression and Immunogenicity of the Mycobacterial Ag85B/ESAT-6 Antigens Produced in Transgenic Plants by Elastin-Like Peptide Fusion Strategy  

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This study explored a novel system combining plant-based production and the elastin-like peptide (ELP) fusion strategy to produce vaccinal antigens against tuberculosis. Transgenic tobacco plants expressing the mycobacterial antigens Ag85B and ESAT-6 fused to ELP (TBAg-ELP) were generated. Purified TBAg-ELP was obtained by the highly efficient, cost-effective, inverse transition cycling (ICT) method and tested in mice. Furthermore, safety and immunogenicity of the crude tobacco leaf extracts ...

Doreen Manuela Floss; Michael Mockey; Galliano Zanello; Damien Brosson; Marie Diogon; Roger Frutos; Bruel, Timoth E.; Valérie Rodrigues; Edwin Garzon; Claire Chevaleyre; Mustapha Berri; Henri Salmon; Udo Conrad; Laurence Dedieu

2010-01-01

249

Transcriptional profiling of mycobacterial antigen-induced responses in infants vaccinated with BCG at birth  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Novel tuberculosis (TB vaccines recently tested in humans have been designed to boost immunity induced by the current vaccine, Mycobacterium bovis Bacille Calmette-Guérin (BCG. Because BCG vaccination is used extensively in infants, this population group is likely to be the first in which efficacy trials of new vaccines will be conducted. However, our understanding of the complexity of immunity to BCG in infants is inadequate, making interpretation of vaccine-induced immune responses difficult. Methods To better understand BCG-induced immunity, we performed gene expression profiling in five 10-week old infants routinely vaccinated with BCG at birth. RNA was extracted from 12 hour BCG-stimulated or purified protein derivative of tuberculin (PPD-stimulated PBMC, isolated from neonatal blood collected 10 weeks after vaccination. RNA was hybridised to the Sentrix® HumanRef-8 Expression BeadChip (Illumina to measure expression of >16,000 genes. Results We found that ex vivo stimulation of PBMC with PPD and BCG induced largely similar gene expression profiles, except that BCG induced greater macrophage activation. The peroxisome proliferator-activated receptor (PPAR signaling pathway, including PPAR-?, involved in activation of the alternative, anti-inflammatory macrophage response was down-regulated following stimulation with both antigens. In contrast, up-regulation of genes associated with the classic, pro-inflammatory macrophage response was noted. Further analysis revealed a decrease in the expression of cell adhesion molecules (CAMs, including integrin alpha M (ITGAM, which is known to be important for entry of mycobacteria into the macrophage. Interestingly, more leukocyte genes were down-regulated than up-regulated. Conclusion Our results suggest that a combination of suppressed and up-regulated genes may be key in determining development of protective immunity to TB induced by vaccination with BCG.

Hill Adrian VS

2009-02-01

250

Characterization of the katG and inhA genes of isoniazid-resistant clinical isolates of Mycobacterium tuberculosis.  

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Resistance to isoniazid in Mycobacterium tuberculosis has been associated with mutations in genes encoding the mycobacterial catalase-peroxidase (katG) and the InhA protein (inhA). Among the 26 isoniazid-resistant clinical isolates evaluated in this study, mutations in putative inhA regulatory sequences were identified in 2 catalase-positive isolates, katG gene alterations were detected in 20 strains, and 4 isolates had wild-type katG and inhA genes. Mutations in the katG gene were detected i...

Rouse, D. A.; Li, Z.; Bai, G. H.; Morris, S. L.

1995-01-01

251

Determination of antigenic binding sites in antibodies directed against a mycobacterial peptide by deletion and substitution of single amino acids.  

Science.gov (United States)

A component of Mycobacterium bovis (BCG) was previously isolated using a monoclonal antibody to BCG and affinity chromatography. It was designated BCG-a. The sequence of N-terminal residues of BCG-a was used to synthesize a 13 amino acid peptide designated BCG-a-P which elicited immunologic responses. The present study was undertaken to identify which amino acid residues were critical for the immunologic characteristics of BCG-a-P. By virtue of analogues synthesized with either amino acid deletions or substitutions along the BCG-a-P sequence, it was possible to identify the regions of the peptide which were responsible for the recognition and binding by antibodies directed to BCG-a-P. In both direct and competition ELISA systems, the deletion of single amino acids caused a change in the binding of BCG-a-P by an antiserum directed to BCG-a-P. Similar results were evident when the amino acid phenylalanine was substituted for individual residues along the sequence of BCG-a-P. The residues responsible for antibody binding had the highest localized hydrophilic index of any hexapeptide stretch of the BCG-a-P sequence. These findings indicate that the activity expressed by BCG-a-P was due to a defined region of the peptide. The techniques used here may have applications in identifying the regions within other mycobacterial antigens which are involved in antibody binding. PMID:2520760

Voegtline, M S; Houghten, R A; Minden, P

1989-01-01

252

A dual conformation of the post-decarboxylation intermediate is associated with distinct enzyme states in mycobacterial KGD (?-ketoglutarate decarboxylase).  

Science.gov (United States)

?-Ketoacid dehydrogenases are large multi-enzyme machineries that orchestrate the oxidative decarboxylation of ?-ketoacids with the concomitant production of acyl-CoA and NADH. The first reaction, catalysed by ?-ketoacid decarboxylases (E1 enzymes), needs a thiamine diphosphate cofactor and represents the overall rate-limiting step. Although the catalytic cycles of E1 from the pyruvate dehydrogenase (E1p) and branched-chain ?-ketoacid dehydrogenase (E1b) complexes have been elucidated, little structural information is available on E1o, the first component of the ?-ketoglutarate dehydrogenase complex, despite the central role of this complex at the branching point between the TCA (tricarboxylic acid) cycle and glutamate metabolism. In the present study, we provide structural evidence that MsKGD, the E1o (?-ketoglutarate decarboxylase) from Mycobacterium smegmatis, shows two conformations of the post-decarboxylation intermediate, each one associated with a distinct enzyme state. We also provide an overall picture of the catalytic cycle, reconstructed by either crystallographic snapshots or modelling. The results of the present study show that the conformational change leading the enzyme from the initial (early) to the late state, although not required for decarboxylation, plays an essential role in catalysis and possibly in the regulation of mycobacterial E1o. PMID:24171907

Wagner, Tristan; Barilone, Nathalie; Alzari, Pedro M; Bellinzoni, Marco

2014-02-01

253

Progressing features of atypical mycobacterial infection in the lung on conventional and high resolution CT (HRCT) images  

International Nuclear Information System (INIS)

The aim of this study was to clarify the localization of abnormalities within secondary pulmonary lobules and the changes in follow-up studies of pulmonary atypical mycobacterial infection (AMI) by conventional and high-resolution computed tomography (HRCT). Forty-six patients (16 men and 30 women; 43-84 years) with pulmonary AMI (M. intracellulare 36; M. avium 10) in the lung were examined by conventional and HRCT. In peripheral zones, all patients had the nodule located in the terminal or lobular bronchiole, and most of the patients also had nodules accompanied with a wedge-shaped or linear shadow connected with the pleura. In the follow-up scans, new centrilobular nodules appeared in other segments, and consolidation or ground-glass pattern appeared newly and was preceded by nodules. Bronchiectasis became more severe in five of 38 follow-up patients. The common HRCT findings of AMI were centrilobular, peribronchovascular nodules, bronchiectasis, consolidation, and pleural thickening/adhesion. The nodules frequently connected with the pleura. The initial and follow-up studies suggest that the disease may begin in the terminal bronchiole or as preexisting bronchiectasis and spread transbronchially along the draining bronchus or towards the pleura to produce lesions such as new nodules, cavities, consolidation, pleuritis, and bronchiectasis, or more severe bronchiectasis. (author)

2001-01-01

254

Progressing features of atypical mycobacterial infection in the lung on conventional and high resolution CT (HRCT) images  

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The aim of this study was to clarify the localization of abnormalities within secondary pulmonary lobules and the changes in follow-up studies of pulmonary atypical mycobacterial infection (AMI) by conventional and high-resolution computed tomography (HRCT). Forty-six patients (16 men and 30 women; 43-84 years) with pulmonary AMI (M. intracellulare 36; M. avium 10) in the lung were examined by conventional and HRCT. In peripheral zones, all patients had the nodule located in the terminal or lobular bronchiole, and most of the patients also had nodules accompanied with a wedge-shaped or linear shadow connected with the pleura. In the follow-up scans, new centrilobular nodules appeared in other segments, and consolidation or ground-glass pattern appeared newly and was preceded by nodules. Bronchiectasis became more severe in five of 38 follow-up patients. The common HRCT findings of AMI were centrilobular, peribronchovascular nodules, bronchiectasis, consolidation, and pleural thickening/adhesion. The nodules frequently connected with the pleura. The initial and follow-up studies suggest that the disease may begin in the terminal bronchiole or as preexisting bronchiectasis and spread transbronchially along the draining bronchus or towards the pleura to produce lesions such as new nodules, cavities, consolidation, pleuritis, and bronchiectasis, or more severe bronchiectasis. (author)

Tanaka, Daizo; Niwatsukino, Hiroshi; Nakajo, Masayuki [Kagoshima Univ. (Japan). Faculty of Medicine; Oyama, Takao

2001-10-01

255

Mycobacterial and nonbacterial pulmonary complications in hospitalized patients with human immunodeficiency virus infection: A prospective, cohort study  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background A prospective observational study was done to describe nonbacterial pulmonary complications in hospitalized patients with human immunodeficiency virus (HIV infection. Methods The study included 1,225 consecutive hospital admissions of 599 HIV-infected patients treated from April 1995 through March 1998. Data included demographics, risk factors for HIV infection, Acute Physiology and Chronic Health Evaluation (APACHE II score, pulmonary complications, CD4+ lymphocyte count, hospital stay and case-fatality rate. Results Patient age (mean ± SD was 38.2 ± 8.9 years, 62% were men, and 84% were African American. The median APACHE II score was 14, and median CD4+ lymphocyte count was 60/?L. Pulmonary complications were Pneumocystis carinii pneumonia (85 in 78 patients, Mycobacterium avium complex (51 in 38, Mycobacterium tuberculosis (40 in 35, Mycobacterium gordonae (11 in 11, Mycobacterium kansasii (10 in 9, Cytomegalovirus (10 in 10, Nocardia asteroides (3 in 3, fungus ball (2 in 2, respiratory syncytial virus (1, herpes simplex virus (1, Histoplasma capsulatum (1, lymphoma (3 in 3, bronchogenic carcinoma (2 in 2, and Kaposi sarcoma (1. The case-fatality rate of patients was 11% with Pneumocystis carinii pneumonia; 5%, Mycobacterium tuberculosis; 6%, Mycobacterium avium complex; and 7%, noninfectious pulmonary complications. Conclusion Most pulmonary complications in hospitalized patients with HIV are from Pneumocystis and mycobacterial infection.

Afessa Bekele

2001-09-01

256

Interaction of the mycobacterial heparin-binding hemagglutinin with actin, as evidenced by single-molecule force spectroscopy.  

Science.gov (United States)

Although Mycobacterium tuberculosis and related species are considered to be typical endosomal pathogens, recent studies have suggested that mycobacteria can be present in the cytoplasm of infected cells and cause cytoskeleton rearrangements, the mechanisms of which remain unknown. Here, we used single-molecule force spectroscopy to demonstrate that the heparin-binding hemagglutinin (HBHA), a surface adhesin from Mycobacterium tuberculosis displaying sequence similarities with actin-binding proteins, is able to bind to actin. Force curves recorded between actin and the coiled-coil, N-terminal domain of HBHA showed a bimodal distribution of binding forces reflecting the detection of single and double HBHA-actin interactions. Force curves obtained between actin and the lysine-rich C-terminal domain of HBHA showed a broader distribution of binding events, suggesting they originate primarily from intermolecular electrostatic bridges between cationic HBHA domains and anionic actin residues. We also explored the dynamics of the HBHA-actin interaction, showing that the binding force and binding frequency increased with the pulling speed and contact time, respectively. Taken together, our data indicate that HBHA is able to specifically bind actin, via both its N-terminal and C-terminal domains, strongly suggesting a role of the HBHA-actin interaction in the pathogenesis of mycobacterial diseases. PMID:18835984

Verbelen, Claire; Dupres, Vincent; Raze, Dominique; Bompard, Coralie; Locht, Camille; Dufrêne, Yves F

2008-12-01

257

The interplay of multiple feedback loops with post-translational kinetics results in bistability of mycobacterial stress response  

Science.gov (United States)

Bacterial persistence is the phenomenon in which a genetically identical fraction of a bacterial population can survive exposure to stress by reduction or cessation of growth. Persistence in mycobacteria has been recently linked to a stress-response network, consisting of the MprA/MprB two-component system and alternative sigma factor ?E. This network contains multiple positive transcriptional feedback loops which may give rise to bistability, making it a good candidate for controlling the mycobacterial persistence switch. To analyze the possibility of bistability, we develop a method that involves decoupling of the network into transcriptional and post-translational interaction modules. As a result we reduce the dimensionality of the dynamical system and independently analyze input-output relations in the two modules to formulate a necessary condition for bistability in terms of their logarithmic gains. We show that neither the positive autoregulation in the MprA/MprB network nor the ?E-mediated transcriptional feedback is sufficient to induce bistability in a biochemically realistic parameter range. Nonetheless, inclusion of the post-translational regulation of ?E by RseA increases the effective cooperativity of the system, resulting in bistability that is robust to parameter variation. We predict that overexpression or deletion of RseA, the key element controlling the ultrasensitive response, can eliminate bistability.

Tiwari, Abhinav; Balázsi, Gábor; Gennaro, Maria Laura; Igoshin, Oleg A.

2010-09-01

258

Conserved mycobacterial lipoglycoproteins activate TLR2 but also require glycosylation for MHC class II-restricted T cell activation.  

Science.gov (United States)

CD4(+) T cell clones derived from a leprosy lesion and patient blood were used to monitor the isolation and identification of an Ag associated with the self-limited form of the disease. Biochemical purification and genetic analysis identified the T cell Ag as a conserved mycobacterial lipoglycoprotein LprG. LprG-mediated activation of CD4(+) T cells required specific MHC class II restriction molecules and intracellular processing. Although LprG activated TLR2, this alone was not sufficient to stimulate or inhibit T cell activation. A striking finding was that the carbohydrate moieties of LprG were required for optimal T cell activation, because recombinant LprG produced in Escherichia coli, or recombinant LprG produced in Mycobacterium smegmatis and digested by alpha-mannosidase, did not activate T cells. This study demonstrates that the universe of bacterial T cell Ags includes lipoglycoproteins, which act as TLR2 ligands but also require glycosylation for MHC class II-restricted T cell activation in vivo. PMID:18424702

Sieling, Peter A; Hill, Preston J; Dobos, Karen M; Brookman, Kerry; Kuhlman, Andrew M; Fabri, Mario; Krutzik, Stephan R; Rea, Thomas H; Heaslip, Darragh G; Belisle, John T; Modlin, Robert L

2008-05-01

259

Conserved mycobacterial lipoglycoproteins activate TLR2 but also require glycosylation for MHC class II-restricted T cell activation1  

Science.gov (United States)

CD4+ T cell clones derived from a leprosy lesion and patient blood were used to monitor the isolation and identification of an antigen associated with the self-limited form of the disease. Biochemical purification and genetic analysis identified the T cell antigen as a conserved mycobacterial lipoglycoprotein LprG. LprG-mediated activation of CD4+T cells required specific MHC class II restriction molecules and intracellular processing. Although LprG activated TLR2, this alone was not sufficient to stimulate nor did it inhibit T cell activation. A striking finding was that the carbohydrate moieties of LprG were required for optimal T cell activation, since recombinant LprG produced in Escherichia coli, or recombinant LprG produced in Mycobacterium smegmatis and digested by ?-mannosidase did not activate T cells. This study demonstrates that the universe of bacterial T cell antigens includes lipoglycoproteins, which act as TLR2 ligands but also require glycosylation for MHC class II-restricted T cell activation in vivo.

Sieling, Peter A.; Hill, Preston J.; Dobos, Karen M.; Brookman, Kerry; Kuhlman, Andrew M.; Fabri, Mario; Krutzik, Stephan R.; Rea, Thomas H.; Heaslip, Darragh G.; Belisle, John T; Modlin, Robert L.

2009-01-01

260

rpoB Gene Mutations and Molecular Characterization of Rifampin-Resistant Mycobacterium tuberculosis Isolates from Shandong Province, China  

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Sixty rifampin (RIF)-resistant and 75 RIF-susceptible Mycobacterium tuberculosis isolates from Shandong Province, China, were analyzed for rpoB gene mutations and genotyped. Mycobacterial interspersed repetitive unit (MIRU) genotype 223325173533 was overrepresented among RIF-resistant isolates. MIRU combined with IS6110 restriction fragment length polymorphism analysis as the second-line genotyping method may reflect epidemiologic links more reliably than each method alone.

Ma, Xin; Wang, Haiying; Deng, Yunfeng; Liu, Zhimin; Xu, Yong; Pan, Xi; Musser, James M.; Graviss, Edward A.

2006-01-01

 
 
 
 
261

Tetracycline-inducible gene regulation in mycobacteria  

Science.gov (United States)

A system for the tetracycline-inducible regulation of gene expression in mycobacteria has been developed. We have sub-cloned the tetRO region from the Corynebacterium glutamicum TetZ locus into a mycobacterial shuttle plasmid, making expression of genes cloned downstream of tetRO responsive to tetracycline. Using the luxAB-encoded luciferase from Vibrio harveyi as a reporter (pMind-Lx), we observed a 40-fold increase in light output from Mycobacterium smegmatis cultures 2 h after adding 20 ng ml?1 of tetracycline. Similarly, exposure to the drug resulted in up to 20-fold increase in relative light units from M.bovis BCG carrying the reporter construct, and a 10-fold increase for M.tuberculosis. Tetracycline induction was demonstrated in log and stationary phase cultures. To evaluate whether this system is amenable to use in vivo, J774 macrophages were infected with M.bovis BCG[pMind-Lx], treated with amikacin to kill extracellular bacteria, and then incubated with tetracycline. A 10-fold increase in light output was measured after 24 h, indicating that intracellular bacteria are accessible and responsive to exogenously added tetracycline. To test the use of the tetracycline-inducible system for conditional gene silencing, mycobacteria were transformed with a pMind construct with tetRO driving expression of antisense RNA for the ftsZ gene. Bacterial cells containing the antisense construct formed filaments after 24 h exposure to tetracycline. These results demonstrate the potential of this tetracycline-regulated system for the manipulation of mycobacterial gene expression inside and outside cells.

Blokpoel, Marian C. J.; Murphy, Helen N.; O'Toole, Ronan; Wiles, Siouxsie; Runn, Ellen S. C.; Stewart, Graham R.; Young, Douglas B.; Robertson, Brian D.

2005-01-01

262

Phylogenetic detection of horizontal gene transfer during the step-wise genesis of Mycobacterium tuberculosis  

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Full Text Available Abstract Background In the past decade, the availability of complete genome sequence data has greatly facilitated comparative genomic research aimed at addressing genetic variability within species. More recently, analysis across species has become feasible, especially in genera where genome sequencing projects of multiple species have been initiated. To understand the genesis of the pathogen Mycobacterium tuberculosis within a genus where the majority of species are harmless environmental organisms, we have used genome sequence data from 16 mycobacteria to look for evidence of horizontal gene transfer (HGT associated with the emergence of pathogenesis. First, using multi-locus sequence analysis (MLSA of 20 housekeeping genes across these species, we derived a phylogeny that serves as the basis for HGT assignments. Next, we performed alignment searches for the 3989 proteins of M. tuberculosis H37Rv against 15 other mycobacterial genomes, generating a matrix of 59835 comparisons, to look for genetic elements that were uniquely found in M. tuberculosis and closely-related pathogenic mycobacteria. To assign when foreign genes were likely acquired, we designed a bioinformatic program called mycoHIT (mycobacterial homologue investigation tool to analyze these data in conjunction with the MLSA-based phylogeny. Results The bioinformatic screen predicted that 137 genes had been acquired by HGT at different phylogenetic strata; these included genes coding for metabolic functions and modification of mycobacterial lipids. For the majority of these genes, corroborating evidence of HGT was obtained, such as presence of phage or plasmid, and an aberrant GC%. Conclusion M. tuberculosis emerged through vertical inheritance along with the step-wise addition of genes acquired via HGT events, a process that may more generally describe the evolution of other pathogens.

Turenne Christine

2009-08-01

263

Mycobacterium bourgelatii sp. nov., a rapidly growing, non-chromogenic species isolated from the lymph nodes of cattle.  

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Three independent strains of a rapidly growing, non-chromogenic member of the genus Mycobacterium were isolated from lymph nodes of French cattle. Identification of the isolates was carried out using a polyphasic approach. The nearly complete SSU rRNA gene sequences (>1200 bp) of the strains MLB-A23, MLB-A30 and MLB-A84(T) were identical. A phylogenetic analysis of these unique SSU rRNA gene sequences showed that these strains were most closely related to Mycobacterium intermedium. Further phylogenetic analysis based on concatenated sequences (2854 bp) of four housekeeping genes (hsp65, rpoB, sodA and tuf), the transfer-messenger RNA (tmRNA) and SSU rRNA genes indicated that these three strains represented a distinct species that shares a common ancestor with M. intermedium. Phylogenetic and phenotypic data strongly indicate that the strains MLB-A23, MLB-A30 and MLB-A84(T) belong to a novel mycobacterial species for which the name Mycobacterium bourgelatii sp. nov. is proposed. The type strain is MLB-A84(T) (?=?CIP 110557(T)?=?DSM 45746(T)). PMID:23990648

Guérin-Faublée, Véronique; Flandrois, Jean-Pierre; Pichat, Catherine; Boschiroli, Maria Laura; Lamy, Brigitte

2013-12-01

264

Tomography high Resolution CT findings of nontuberculous mycobacterial pulmonary disease: Comparison between the first treatment and the re treatment group  

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To analyze and compare the thin section CT findings of first and re treatment nontuberculous mycobacterial (NTM) pulmonary disease. Between January 2005 and April 2010, 121 patients with positive sputum culture for NTM were recruited. We included only 32 patients underwent high resolution chest CT and were confirmed by American Thoracic Society criteria NTM pulmonary infection (first treatment 15, re treatment 17 patients). CT images of 32 patients were reviewed retrospectively. We evaluated the frequency and laterality of the followings; nodule, increased density, bronchial change, parenchymal change. The significantly frequent CT findings of the re treatment NTM group were well defined nodules (retreatment 82.4%, first treatment 33.3%, p = 0.00), consolidations (retreatment 88.2%, first treatment 53.3%, p = 0.03), bronchial changes (bronchiectasis; retreatment 100%, first treatment 66.6%, p = 0.01, bronchial narrowing; retreatment 23.5%, first treatment 0%, p = 0.04 and mucoid impaction; retreatment-58.8%, first treatment-20.0%, p = 0.03) and atelectasis with bronchiectasis (retreatment-88.2%, first treatment 26.7%, p = 0.00). However, most of the evaluated thin section CT findings, such as centrilobular and ill defined nodules, lobular, segmental and subpleural consolidations, ground glass attenuation, bronchial wall thickening, cavities, pleural lesions, fibrotic band, emphysema and laterality of lesions, have not shown significant differences between first treatment and the re treatment group. Thin section CT findings of well defined nodules, consolidations, bronchial changes (bronchiectasis, bronchial narrowing and mucoid impaction) and atelectasis with bronchiectasis are highly suggestive of re treatment NTM pulmonary disease

2012-06-01

265

Tomography high Resolution CT findings of nontuberculous mycobacterial pulmonary disease: Comparison between the first treatment and the re treatment group  

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To analyze and compare the thin section CT findings of first and re treatment nontuberculous mycobacterial (NTM) pulmonary disease. Between January 2005 and April 2010, 121 patients with positive sputum culture for NTM were recruited. We included only 32 patients underwent high resolution chest CT and were confirmed by American Thoracic Society criteria NTM pulmonary infection (first treatment 15, re treatment 17 patients). CT images of 32 patients were reviewed retrospectively. We evaluated the frequency and laterality of the followings; nodule, increased density, bronchial change, parenchymal change. The significantly frequent CT findings of the re treatment NTM group were well defined nodules (retreatment 82.4%, first treatment 33.3%, p = 0.00), consolidations (retreatment 88.2%, first treatment 53.3%, p = 0.03), bronchial changes (bronchiectasis; retreatment 100%, first treatment 66.6%, p = 0.01, bronchial narrowing; retreatment 23.5%, first treatment 0%, p = 0.04 and mucoid impaction; retreatment-58.8%, first treatment-20.0%, p = 0.03) and atelectasis with bronchiectasis (retreatment-88.2%, first treatment 26.7%, p = 0.00). However, most of the evaluated thin section CT findings, such as centrilobular and ill defined nodules, lobular, segmental and subpleural consolidations, ground glass attenuation, bronchial wall thickening, cavities, pleural lesions, fibrotic band, emphysema and laterality of lesions, have not shown significant differences between first treatment and the re treatment group. Thin section CT findings of well defined nodules, consolidations, bronchial changes (bronchiectasis, bronchial narrowing and mucoid impaction) and atelectasis with bronchiectasis are highly suggestive of re treatment NTM pulmonary disease.

Gwak, Soon Hyuk; Cho, Bum Sang; Jeon, Min Hee; Kim, Eun Young; Kang, Min Ho; Yi, Kyung Sik; Lee, Seung Young; Kim, Sung Jin; Lee, Ki Man [Chungbuk National Univ., Cheongju, (Korea, Republic of)

2012-06-15

266

CD3+ cells transfer the hypersensitive granulomatous response to mycobacterial glycolipid trehalose 6,6'-dimycolate in mice.  

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The granulomatous response is the characteristic histological feature of Mycobacterium tuberculosis infection that is essential for organism containment. Trehalose 6,6-dimycolate (TDM), a cell-wall glycolipid present on most mycobacterial species, has been implicated in the pathogenesis of M. tuberculosis infection. TDM has potent immunoregulatory and inflammatory properties, and can be used to model granulomatous reactions that mimic, in part, pathology caused during active infection. This study examined the hypersensitive granulomatous response, focusing on cellular responses specific to TDM. Lungs from mice immunized with TDM emulsion demonstrated exacerbated histological damage, inflammation, and lymphocytic infiltration upon subsequent challenge with TDM. Splenocytes recovered from these mice demonstrated significant interferon (IFN)-gamma production during recall response to TDM, as well as increased production of proinflammatory mediators (tumour necrosis factor-alpha, interleukin-6 and macrophage inflammatory protein-1alpha). The exacerbated response could be adoptively transferred to naïve mice. Administration of non-adherent lymphocytes or purified CD3(+) cells from TDM-immunized mice led to increased inflammation, lymphocytic infiltration, and vascular endothelial cell damage upon challenge with TDM. Recipient mice that received immunized CD3(+) lymphocytes demonstrated significant increases in Th1-type cytokines and proinflammatory mediators in lung tissue following TDM challenge. When CD1d(-/-) mice were immunized with TDM, they failed to generate a specific IFN-gamma response, suggesting a role for this molecule in the generation of hypersensitivity. These experiments provide further evidence for the involvement of TDM-specific CD3(+) T cells in pathological damage elicited during M. tuberculosis infection. PMID:17159227

Guidry, Tera V; Hunter, Robert L; Actor, Jeffrey K

2006-12-01

267

Does Neutralization of Gastric Aspirates from Children with Suspected Intrathoracic Tuberculosis Affect Mycobacterial Yields on MGIT Culture?  

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The microbiological confirmation of pulmonary tuberculosis in children relies on cultures of gastric aspirate (GA) specimens. Conventionally, GAs are neutralized to improve culture yields of mycobacteria. However, there are limited data to support this practice. To study the utility of neutralization of GAs with sodium bicarbonate in children with intrathoracic tuberculosis, a total of 116 children of either sex, aged 6 months to 14 years (median age, 120 months; interquartile range [IQR], 7 to 192 months), underwent gastric aspiration on 2 consecutive days. Gastric aspirates were divided into two aliquots, and only one aliquot was neutralized with 1% sodium bicarbonate. Both aliquots were processed for smear and culture examinations. Out of the 232 gastric aspirates, 12 (5.17%) were acid-fast bacilli (AFB) smear positive. There were no differences in smear positivity rates from samples with or without neutralization. The yield of Mycobacterium tuberculosis on a Bactec MGIT 960 culture system was significantly lower in the neutralized samples (16.3% [38/232]) than in the nonneutralized samples (21.5% [50/232]) (P = 0.023). There was no significant difference between the neutralized and the nonneutralized samples in time to detection using the MGIT 960 system (average, 24.6 days; IQR, 12 to 37 days) (P = 0.9). The contamination rates were significantly higher in the neutralized samples than in the nonneutralized samples (17.2% [40/232] versus 3.9% [9/232]) (P = 0.001). The agreement for positive mycobacterial culture between the two approaches was 66.5% (P = 0.001). Hence, we recommend that gastric aspirate samples not be neutralized with sodium bicarbonate prior to culture for M. tuberculosis.

Parashar, Deepak; Kabra, Sushil K.; Lodha, Rakesh; Singh, Varinder; Mukherjee, Aparna; Arya, Tina; Grewal, Harleen M. S.

2013-01-01

268

Does neutralization of gastric aspirates from children with suspected intrathoracic tuberculosis affect mycobacterial yields on MGIT culture?  

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The microbiological confirmation of pulmonary tuberculosis in children relies on cultures of gastric aspirate (GA) specimens. Conventionally, GAs are neutralized to improve culture yields of mycobacteria. However, there are limited data to support this practice. To study the utility of neutralization of GAs with sodium bicarbonate in children with intrathoracic tuberculosis, a total of 116 children of either sex, aged 6 months to 14 years (median age, 120 months; interquartile range [IQR], 7 to 192 months), underwent gastric aspiration on 2 consecutive days. Gastric aspirates were divided into two aliquots, and only one aliquot was neutralized with 1% sodium bicarbonate. Both aliquots were processed for smear and culture examinations. Out of the 232 gastric aspirates, 12 (5.17%) were acid-fast bacilli (AFB) smear positive. There were no differences in smear positivity rates from samples with or without neutralization. The yield of Mycobacterium tuberculosis on a Bactec MGIT 960 culture system was significantly lower in the neutralized samples (16.3% [38/232]) than in the nonneutralized samples (21.5% [50/232]) (P = 0.023). There was no significant difference between the neutralized and the nonneutralized samples in time to detection using the MGIT 960 system (average, 24.6 days; IQR, 12 to 37 days) (P = 0.9). The contamination rates were significantly higher in the neutralized samples than in the nonneutralized samples (17.2% [40/232] versus 3.9% [9/232]) (P = 0.001). The agreement for positive mycobacterial culture between the two approaches was 66.5% (P = 0.001). Hence, we recommend that gastric aspirate samples not be neutralized with sodium bicarbonate prior to culture for M. tuberculosis. PMID:23536406

Parashar, Deepak; Kabra, Sushil K; Lodha, Rakesh; Singh, Varinder; Mukherjee, Aparna; Arya, Tina; Grewal, Harleen M S; Singh, Sarman

2013-06-01

269

The antituberculous Mycobacterium bovis BCG vaccine is an attenuated mycobacterial producer of phosphorylated nonpeptidic antigens for human gamma delta T cells.  

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The mycobacterial antigens stimulating human gamma delta T lymphocytes (R. L. Modlin, C. Permitz, F. M. Hofman, V. Torigian, K. Uemura, T. H. Rea, B. R. Bloom, and M. B. Brenner, Nature (London) 339:544-548, 1989; D. H. Raulet, Annu. Rev. Immunol. 7:175-207, 1989) have been characterized recently in Mycobacterium tuberculosis H37Rv as a group of four structurally related nucleotidic or phosphorylated molecules, termed TUBag1 to -4 (tuberculous antigens 1 to 4) (P. Constant, F. Davodeau, M. A....

Constant, P.; Poquet, Y.; Peyrat, M. A.; Davodeau, F.; Bonneville, M.; Fournie?, J. J.

1995-01-01

270

Expression, essentiality, and a microtiter plate assay for mycobacterial GlmU, the bifunctional glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase  

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UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor of peptidoglycan and the rhamnose-GlcNAc linker region of mycobacterial cell wall. In Mycobacterium tuberculosis H37Rv genome, Rv1018c shows strong homology to the GlmU protein involved in the formation of UDP-GlcNAc from other bacteria. GlmU is a bifunctional enzyme that catalyzes two sequential steps in UDP-GlcNAc biosynthesis. Glucosamine-1-phosphate acetyl transferase catalyzes the formation of N-acetylglucosamine-1-phospha...

Zhang, Wenli; Jones, Victoria C.; Scherman, Michael S.; Mahapatra, Sebabrata; Crick, Dean; Bhamidi, Suresh; Xin, Yi; Mcneil, Michael R.; Ma, Yufang

2008-01-01

271

The DosS-DosT/DosR Mycobacterial Sensor System  

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DosS/DosR is a two-component regulatory system in which DosS, a heme-containing sensor also known as DevS, under certain conditions undergoes autophosphorylation and then transfers the phosphate to DosR, a DNA-binding protein that controls the entry of Mycobacterium tuberculosis and other mycobacteria into a latent, dormant state. DosT, a second sensor closely related to DosS, is present in M. tuberculosis and participates in the control of the dormancy response mediated by DosR. The binding of phosphorylated DosR to DNA initiates the expression of approximately fifty dormancy-linked genes. DosT is accepted to be a gas sensor that is activated in the ferrous state by the absence of an oxygen ligand or by the binding of NO or CO. DosS functions in a similar fashion as a gas sensor, but contradictory evidence has led to the suggestion that it also functions as a redox state sensor. This review focuses on the structure, biophysical properties, and function of the DosS/DosT heme sensors.

Sivaramakrishnan, Santhosh; de Montellano, Paul R. Ortiz

2014-01-01

272

The DosS-DosT/DosR Mycobacterial Sensor System  

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Full Text Available DosS/DosR is a two-component regulatory system in which DosS, a heme-containing sensor also known as DevS, under certain conditions undergoes autophosphorylation and then transfers the phosphate to DosR, a DNA-binding protein that controls the entry of Mycobacterium tuberculosis and other mycobacteria into a latent, dormant state. DosT, a second sensor closely related to DosS, is present in M. tuberculosis and participates in the control of the dormancy response mediated by DosR. The binding of phosphorylated DosR to DNA initiates the expression of approximately fifty dormancy-linked genes. DosT is accepted to be a gas sensor that is activated in the ferrous state by the absence of an oxygen ligand or by the binding of NO or CO. DosS functions in a similar fashion as a gas sensor, but contradictory evidence has led to the suggestion that it also functions as a redox state sensor. This review focuses on the structure, biophysical properties, and function of the DosS/DosT heme sensors.

Santhosh Sivaramakrishnan

2013-07-01

273

TB tools to tell the tale-molecular genetic methods for mycobacterial research.  

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In spite of the availability of drugs and a vaccine, tuberculosis--one of man's medical nemeses--remains a formidable public health problem, particularly in the developing world. The persistent nature of the tubercle bacillus, with one third of the world's population is estimated to be infected, combined with the emergence of multi drug-resistant strains and the exquisite susceptibility of HIV-positive individuals, has underscored the urgent need for in-depth study of the biology of Mycobacterium tuberculosis address the resurgence of TB. In aiming to understand the mechanisms by which mycobacteria react to their immediate environments, molecular genetic tools have been developed from naturally occurring genetic elements. These include protein expressing genes, and episomal and integrating elements, which have been derived mainly from prokaryotic but also from eukaryotic organisms. Molecular genetic tools that had been established as routine procedures in other prokaryotic genera were thus mimicked. Knowledge of the underlying mechanisms greatly expedited the harnessing of these elements for mycobacteriological research and has brought us to a point where these molecular genetic tools are now employed routinely in laboratories worldwide. PMID:15381150

Machowski, Edith E; Dawes, Stephanie; Mizrahi, Valerie

2005-01-01

274

[The development of novel vaccines against tuberculosis].  

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We have developed a novel tuberculosis (TB) vaccine ; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-liposome or-envelope (HSP65+IL-12/HVJ). This vaccine provided remarkable protective efficacy in mouse and guinea pig models compared to the BCG vaccine, on the basis of an induction of the CD8 positive CTL activity against TB antigens and improvement of the histopathological tuberculosis lesions, respectively. The Elispot assay showed that HSP65+IL-12 DNA/ HVJ vaccine induced a greater number of IFN-gamma producing T cells than BCG in the mouse model. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR, body weight, chest X-ray findings and immune responses (IFN-gamma, IL-2, IL-6 production , and lymphocyte proliferation of cynomolgus monkey). The combination of HSP65+IL-12/HVJ and BCG by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). In contrast, 33% of monkeys from BCG Tokyo alone group were alive (33% survival). These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis for human clinical trials. PMID:18974619

Okada, Masaji

2008-10-01

275

Naive helper T cells from BCG-vaccinated volunteers produce IFN-gamma and IL-5 to mycobacterial antigen-pulsed dendritic cells.  

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Full Text Available Mycobacterium bovis bacillus Calmette-Gu?Šrin (BCG is a live vaccine that has been used in routine vaccination against tuberculosis for nearly 80 years. However, its efficacy is controversial. The failure of BCG vaccination may be at least partially explained by the induction of poor or inappropriate host responses. Dendritic cells (DCs are likely to play a key role in the induction of immune response to mycobacteria by polarizing the reactivity of T lymphocytes toward a Th1 profile, contributing to the generation of protective cellular immunity against mycobacteria. In this study we aimed to investigate the production of Th1 and Th2 cytokines by naive CD4+ T cells to mycobacterial antigen-pulsed DCs in the group of young, healthy BCG vaccinated volunteers. The response of naive helper T cells was compared with the response of total blood lymphocytes. Our present results clearly showed that circulating naive CD45RA+CD4+ lymphocytes from BCG-vaccinated subjects can become effector helper cells producing IFN-gamma and IL-5 under the stimulation by autologous dendritic cells presenting mycobacterial protein antigen-PPD or infected with live M. bovis BCG bacilli.

Jo??l Pestel

2008-06-01

276

Cloning, Expression, and Site-Directed Mutagenesis of the Propene Monooxygenase Genes from Mycobacterium sp. Strain M156  

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Propene monooxygenase has been cloned from Mycobacterium sp. strain M156, based on hybridization with the amoABCD genes of Rhodococcus corallinus B276. Sequencing indicated that the mycobacterial enzyme is a member of the binuclear nonheme iron monooxygenase family and, in gene order and sequence, is most similar to that from R. corallinus B-276. Attempts were made to express the pmoABCD operon in Escherichia coli and Mycobacterium smegmatis mc2155. In the former, there appeared to be a probl...

Chan Kwo Chion, Chan K.; Askew, Sarah E.; Leak, David J.

2005-01-01

277

Mycobacterial infections in AIDS  

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Tuberculosis (TB) remains uniquely important among acquired immune deficiency syndrome (AIDS)-associated opportunistic infections: it presents the greatest public health hazard worldwide, is the most readily curable, and is largely preventable with existing means. Given the expanding pool of human immunodeficiency virus (HIV) seropositive persons, particularly in developing nations where Mycobacterium tuberculosis remains a leading health problem, one can expect a continued rise in TB cases d...

Hill, A. Ross

1991-01-01

278

Mycobacterial lipid logic.  

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During infection of the lung epithelium, Mycobacterium tuberculosis must infect and survive within macrophages long enough to be transported into deeper lung tissues. Cambier et al. (2013) show that pathogenic mycobacteria use the coordinated action of two cell wall glycolipids to regulate macrophage recruitment to initial infection sites. PMID:24439891

Siegrist, M Sloan; Bertozzi, Carolyn R

2014-01-15

279

Both Corynebacterium diphtheriae DtxR(E175K) and Mycobacterium tuberculosis IdeR(D177K) Are Dominant Positive Repressors of IdeR-Regulated Genes in M. tuberculosis  

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The diphtheria toxin repressor (DtxR) is an important iron-dependent transcriptional regulator of known virulence genes in Corynebacterium diphtheriae. The mycobacterial iron-dependent repressor (IdeR) is phylogenetically closely related to DtxR, with high amino acid similarity in the DNA binding and metal ion binding site domains. We have previously shown that an iron-insensitive, dominant-positive dtxR(E175K) mutant allele from Corynebacterium diphtheriae can be expressed in Mycobacterium t...

Manabe, Yukari C.; Hatem, Christine L.; Kesavan, Anup K.; Durack, Justin; Murphy, John R.

2005-01-01

280

Rapid Rebound of the Treg Compartment in DEREG Mice Limits the Impact of Treg Depletion on Mycobacterial Burden, but Prevents Autoimmunity  

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The development of an effective vaccine against tuberculosis (Tb) represents one of the major medical challenges of this century. Mycobacterium bovis Bacille Calmette-Guerin (BCG), the only vaccine available at present, is mostly effective at preventing disseminated Tb in children, but shows variable protection against pulmonary Tb, the most common form in adults. The reasons for this poor efficacy are not completely understood, but there is evidence that T regulatory cells (Tregs) might be involved. Similarly, Tregs have been associated with the immunosuppression observed in patients infected with Tb and are therefore believed to play a role in pathogen persistence. Thus, Treg depletion has been postulated as a novel strategy to potentiate M. bovis BCG vaccination on one side, while on the other, employed as a therapeutic approach during chronic Tb infection. Yet since Tregs are critically involved in controlling autoimmune inflammation, elimination of Tregs may therefore also incur the danger of an excessive inflammatory immune response. Thus, understanding the dynamics and function of Tregs during mycobacterial infection is crucial to evaluate the potential of Treg depletion as a medical option. To address this, we depleted Tregs after infection with M. bovis BCG or Mycobacterium tuberculosis (Mtb) using DEREG mice, which express the diphtheria toxin (DT) receptor under the control of the FoxP3 locus, thereby allowing the selective depletion of FoxP3+ Tregs. Our results show that after depletion, the Treg niche is rapidly refilled by a population of DT-insensitive Tregs (diTregs) and bacterial load remains unchanged. On the contrary, impaired rebound of Tregs in DEREG × FoxP3GFP mice improves pathogen burden, but is accompanied by detrimental autoimmune inflammation. Therefore, our study provides the proof-of-principle that, although a high degree of Treg depletion may contribute to the control of mycobacterial infection, it carries the risk of autoimmunity.

Varela, Filipa; Behrends, Jochen; Swallow, Maxine; Kruse, Friederike; Krull, Freyja; Ghorbani, Peyman; Mayer, Christian T.

2014-01-01

 
 
 
 
281

Evaluation of INNO-LiPA mycobacteria v2 assay for identification of rapidly growing mycobacteria  

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Full Text Available A total of 54 rapidly growing mycobacteria (RGM isolated from patients attended in the two hospitals of Cádiz Bay (Spain were selected during a seven-year-period (2000-2006 in order to evaluate the INNO-LiPA Mycobacteria v2 assay for mycobacterial identification, based on the reverse hybridization principle. The strains were cultured in Löwenstein-Jensen and Middlebrook 7H9 media and identified to the species level by sequencing of the 16S rRNA, PCR-restriction enzyme analysis of the hsp65 gene, conventional tests and INNO-LiPA Mycobacteria v2 assay. By the molecular methods we identified a total of 12 different species: 23 Mycobacterium fortuitum, 11 M. chelonae, 10 M. abscessus, 2 M. senegalense, 1 M. alvei, 1 M. brumae, 1 M. mageritense, 1 M. mucogenicum, 1 M. neoaurum, 1 M. peregrinum, 1 M. septicum and 1 M. smegmatis. Fifty two strains (96.3% were correctly identified by conventional techniques and 47 strains (87.0% by INNO-LiPA Mycobacteria v2 assay. We find INNO-LiPA Mycobacteria v2 assay simple to perform but it provides few advantages in comparison with conventional methods and sometimes needs complementary tests to identify Mycobacterium fortuitum complex, M. chelonae complex and specific species due to the great heterogeneity in the RGM group.

Lidia García-Agudo

2011-09-01

282

Disseminated infection due to Mycobacterium avium subsp. avium in an Asian elephant (Elephas maximus).  

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A disseminated infection caused by Mycobacterium avium subspecies avium (MAA) was diagnosed in a 57-yr-old male Asian elephant (Elephas maximus) housed at the Seoul Zoo, Gyeonggi, Republic of Korea. An apparent granulomatous inflammation with central caseous necrosis was evident in the lung sections. To confirm mycobacterial infection, polymerase chain reaction-restriction enzyme polymorphism analysis (PCR-RFLP) of the rpoB and hsp65 genes was performed from multiple organs and cultured bacteria. The PCR-RFLP revealed a M. avium subspecies. MAA was identified by multiplex PCR for detection of IS901 and IS1311. Thus, it is believed that MAA caused the disseminated infection in this case. Although the source of infection was not determined, the elephant may have become infected through contamination of soil and feed by free-living birds infected with MAA. This is the first reported case of disseminated infection due to MAA in a captive elephant in the Republic of Korea. PMID:22204075

Yong, Hwanyul; Choi, Go-Eun; Lee, Byung Soo; Whang, Jake; Shin, Sung Jae

2011-12-01

283

Gene Replacement in Mycobacterium chelonae: Application to the Construction of Porin Knock-Out Mutants  

Science.gov (United States)

Mycobacterium chelonae is a rapidly growing mycobacterial opportunistic pathogen closely related to Mycobacterium abscessus that causes cornea, skin and soft tissue infections in humans. Although M. chelonae and the emerging mycobacterial pathogen M. abscessus have long been considered to belong to the same species, these two microorganisms considerably differ in terms of optimum growth temperature, drug susceptibility, pathogenicity and the types of infection they cause. The whole genome sequencing of clinical isolates of M. chelonae and M. abscessus is opening the way to comparative studies aimed at understanding the biology of these pathogens and elucidating the molecular bases of their pathogenicity and biocide resistance. Key to the validation of the numerous hypotheses that this approach will raise, however, is the availability of genetic tools allowing for the expression and targeted mutagenesis of genes in these species. While homologous recombination systems have recently been described for M. abscessus, genetic tools are lacking for M. chelonae. We here show that two different allelic replacement methods, one based on mycobacteriophage-encoded recombinases and the other on a temperature-sensitive plasmid harboring the counterselectable marker sacB, can be used to efficiently disrupt genes in this species. Knock-out mutants for each of the three porin genes of M. chelonae ATCC 35752 were constructed using both methodologies, one of which displays a significantly reduced glucose uptake rate consistent with decreased porin expression.

Calado Nogueira de Moura, Vinicius; Gibbs, Sara; Jackson, Mary

2014-01-01

284

Discordance between Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Typing and IS6110 Restriction Fragment Length Polymorphism Genotyping for Analysis of Mycobacterium tuberculosis Beijing Strains in a Setting of High Incidence of Tuberculosis? †  

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IS6110 restriction fragment length polymorphism (RFLP) genotyping is the most widely used genotyping method to study the epidemiology of Mycobacterium tuberculosis. However, due to the complexity of the IS6110 RFLP genotyping technique, and the interpretation of RFLP data, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping has been proposed as the new genotyping standard. This study aimed to determine the discriminatory power of different MIRU-VNTR...

Hanekom, M.; Spuy, G. D.; Pittius, N. C. Gey; Mcevoy, C. R. E.; Hoek, K. G. P.; Ndabambi, S. L.; Jordaan, A. M.; Victor, T. C.; Helden, P. D.; Warren, R. M.

2008-01-01

285

Determination of Genotypic Diversity of Mycobacterium avium Subspecies from Human and Animal Origins by Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat and IS1311 Restriction Fragment Length Polymorphism Typing Methods ? †  

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Members of the Mycobacterium avium complex (MAC) are ubiquitous bacteria that can be found in water, food, and other environmental samples and are considered opportunistic pathogens for numerous animal species, mainly birds and pigs, as well as for humans. We have recently demonstrated the usefulness of a PCR-based mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing for the molecular characterization of M. avium subsp. paratuberculosis and M. avium stra...

Radomski, Nicolas; Thibault, Virginie C.; Karoui, Claudine; Cruz, Krystel; Cochard, Thierry; Gutie?rrez, Cristina; Supply, Philip; Biet, Frank; Boschiroli, Mari?a Laura

2010-01-01

286

Characterization of human cellular immune responses to Mycobacterium tuberculosis proteins encoded by genes predicted in RD15 genomic region that is absent in Mycobacterium bovis BCG.  

Science.gov (United States)

RD15 is a genomic region of difference (RD) present in Mycobacterium tuberculosis H37Rv but absent in all strains of Mycobacterium bovis BCG. RD15 contains genes encoding proteins of mammalian cell entry (Mce3A-F), important for the invasion and survival of M. tuberculosis in host cells. In this study, we have evaluated cellular immune responses to RD15 proteins using peripheral blood mononuclear cells (PBMC) from pulmonary tuberculosis patients and M. bovis BCG-vaccinated healthy subjects. PBMC were tested for T-helper (Th) type 1 [antigen-induced proliferation and interferon (IFN)-gamma secretion] and anti-inflammatory [interleukin (IL)-10 secretion] responses to complex mycobacterial antigens and peptides corresponding to proteins of RD1 and RD15. In Th1 assays, complex mycobacterial antigens induced strong responses in both donor groups, and RD1 induced strong responses in tuberculosis patients and moderate responses in healthy subjects, whereas RD15 induced weak responses in tuberculosis patients and strong to moderate responses in healthy subjects. IL-10 secretion in both donor groups was strong to moderate in response to complex mycobacterial antigens, but weak in response to RD1 and RD15. Analysis of IFN-gamma : IL-10 ratios showed strong Th1 biases to complex mycobacterial antigens and RD1 in both donor groups, and to RD15 and RD1504 (Mce3A) in healthy subjects only. These results suggest that RD1504 is the best Th1-stimulating antigen present in RD15, and therefore may be a potential vaccine candidate against TB. PMID:20482628

Al-Attiyah, Raja'a; Mustafa, Abu Salim

2010-07-01

287

Mycobacterium thermoresistibile: Case report of a rarely isolated mycobacterium from Europe and review of literature  

Directory of Open Access Journals (Sweden)

Full Text Available Mycobacterium thermoresistibile is a non-tuberculous mycobacterium strongly associated with human infections. Since 1966, there have only been six reports of its isolation from clinical samples. We report on the first case from Europe and review all the previous cases. Identification was achieved with sequencing of the 16S rRNA and hsp65 genes. This study presents its phenotypic and biochemical profile, susceptibilities to selected antibiotics and hsp65 polymerase chain reaction-restriction fragment length polymorphism profile with BsteII and Hae III .

Neonakis I

2009-01-01

288

Screening of Highly Expressed Mycobacterial Genes Identifies Rv3615c as a Useful Differential Diagnostic Antigen for the Mycobacterium tuberculosis Complex ? †  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Tuberculous infections caused by mycobacteria, especially tuberculosis of humans and cattle, are important both clinically and economically. Human populations can be vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG), and control measures for cattle involving vaccination are now being actively considered. However, diagnostic tests based on tuberculin cannot distinguish between genuine infection and vaccination with BCG. Therefore, identification of differential diagnostic anti...

Sidders, Ben; Pirson, Chris; Hogarth, Philip J.; Hewinson, R. Glyn; Stoker, Neil G.; Vordermeier, H. Martin; Ewer, Katie

2008-01-01

289

Rapid mycobacterial liquid culture-screening method for Mycobacterium avium complex based on secreted antigen-capture enzyme-linked immunosorbent assay.  

Science.gov (United States)

Sensors in automated liquid culture systems for mycobacteria, such as MGIT, BacT/Alert 3D, and Trek ESP II, flag growth of any type of bacteria; a positive signal does not mean that the target mycobacteria are present. All signal-positive cultures thus require additional and often laborious testing. An immunoassay was developed to screen liquid mycobacterial cultures for evidence of Mycobacterium avium complex (MAC). The method, called the MAC-enzyme-linked immunosorbent assay (ELISA), relies on detection of MAC-specific secreted antigens in liquid culture. Secreted MAC antigens were captured by the MAC-ELISA with polyclonal anti- Mycobacterium avium subsp. paratuberculosis chicken immunoglobulin Y (IgY), detected using rabbit anti-MAC IgG, and then revealed using horseradish peroxidase-conjugated goat anti-rabbit IgG. When the MAC-ELISA was evaluated using pure cultures of known mycobacterial (n = 75) and nonmycobacterial (n = 17) organisms, no false-positive or false-negative MAC-ELISA results were found. By receiver operator characteristic (ROC) analysis of 1,275 previously identified clinical isolates, at the assay optimal cutoff the diagnostic sensitivity and specificity of the MAC-ELISA were 92.6% (95% confidence interval [95% CI], 90.3 to 94.5) and 99.9% (95% CI, 99.2 to 100), respectively, with an area under the ROC curve of 0.992. Prospective evaluation of the MAC-ELISA with an additional 652 clinical samples inoculated into MGIT ParaTB medium and signaling positive per the manufacturer's instructions found that the MAC-ELISA was effective in determining those cultures that actually contained MAC species and warranting the resources required to identify the organism by PCR. Of these 652 MGIT-positive cultures, the MAC-ELISA correctly identified 96.8% (of 219 MAC-ELISA-positive cultures) as truly containing MAC mycobacteria, based on PCR or high-performance liquid chromatography (HPLC) as reference tests. Only 6 of 433 MGIT signal-positive cultures (1.4%) were MAC-ELISA false negative, and only 7 of 219 MGIT signal-negative cultures (3.2%) were false positive. The MAC-ELISA is a low-cost, rapid, sensitive, and specific test for MAC in liquid cultures. It could be used in conjunction with or independent of automated culture reading instrumentation. For maximal accuracy and subspecies-specific identification, use of a confirmatory multiplex MAC PCR is recommended. PMID:19261776

Shin, Sung Jae; Anklam, Kelly; Manning, Elizabeth J B; Collins, Michael T

2009-05-01

290

Development of a new generation of vectors for gene expression, gene replacement, and protein-protein interaction studies in mycobacteria.  

Science.gov (United States)

Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research. PMID:23315736

Parikh, Amit; Kumar, Devanand; Chawla, Yogesh; Kurthkoti, Krishna; Khan, Shazia; Varshney, Umesh; Nandicoori, Vinay K

2013-03-01

291

Development of a New Generation of Vectors for Gene Expression, Gene Replacement, and Protein-Protein Interaction Studies in Mycobacteria  

Science.gov (United States)

Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.

Parikh, Amit; Kumar, Devanand; Chawla, Yogesh; Kurthkoti, Krishna; Khan, Shazia; Varshney, Umesh

2013-01-01

292

Increased frequency of {gamma}{delta} T cells in cerebrospinal fluid and peripheral blood of patients with multiple sclerosis: Reactivity, cytotoxicity, and T cell receptor V gene rearrangements  

Energy Technology Data Exchange (ETDEWEB)

Infiltrating {gamma}{delta} T cells are potentially involved in the central nervous system demyelination in multiple sclerosis (MS). To further study this hypothesis, we analyzed the frequency and functional properties of {gamma}{delta} T cells in peripheral blood (PB) and paired cerebrospinal fluid (CSF) of patients with MS and control subjects, including patients with other neurologic diseases (OND) and healthy individuals. The frequency analysis was performed under limiting dilution condition using rIL-2 and PHA. After PHA stimulation, a significantly increased frequency of {gamma}{delta} T cells was observed in PB and in CSF of MS patients as compared with PB and CSF of patients with OND. The frequency was represented equally in OND patients and normal individuals. Similarly, the IL-2-responsive {gamma}{delta} T cells occurred at a higher frequency in PB of MS than of control subjects. Forty-three percent of the {gamma}{delta} T cell clones isolates from PB and CSF of MS patients responded to heat shock protein (HSP70) but not HSP65, whereas only 2 of 30 control {gamma}{delta} T cell clones reacted to the HSP. The majority of the {gamma}{delta} T cell clones were able to induce non-MHC-restricted cytolysis of Daudi cells. All clones displayed a substantial reactivity to bacterial superantigens staphylococcal enterotoxin B and toxic shock syndrome toxin-1, irrespective of their {gamma}{delta} V gene usage. Furthermore, the {gamma}{delta} T cell clones expressed predominantly TCRDV2 and GV2 genes, whereas the clones derived from CSF of MS patients expressed either DV1 or DV2 genes. The obtained {gamma}{delta} clones, in general, represented rather heterogeneous clonal origins, even though a predominant clonal origin was found in a set of 10 {gamma}{delta} clones derived from one patient with MS. The present study provides new evidence supporting a possible role of {gamma}{delta} T cells in the secondary inflammatory processes in MS. 39 refs., 5 figs., 4 tabs.

Stinissen, P.; Vandevyver, C.; Medaer, R. [Dr. L. Willems Institute, Diepenbeek (Belgium)] [and others

1995-05-01

293

Cytotoxic human HLA class II restricted purified protein derivative-reactive T-lymphocyte clones. IV. Analysis of HLA restriction pattern and mycobacterial antigen specificity  

DEFF Research Database (Denmark)

Human T-lymphocyte clones specific for antigenic components of purified protein derivative (PPD) of tuberculin were generated by limiting dilution using in vitro PPD-activated peripheral blood mononuclear cells from a single donor. The HLA restriction specificity of eight clones that were cytotoxic against autologous PPD-pulsed monocyte targets, was examined against a panel of allogeneic PPD pulsed targets. In agreement with our findings with bulk-expanded PPD-reactive cytotoxic T lymphocytes, all clones were restricted by HLA class II antigens: seven by HLA-DR 2 and one by HLA-DRw10--the other HLA-DR antigen of the donor. All clones were CD3+, CD4+, CD8-. One clone exhibited, in addition to HLA-DR2 restriction, unrestricted cytotoxic alloreactivity against HLA-DR1. In monoclonal antibody-blocking experiments the latter clone was the only one that was blocked. Its lytic ability was abolished by two monoclonal antibodies against monomorphic HLA-DR determinants. The antigen specificity of the clones was studiedby using autologous monocyte targets pulsed with antigens prepared from a range of different mycobacterial species. All seven HLA-DR2-restricted clones reacted with the majority of antigens tested. In contrast, the HLA-DRw10-restricted clone reacted exclusively with an antigen unique to PPD.

Hansen, P W; Petersen, C M

1987-01-01

294

Reactividad serológica y celular frente a proteínas micobacterianas en la enfermedad de Hansen / Serological and cellular reactivity to mycobacterial proteins in Hansen´s disease  

Scientific Electronic Library Online (English)

Full Text Available SciELO Venezuela | Language: Spanish Abstract in spanish Se diseñó un estudio para evaluar la reactividad inmunológica frente a diferentes preparaciones proteicas micobacterianas utilizando pruebas serológicas y de inmunidad celular. Para el estudio fueron incluídos pacientes con manifestaciones clínicas de lepra predominantemente de la forma multibacilar [...] . Todos los pacientes fueron adultos con edad comprendida entre 20 y 39 años. El 58% correspondía a la forma clínica de Lepra Lepromatosa (LL) n= 81, el 29% a la forma Borderline Lepromatosa (BL) n=41 y 10% a Borderline Borderline (BB) n=14. Solo el 3% fueron pacientes Borderline Tuberculoide (BT): 74% masculino y 26% femenino. El fenómeno reaccional más frecuente fue del tipo eritema nodoso leproso (ENL). Las proteínas micobacterianas ensayadas fueron: antígenos proteicos crudos totales de Mycobacterium leprae (MlSA), Mycobacterium bovis (MbSA y MbSA de excreción), antígeno proteico de excreción parcialmente purificado con una movilidad relativa de 30 kDa (Ml 30) y proteínas recombinantes de Mycobacterium (Mt70, Mb 65, Ml 36, 28, 18 y 10 kDa) encontrandose que las proteínas recombinantes (Ml10 kDa, Ml 36 kDa) a mayor carga bacilar presentaban una mayor reactividad serológica estadísticamente significativa (p= 0,0051 y 0,050 respectivamente). La proteína de 30 kDa fue predominantemente reconocida por anticuerpos de los pacientes multibacilares. Los resultados demuestran que el promedio de los valores de anticuerpos en pacientes no reaccionales fueron superiores en presencia de proteínas completas (MbSA y MbSA de exc) en comparación con el grupo de pacientes que presentaron fenómenos reaccionales (p=0,000567 y 0,000061 respectivamente) Este mismo comportamiento se observó frente a las proteínas micobacterianas individuales (30 kDa, 10 kDa y 36 kDa). La respuesta proliferativa de los linfocitos T en los pacientes multibacilares reaccionales y no reaccionales frente a las proteínas micobacterianas (MlSA, Ml 10 kDa, MbSA, MbSA de excreción) fue negativa en ambos grupos. Abstract in english The study was designed for evaluating immunological reactivity to various mycobacterial protein preparations using serological and cell-mediated immunological tests in patients with clinical leprosy signs, predominantly, with the multibacillary forms. All patients were adults with ages between 20 an [...] d 30 years. Fifty eight (n= 81) percent corresponded to Lepromatous Leprosy (LL), 29% (n= 41) to Borderline Lepromatous Leprosy (BL) and 10% (n=41) to Borderline Borderline Leprosy (BB); only 3% were Borderline Tuberculoid (BT) patients: 74% males and 26% females. The most frequent reactional phenomenon was of the Erythema Nodosum (ENL) type. The mycobacterial proteins tested were: total crude Mycobacterium leprae antigens (MISA); Mycobacterium bovis (MbSA and excretion MbSA); partially purified excretion protein antigen, with a 30kDa relative movility (Ml30); and recombinant M. leprae proteins (Mt70, Mb 65, Ml 36, 28, 18 and 10 kDa). Two of the recombinant proteins (Ml10 and Ml 36 kDa) presented a statiscally significant higher serological reactivity, directly related with a larger bacillary load (p= 0.0051 and 0.050 respectively). The 30 kDa protein was predominantly recognized by antibodies from multibacillary patients. Results show that mean antibody values were higher in non reactional patients when tested against complete proteins (MbSA and ex MbSA) when compared with the group of patients who presented reactional phenomena (p= 0.000567 and 0.000061, respectively). Comparing reactional with non reactional patients, it was seen that mean antibody values against complete proteins (MbSA and ex MbSA) were higher in non reactional individuals (p= 0.000567 and 0.000061, respectively). This same behavior occurred towards individual mycobacterial proteins (30, 10 and 36 kDa). The T lymphocyte prolypherative response in reactional and non reactional patients towards mycobacterial proteins (MlSA, Ml 10 kDa, MbSA, ex MbSA) was negative.

Elsa, Rada; Nacarid, Aranzazu; Vestalia, Rodríguez; Rafael, Borges; Jacinto, Convit.

295

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India  

Directory of Open Access Journals (Sweden)

Full Text Available The aim of the present study was to compare polymerase chain reaction (PCR-based methods - spoligotyping and mycobacterial interspersed repetitive units (MIRU typing - with the gold-standard IS6110 restriction fragment length polymorphism (RFLP analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters; the largest cluster was composed of Spoligotype International Types (SITs26 [Central-Asian (CAS1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI 3-Indian lineage]. A large number of isolates (75% belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

Mandira Varma-Basil

2011-08-01

296

Three Mycobacterium tuberculosis Rel Toxin-Antitoxin Modules Inhibit Mycobacterial Growth and Are Expressed in Infected Human Macrophages?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Mycobacterium tuberculosis protein pairs Rv1246c-Rv1247c, Rv2865-Rv2866, and Rv3357-Rv3358, here named RelBE, RelFG, and RelJK, respectively, were identified based on homology to the Escherichia coli RelBE toxin:antitoxin (TA) module. In this study, we have characterized each Rel protein pair and have established that they are functional TA modules. Overexpression of individual M. tuberculosis rel toxin genes relE, relG, and relK induced growth arrest in Mycobacterium smegmatis; a phenotype t...

Korch, Shaleen B.; Contreras, Heidi; Clark-curtiss, Josephine E.

2009-01-01

297

The Differential Gene Expression Pattern of Mycobacterium tuberculosis in Response to Capreomycin and PA-824 versus First-Line TB Drugs Reveals Stress- and PE/PPE-Related Drug Targets  

Directory of Open Access Journals (Sweden)

Full Text Available Tuberculosis is a leading infectious disease causing millions of deaths each year. How to eradicate mycobacterial persistence has become a central research focus for developing next-generation TB drugs. Yet, the knowledge in this area is fundamentally limited and only a few drugs, notably capreomycin and PA-824, have been shown to be active against non-replicating persistent TB bacilli. In this study, we performed a new bioinformatics analysis on microarray-based gene expression data obtained from the public domain to explore genes that were differentially induced by drugs between the group of capreomycin and PA-824 and the group of mainly the first-line TB drugs. Our study has identified 42 genes specifically induced by capreomycin and PA-824. Many of these genes are related to stress responses. In terms of the distribution of identified genes in a specific category relative to the whole genome, only the categories of PE/PPE and conserved hypotheticals have statistical significance. Six among the 42 genes identified in this study are on the list of the top 100 persistence targets selected by the TB Structural Genomics Consortium. Further biological elucidation of their roles in mycobacterial persistence is warranted.

Li M. Fu

2009-01-01

298

Cytomegalovirus infection modulates the phenotype and functional profile of the T-cell immune response to mycobacterial antigens in older life.  

Science.gov (United States)

Infection with Cytomegalovirus is associated with accelerated immunosenescence. Expansions of CMV-specific T cell responses have previously been demonstrated to affect the ability of T cells to respond to other infections. Most people above 60years of age display M. tuberculosis-specific immunity because of vaccination, exposure, or both. T-cell responses can be assessed by measuring intracellular IFN-? in vitro after tuberculin stimulation. Here we investigated tuberculin-specific CD4 T-cell responses in independently living healthy older people in the South of England using flow-cytometry. Individuals were investigated for tuberculin and CMV-specific T-cell immunity using in vitro antigen stimulation followed by intracellular staining for IFN-?, TNF-?, IL2, as well as degranulation and CD154 upregulation. We also examined a control group of younger individuals (20-35years of age). There was no significant difference between older and young people in regards to tuberculin responsiveness of CD4 T-cells; however, older people seemed to show more outliers. Increased responsiveness to tuberculin was significantly correlated to CMV responsiveness but not age. In older donors, the memory phenotype of tuberculin-induced T-cells was significantly skewed towards a more terminal differentiation phenotype in CMV-infected compared to uninfected individuals and the degree of skewing correlated quantitatively with the size of the CMV-specific CD4 T-cell response. This is a fundamental advance over previous reports of changes of the tuberculin-specific CD4 T-cell response with CMV serostatus. Our results show that how the immune system responds to CMV has a fundamental impact on the phenotype and function of the immune response to mycobacterial antigens in older life. PMID:24370373

Terrazzini, Nadia; Bajwa, Martha; Vita, Serena; Thomas, David; Smith, Helen; Vescovini, Rosanna; Sansoni, Paolo; Kern, Florian

2014-06-01

299

Mycobacterial and fungal skin sensitivity patterns among remote population groups in Papua New Guinea, and in the New Hebrides, Solomon, and Caroline Islands.  

Science.gov (United States)

Simultaneous intradermal sensitivity testing to Mycobacterium tuberculosis (PPD-S), six different atypical mycobacteria (PPD-A, B, F, G, T, and Y), Coccidiosides immitis (coccidioidin), and Histoplasma capsulatum (histoplasmin) was performed on 560 subjects among the relatively isolated island populations of Ifaluk (Caroline Islands), Loh and Merig (New Hebrides), Anuta (Solomon Islands), and in the Mala (Amdei) and Iwane (Simbari) villages of the Anga linguistic groups in the Marawaka area of the Eastern Highlands Province of Papua New Guinea. At the time these tests were performed (Mala and Iwane villages, and Ifaluk Atoll in 1967, and Loh, Merig, and Anuta Islands in 1972), all island populations had already had a long history of sporadic European contact, whereas the New Guinean villages of Mala and Iwane had remained virtually unexposed to the outside world. Using a dose of 0.001 mg PPD per 0.0 ml (5 TU), and considering induration of at least 10 mm at 48 hours to represent a positive reaction, skin sensitivity to M. tuberculosis was found to be absent among the Anga, and to have an incidence of 11% on Loh, 17% on Merig, 34% on Ifaluk, and 55% on Anuta. Data on the prevalence of tuberculous infection obtained by skin tests reflected the reported prevalence of symptomatic tuberculosis in all groups. The frequency of reactions to 0.0001 mg doses of the atypical mycobacterial antigens corresponded to tuberculin sensitivity in the different groups: none among the Anga, sporadic on Loh and Merig, and common on Ifaluk and Anuta. However, analysis of PPD profiles suggests that actions greater than or equal to 5 mm to a 0.1 ml dose of 1:100 coccidioidin were observed in 26% of the population of Ifaluk, and of 1:100 histoplasmin in 8% of the population of Anuta, and 14-16% of the populations of the two New Guinean villages. PMID:6792936

Brown, P; Cathala, F; Gajdusek, D C

1981-09-01

300

Optimization of Pyrrolamides as Mycobacterial GyrB ATPase Inhibitors: Structure-Activity Relationship and In Vivo Efficacy in a Mouse Model of Tuberculosis  

Science.gov (United States)

Moxifloxacin has shown excellent activity against drug-sensitive as well as drug-resistant tuberculosis (TB), thus confirming DNA gyrase as a clinically validated target for discovering novel anti-TB agents. We have identified novel inhibitors in the pyrrolamide class which kill Mycobacterium tuberculosis through inhibition of ATPase activity catalyzed by the GyrB domain of DNA gyrase. A homology model of the M. tuberculosis H37Rv GyrB domain was used for deciphering the structure-activity relationship and binding interactions of inhibitors with mycobacterial GyrB enzyme. Proposed binding interactions were later confirmed through cocrystal structure studies with the Mycobacterium smegmatis GyrB ATPase domain. The most potent compound in this series inhibited supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC50) of <5 nM, an MIC of 0.03 ?g/ml against M. tuberculosis H37Rv, and an MIC90 of <0.25 ?g/ml against 99 drug-resistant clinical isolates of M. tuberculosis. The frequency of isolating spontaneous resistant mutants was ?10?6 to 10?8, and the point mutation mapped to the M. tuberculosis GyrB domain (Ser208 Ala), thus confirming its mode of action. The best compound tested for in vivo efficacy in the mouse model showed a 1.1-log reduction in lung CFU in the acute model and a 0.7-log reduction in the chronic model. This class of GyrB inhibitors could be developed as novel anti-TB agents.

P, Shahul Hameed; Mukherjee, Kakoli; Nandi, Vrinda; Waterson, David; Shandil, Radha; Balganesh, Meenakshi; Sambandamurthy, Vasan K.; Raichurkar, Anand Kumar; Deshpande, Abhijeet; Ghosh, Anirban; Awasthy, Disha; Shanbhag, Gajanan; Sheikh, Gulebahar; McMiken, Helen; Puttur, Jayashree; Reddy, Jitendar; Werngren, Jim; Read, Jon; Kumar, Mahesh; R, Manjunatha; Chinnapattu, Murugan; Madhavapeddi, Prashanti; Manjrekar, Praveena; Basu, Reetobrata; Gaonkar, Sheshagiri; Sharma, Sreevalli; Hoffner, Sven; Humnabadkar, Vaishali; Subbulakshmi, Venkita; Panduga, Vijender

2014-01-01

 
 
 
 
301

Optimization of pyrrolamides as mycobacterial GyrB ATPase inhibitors: structure-activity relationship and in vivo efficacy in a mouse model of tuberculosis.  

Science.gov (United States)

Moxifloxacin has shown excellent activity against drug-sensitive as well as drug-resistant tuberculosis (TB), thus confirming DNA gyrase as a clinically validated target for discovering novel anti-TB agents. We have identified novel inhibitors in the pyrrolamide class which kill Mycobacterium tuberculosis through inhibition of ATPase activity catalyzed by the GyrB domain of DNA gyrase. A homology model of the M. tuberculosis H37Rv GyrB domain was used for deciphering the structure-activity relationship and binding interactions of inhibitors with mycobacterial GyrB enzyme. Proposed binding interactions were later confirmed through cocrystal structure studies with the Mycobacterium smegmatis GyrB ATPase domain. The most potent compound in this series inhibited supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC50) of <5 nM, an MIC of 0.03 ?g/ml against M. tuberculosis H37Rv, and an MIC90 of <0.25 ?g/ml against 99 drug-resistant clinical isolates of M. tuberculosis. The frequency of isolating spontaneous resistant mutants was ?10(-6) to 10(-8), and the point mutation mapped to the M. tuberculosis GyrB domain (Ser208 Ala), thus confirming its mode of action. The best compound tested for in vivo efficacy in the mouse model showed a 1.1-log reduction in lung CFU in the acute model and a 0.7-log reduction in the chronic model. This class of GyrB inhibitors could be developed as novel anti-TB agents. PMID:24126580

P, Shahul Hameed; Solapure, Suresh; Mukherjee, Kakoli; Nandi, Vrinda; Waterson, David; Shandil, Radha; Balganesh, Meenakshi; Sambandamurthy, Vasan K; Raichurkar, Anand Kumar; Deshpande, Abhijeet; Ghosh, Anirban; Awasthy, Disha; Shanbhag, Gajanan; Sheikh, Gulebahar; McMiken, Helen; Puttur, Jayashree; Reddy, Jitendar; Werngren, Jim; Read, Jon; Kumar, Mahesh; R, Manjunatha; Chinnapattu, Murugan; Madhavapeddi, Prashanti; Manjrekar, Praveena; Basu, Reetobrata; Gaonkar, Sheshagiri; Sharma, Sreevalli; Hoffner, Sven; Humnabadkar, Vaishali; Subbulakshmi, Venkita; Panduga, Vijender

2014-01-01

302

Proposal of a Consensus Set of Hypervariable Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Loci for Subtyping of Mycobacterium tuberculosis Beijing Isolates  

Science.gov (United States)

Mycobacterium tuberculosis Beijing strains represent targets of special importance for molecular surveillance of tuberculosis (TB), especially because they are associated with spread of multidrug resistance in some world regions. Standard 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing lacks resolution power for accurately discriminating closely related clones that often compose Beijing strain populations. Therefore, we evaluated a set of 7 additional, hypervariable MIRU-VNTR loci for better resolution and tracing of such strains, using a collection of 535 Beijing isolates from six world regions where these strains are known to be prevalent. The typeability and interlaboratory reproducibility of these hypervariable loci were lower than those of the 24 standard loci. Three loci (2163a, 3155, and 3336) were excluded because of their redundant variability and/or more frequent noninterpretable results compared to the 4 other markers. The use of the remaining 4-locus set (1982, 3232, 3820, and 4120) increased the number of types by 52% (from 223 to 340) and reduced the clustering rate from 58.3 to 36.6%, when combined with the use of the standard 24-locus set. Known major clonal complexes/24-locus-based clusters were all subdivided, although the degree of subdivision varied depending on the complex. Only five single-locus variations were detected among the hypervariable loci of an additional panel of 92 isolates, representing 15 years of clonal spread of a single Beijing strain in a geographically restricted setting. On this calibrated basis, we propose this 4-locus set as a consensus for subtyping Beijing clonal complexes and clusters, after standard typing.

Allix-Beguec, Caroline; Wahl, Celine; Hanekom, Madeleine; Nikolayevskyy, Vladyslav; Drobniewski, Francis; Maeda, Shinji; Campos-Herrero, Isolina; Mokrousov, Igor; Niemann, Stefan; Kontsevaya, Irina; Rastogi, Nalin; Samper, Sofia; Sng, Li-Hwei; Warren, Robin M.

2014-01-01

303

The twin-arginine translocation pathway of Mycobacterium smegmatis is functional and required for the export of mycobacterial beta-lactamases.  

Science.gov (United States)

The twin-arginine translocation (Tat) pathway exports folded proteins across the bacterial cytoplasmic membrane and is responsible for the proper extracytoplasmic localization of proteins involved in a variety of cellular functions, including pathogenesis. The Mycobacterium tuberculosis and Mycobacterium smegmatis genomes contain open reading frames with homology to components of the Tat export system (TatABC) as well as potential Tat-exported proteins possessing N-terminal signal sequences with the characteristic twin-arginine motif. Due to the importance of exported virulence factors in the pathogenesis of M. tuberculosis and the limited understanding of mycobacterial protein export systems, we sought to determine the functional nature of the Tat export pathway in mycobacteria. Here we describe phenotypic analyses of DeltatatA and DeltatatC deletion mutants of M. smegmatis, which demonstrated that tatA and tatC encode components of a functional Tat system capable of exporting characteristic Tat substrates. Both mutants displayed a growth defect on agar medium and hypersensitivity to sodium dodecyl sulfate. The mutants were also defective in the export of active beta-lactamases of M. smegmatis (BlaS) and M. tuberculosis (BlaC), both of which possess twin-arginine signal sequences. The Tat-dependent nature of BlaC was further revealed by mutation of the twin-arginine motif. Finally, we demonstrated that replacement of the native signal sequence of BlaC with the predicted Tat signal sequences of M. tuberculosis phospholipase C proteins (PlcA and PlcB) resulted in the Tat-dependent export of an enzymatically active 'BlaC. Thus, 'BlaC can be used as a genetic reporter for Tat-dependent export in mycobacteria. PMID:16267291

McDonough, Justin A; Hacker, Kari E; Flores, Anthony R; Pavelka, Martin S; Braunstein, Miriam

2005-11-01

304

Cytomegalovirus infection modulates the phenotype and functional profile of the T-cell immune response to mycobacterial antigens in older life?  

Science.gov (United States)

Infection with Cytomegalovirus is associated with accelerated immunosenescence. Expansions of CMV-specific T cell responses have previously been demonstrated to affect the ability of T cells to respond to other infections. Most people above 60 years of age display M. tuberculosis-specific immunity because of vaccination, exposure, or both. T-cell responses can be assessed by measuring intracellular IFN-? in vitro after tuberculin stimulation. Here we investigated tuberculin-specific CD4 T-cell responses in independently living healthy older people in the South of England using flow-cytometry. Individuals were investigated for tuberculin and CMV-specific T-cell immunity using in vitro antigen stimulation followed by intracellular staining for IFN-?, TNF-?, IL2, as well as degranulation and CD154 upregulation. We also examined a control group of younger individuals (20–35 years of age). There was no significant difference between older and young people in regards to tuberculin responsiveness of CD4 T-cells; however, older people seemed to show more outliers. Increased responsiveness to tuberculin was significantly correlated to CMV responsiveness but not age. In older donors, the memory phenotype of tuberculin-induced T-cells was significantly skewed towards a more terminal differentiation phenotype in CMV-infected compared to uninfected individuals and the degree of skewing correlated quantitatively with the size of the CMV-specific CD4 T-cell response. This is a fundamental advance over previous reports of changes of the tuberculin-specific CD4 T-cell response with CMV serostatus. Our results show that how the immune system responds to CMV has a fundamental impact on the phenotype and function of the immune response to mycobacterial antigens in older life.

Terrazzini, Nadia; Bajwa, Martha; Vita, Serena; Thomas, David; Smith, Helen; Vescovini, Rosanna; Sansoni, Paolo; Kern, Florian

2014-01-01

305

The radiology of IRIS (immune reconstitution inflammatory syndrome) in patients with mycobacterial tuberculosis and HIV co-infection: appearances in 11 patients  

International Nuclear Information System (INIS)

Aim: To determine the radiological manifestations of IRIS (immune reconstitution inflammatory syndrome) in patients with HIV and mycobacterium tuberculosis co-infection, in the context of their demographic and clinical data. Materials and methods: The radiological imaging, demographic and clinical data of 11 patients diagnosed with IRIS associated with HIV and mycobacterial tuberculosis co-infection were studied retrospectively. Where available, follow-up imaging studies were also reviewed. Results: The most common radiological feature of IRIS was lymph node enlargement (73%), with central low attenuation centres, in keeping with necrosis, present in most of these cases (88%). Most commonly affected were intra-abdominal nodes (70%), followed by axillary (40%) and mediastinal lymph nodes (36%). Within the lung parenchyma, diffuse, bilateral pulmonary nodules were seen in 55% of cases. Unilateral small volume pleural effusions were seen in two cases with associated parenchymal changes seen in only one. Small volume ascites was seen in two cases. Thirty-six percent of cases presented with new or worsening abscesses despite treatment. In this context, image-guided radiological drainage proved a useful adjunct to the conventional medical therapy for IRIS. The most common clinical signs of IRIS included fever (64%), abdominal pain (36%) and cough (27%). Conclusion: We have described the radiological features that are characteristic in IRIS and the importance of putting these into context with the clinical and pathological findings as part of a multidisciplinary approach in making the diagnosis. The role of the radiologist is central in diagnosis, monitoring of disease progression and management of complications in patients with IRIS

2006-10-01

306

Discovery of Pyrazolopyridones as a Novel Class of Noncovalent DprE1 Inhibitor with Potent Anti-Mycobacterial Activity.  

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A novel pyrazolopyridone class of inhibitors was identified from whole cell screening against Mycobacterium tuberculosis (Mtb). The series exhibits excellent bactericidality in vitro, resulting in a 4 log reduction in colony forming units following compound exposure. The significant modulation of minimum inhibitory concentration (MIC) against a Mtb strain overexpressing the Rv3790 gene suggested the target of pyrazolopyridones to be decaprenylphosphoryl-?-d-ribose-2'-epimerase (DprE1). Genetic mapping of resistance mutation coupled with potent enzyme inhibition activity confirmed the molecular target. Detailed biochemical characterization revealed the series to be a noncovalent inhibitor of DprE1. Docking studies at the active site suggest that the series can be further diversified to improve the physicochemical properties without compromising the antimycobacterial activity. The pyrazolopyridone class of inhibitors offers an attractive non-nitro lead series targeting the essential and vulnerable DprE1 enzyme for the discovery of novel antimycobacterial agents to treat both drug susceptible and drug resistant strains of Mtb. PMID:24818517

Panda, Manoranjan; Ramachandran, Sreekanth; Ramachandran, Vasanthi; Shirude, Pravin S; Humnabadkar, Vaishali; Nagalapur, Kavitha; Sharma, Sreevalli; Kaur, Parvinder; Guptha, Supreeth; Narayan, Ashwini; Mahadevaswamy, Jyothi; Ambady, Anisha; Hegde, Naina; Rudrapatna, Suresh S; Hosagrahara, Vinayak P; Sambandamurthy, Vasan K; Raichurkar, Anandkumar

2014-06-12

307

Functional Complementation of the Essential Gene fabG1 of Mycobacterium tuberculosis by Mycobacterium smegmatis fabG but Not Escherichia coli fabG?  

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Mycolic acids are a key component of the mycobacterial cell wall, providing structure and forming a major permeability barrier. In Mycobacterium tuberculosis mycolic acids are synthesized by type I and type II fatty acid synthases. One of the enzymes of the type II system is encoded by fabG1. We demonstrate here that this gene can be deleted from the M. tuberculosis chromosome only when another functional copy is provided elsewhere, showing that under normal culture conditions fabG1 is essent...

Parish, Tanya; Roberts, Gretta; Laval, Francoise; Schaeffer, Merrill; Daffe?, Mamadou; Duncan, Ken

2007-01-01

308

Horizontal transfer of PAH catabolism genes in Mycobacterium: evidence from comparative genomics and isolated pyrene-degrading bacteria.  

Science.gov (United States)

Biodegradation of high molecular weight polycyclic aromatic hydrocarbons (PAHs), such as pyrene and benzo[a]pyrene, has only been observed in a few genera, namely fast-growing Mycobacterium and Rhodococcus. In M. vanbaalenii PYR-1, multiple aromatic ring hydroxylating dioxygenase (ARHDOs) genes including pyrene dioxygenases nidAB and nidA3B3 are localized in one genomic region. Here we examine the homologous genomic regions in four other PAH-degrading Mycobacterium (strains JLS, KMS, and MCS, and M. gilvum PYR-GCK), presenting evidence for past horizontal gene transfer events. Seven distinct types of ARHDO genes are present in all five genomes, and display conserved syntenic architecture with respect to gene order, orientation, and association with other genes. Duplications and putative integrase and transposase genes suggest past gene shuffling. To corroborate these observations, pyrene-degrading strains were isolated from two PAH-contaminated sediments: Chattanooga Creek (Tennessee) and Lake Erie (western basin). Some were related to fast-growing Mycobacterium spp. and carried both nidA and nidA3 genes. Other isolates belonged to Microbacteriaceae and Intrasporangiaceae presenting the first evidence of pyrene degradation in these families. These isolates had nidA (and some, nidA3) genes that were homologous to Mycobacterial ARHDO genes, suggesting that horizontal gene transfer events have occurred. PMID:21899303

DeBruyn, Jennifer M; Mead, Thomas J; Sayler, Gary S

2012-01-01

309

A randomised controlled trial of the effects of albendazole in pregnancy on maternal responses to mycobacterial antigens and infant responses to bacille Calmette-Guérin (BCG immunisation [ISRCTN32849447  

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Full Text Available Abstract Background Maternal schistosomiasis and filariasis have been shown to influence infant responses to neonatal bacille Calmette-Guérin (BCG immunisation but the effects of maternal hookworm, and of de-worming in pregnancy, are unknown. Methods In Entebbe, Uganda, we conducted a randomised, double-blind, placebo-controlled trial of a single dose of 400 mg of albendazole in the second trimester of pregnancy. Neonates received BCG. Interferon-gamma (IFN-? and interleukin (IL-5 responses to a mycobacterial antigen (crude culture filtrate proteins (CFP of Mycobacterium tuberculosis were measured in a whole blood assay. We analysed results for binary variables using ?2 tests and logistic regression. We analysed continuous variables using Wilcoxon's tests. Results Maternal hookworm was associated with reduced maternal IFN-? responses to CFP (adjusted odds ratio for IFN-? > median response: 0.14 (95% confidence interval 0.02–0.83, p = 0.021. Conversely, maternal hookworm was associated with subsequent increased IFN-? responses in their one-year-old infants (adjusted OR 17.65 (1.20–258.66; p = 0.013. Maternal albendazole tended to reduce these effects. Conclusion Untreated hookworm infection in pregnancy was associated with reduced maternal IFN-? responses to mycobacterial antigens, but increased responses in their infants one year after BCG immunisation. The mechanisms of these effects, and their implications for protective immunity remain, to be determined.

Nampijja Margaret

2005-12-01

310

Inositol monophosphate phosphatase genes of Mycobacterium tuberculosis  

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Full Text Available Abstract Background Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM, lipomannan (LM and phosphatidylinosotol mannosides (PIMs in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome. Results Mutants lacking either impA (Rv1604 or suhB (Rv2701c were isolated in the absence of exogenous inositol, and no differences in levels of PIMs, LM, LAM or mycothiol were observed. Mutagenesis of cysQ (Rv2131c was initially unsuccessful, but was possible when a porin-like gene of Mycobacterium smegmatis was expressed, and also by gene switching in the merodiploid strain. In contrast, we could only obtain mutations in impC (Rv3137 when a second functional copy was provided in trans, even when exogenous inositol was provided. Experiments to obtain a mutant in the presence of a second copy of impC containing an active-site mutation, in the presence of porin-like gene of M. smegmatis, or in the absence of inositol 1-phosphate synthase activity, were also unsuccessful. We showed that all four genes are expressed, although at different levels, and levels of inositol phosphatase activity did not fall significantly in any of the mutants obtained. Conclusions We have shown that neither impA, suhB nor cysQ is solely responsible for inositol synthesis. In contrast, we show that impC is essential for mycobacterial growth under the conditions we used, and suggest it may be required in the early stages of mycothiol synthesis.

Parish Tanya

2010-02-01

311

GeneXpert MTB/RIF Testing in the Management of Patients with Active Tuberculosis; A Real Life Experience from Saudi Arabia  

Science.gov (United States)

Background GeneXpert MTB/RIF is a real-time PCR assay with established diagnostic performance in pulmonary and extra-pulmonary forms of tuberculosis. The aim of this study was to assess the contribution of GeneXpert MTB/RIF assay to the management of patients with any form of active tuberculosis in a single large tertiary center in Saudi Arabia, with a special focus on the impact on time to start of antituberculous therapy compared with Ziehl-Neelsen (ZN) smears and mycobacterial cultures. Materials and Methods Clinical, radiological and laboratory records for all patients who were commenced on antituberculous therapy between March 2011 and February 2013 were retrospectively reviewed. Results A total of 140 patients were included, 38.6% of which had pulmonary tuberculosis. GeneXpert MTB/RIF was requested for only 39.2% of patients and was the only reason for starting antituberculous therapy for only 12.1%. The median time to a positive GeneXpert MTB/RIF result was 0 days (IQR 3) compared with 0 day (IQR 1) for smear microscopy (P > 0.999) and 22 days (IQR 21) for mycobacterial cultures (P < 0.001). No patients discontinued antituberculous therapy because of a negative GeneXpert MTB/RIF result. Conclusions In a setting wherein physicians are highly experienced in the diagnosis and treatment of tuberculosis, GeneXpert MTB/RIF was remarkably under-utilized and had only a limited impact on decisions related to starting or stopping antituberculous therapy. Cost-effectiveness and clinical utility of routine testing of all smear-negative clinical samples submitted for tuberculosis investigations by GeneXpert MTB/RIF warrant further study.

Al-Otaibi, Mohammed F.; Al-Ateah, Souad M.; Al-Onazi, Fahad M.; Baig, Kamran; El-Khizzi, Noura A.; Albarrak, Ali M.

2014-01-01

312

Immunoglobulin genes  

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This book reports on the structure, function, and expression of the genes encoding antibodies in normal and neoplastic cells. Topics covered are: B Cells; Organization and rearrangement of immunoglobin genes; Immunoglobin genes in disease; Immunoglobin gene expression; and Immunoglobin-related genes.

Honjo, T. (Kyoto Univ. (Japan)); Alt, F.W. (Columbia Univ., Dobbs Ferry, NY (USA). Hudson Labs.); Rabbitts, T.H. (Medical Research Council, Cambridge (UK))

1989-01-01

313

Mycobacterial Aerosols and Respiratory Disease  

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Environmental opportunistic mycobacteria, including Mycobacterium avium, M. terrae, and the new species M. immunogenum, have been implicated in outbreaks of hypersensitivity pneumonitis or respiratory problems in a wide variety of settings. One common feature of the outbreaks has been exposure to aerosols. Aerosols have been generated from metalworking fluid during machining and grinding operations as well as from indoor swimming pools, hot tubs, and water-damaged buildings. Environmental opp...

Falkinham, Joseph O.

2003-01-01

314

Diagnosis of nontuberculous mycobacterial infections.  

Science.gov (United States)

The nontuberculous mycobacteria (NTM) are typically environmental organisms residing in soil and water. Although generally of low pathogenicity to humans, NTM can cause a wide array of clinical diseases; pulmonary disease is most frequent, followed by lymphadenitis in children, skin disease by M. marinum (particularly in fish tank fanciers), and other extrapulmonary or disseminated infections in severely immunocompromised patients. Of the >140 NTM species reported in the literature, 25 species have been strongly associated with NTM diseases; the remainder are environmental organisms rarely encountered in clinical samples. Correct species identification is very important because NTM species differ in their clinical relevance. Further, NTM differ strongly in their growth rate, temperature tolerance, and drug susceptibility. The diagnosis of NTM disease is complex and requires good communication between clinicians, radiologists, and microbiologists. Isolation of M. kansasii and (in northwestern Europe) M. malmoense from pulmonary specimens usually indicates disease, whereas Mycobacterium gordonae and, to a lesser extent, M. simiae or M. chelonae are typically contaminants rather than causative agents of true disease. Mycobacterium avium complex (MAC), M. xenopi, and M. abscessus form an intermediate category between these two extremes. This review covers the clinical and laboratory diagnosis of NTM diseases and particularities for the different disease types and patient populations. Because of limited sensitivity and specificity of symptoms, radiology, and direct microscopy of clinical samples, culture remains the gold standard. Yet culture is time consuming and demands the use of multiple media types and incubation temperatures to optimize the yield. Outside of reference centers, such elaborate culture algorithms are scarce. PMID:23460010

van Ingen, Jakko

2013-02-01

315

Infecciones micobacterianas en pacientes infectados por el virus de la inmunodeficiencia humana en Cali, Colombia / Mycobacterial infections in patients infected with human immunodeficiency virus in Cali, Colombia  

Scientific Electronic Library Online (English)

Full Text Available SciELO Public Health | Language: Spanish Abstract in spanish Se determinó la prevalencia de las infecciones por micobacterias en una muestra de 155 individuos infectados por el virus de la inmunodeficiencia humana (VIH) tratados en el Instituto de los Seguros Sociales (ISS) de Cali, Colombia. Se les realizó la prueba de la tuberculina (PPD 2UT RT23) y se inve [...] stigó activamente la presencia de micobacterias mediante microscopia directa y cultivo de sangre, orina, heces y aspirado gástrico; cuando así lo indicó el cuadro clínico, también se examinaron y cultivaron muestras de líquido cefalorraquídeo, médula ósea y esputo. La ausencia de reactividad a la tuberculina fue significativamente más frecuente en los pacientes que en los controles (91,3%, frente a 57,4%. ji² = 33; P = 0). La prevalencia de la tuberculosis fue de 6,5%, en comparación con 0,04% en los afiliados al ISS VIH-negativos (intervalo de confianza binomial exacto de 95%: 0,0313 a 0,1154%). Las micobacterias no tuberculosas (MNT), presentes en 43 pacientes, fueron significativamente más frecuentes que Mycobacterium tuberculosis (27,7% frente a 6,5%. ji² = 24,78; P = 0,000 001), pero solo fueron causa de enfermedad en algunos casos. Las especies más frecuentes fueron las del complejo M. avium-intracellulare. M. avium-intracellulare y M. fortuitum tuvieron una prevalencia total de 7,1% y fueron las MNT de mayor prevalencia como causantes de enfermedad en estos pacientes (4,5%); además fueron responsables de tres casos de infección diseminada. La enfermedad clínica por M. tuberculosis o MNT y la anergia completa a la tuberculina se asociaron al estadio IV de la infección por VIH y a los recuentos de linfocitos CD4 Abstract in english The prevalence of mycobacterial infections was determined in a sample of 155 individuals infected with human immunodeficiency virus (HIV) who were treated in the Social Security Institute (SSI) of Cali, Colombia. A tuberculin test (2 TU PPD RT23) was used, and the presence of mycobacteria was checke [...] d through direct microscopy and culturing blood, urine, feces, and gastric aspirate. When clinically indicated, samples of cerebrospinal fluid, bone marrow, and sputum were also examined and cultivated. The absence of reactivity to tuberculin was significantly more frequent in the patients than in the controls (91.3%, compared to 57.4%; chi² = 33, P = 0). The prevalence of tuberculosis was 6.5%, in comparison with 0.04% among a group of HIV-negative ISS members (exact binomial 95% confidence interval: 0.0313% to 0.1154%). Non- tuberculous mycobacteria (NTM), present in 43 patients, were significantly more frequent than Mycobacterium tuberculosis (27.7%, versus 6.5%; chi² = 24.78, P = 0.000 001), but they caused illness only in some cases. The most common species were those of the M. avium-intracellulare complex. M. avium-intracellulare and M. fortuitum had a total prevalence of 7.1% and were the most-prevalent NTM that caused disease in these patients (4.5%); they were also responsible for three cases of disseminated infection. Clinical disease caused by M. tuberculosis or NTM and complete tuberculin anergy were associated with stage-IV HIV infection and with CD4 lymphocyte counts

María del Pilar, Crespo; Raúl, Heli Corral; Alberto, Alzate; Gabriel, Carrasquilla; Nory, Sánchez.

316

É possível uma vacina gênica auxiliar no controle da tuberculose? / Could a DNA vaccine be useful in the control of tuberculosis?  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: Portuguese Abstract in portuguese Vacinas de DNA, ainda em fase de experimentação e testes clínicos, podem se tornar uma importante ferramenta de combate a doenças infecciosas para as quais, até hoje, não existe prevenção segura e eficaz, como a tuberculose. Nos últimos anos vários estudos têm sido dedicados ao desenvolvimento de va [...] cinas de DNA que codificam proteínas de micobactérias, entre as quais destacam-se as que codificam o antígeno 85 (Ag 85) e a proteína de choque térmico de 65 kDa (hsp65). Estes dois antígenos foram os mais estudados apresentando resultados bastante satisfatórios em ensaios pré-clínicos e com grande volume de dados registrados na literatura. Além de proteger contra infecção experimental por Mycobacterium tuberculosis virulenta, a vacina DNA-hsp65 também apresenta atividade terapêutica, ou seja, é capaz de curar os animais previamente infectados, inclusive aqueles com bacilos resistentes a múltiplas drogas. Esta vacina, hoje em avaliação clínica no Brasil também para o tratamento de câncer, é capaz de induzir a produção de citocinas de padrão Th1 tal como IFN- interferon-gama, associadas ao controle da doença. Além disso, a vacina de DNA-hsp65 é capaz de estimular clones de células CD8 citotóxicos e CD4 que podem ser caracterizados como células de memória sendo responsáveis por conferir imunidade duradoura contra a infecção. Quando utilizada na terapia da infecção, a vacina de DNA-hsp65 faz com que haja uma mudança no padrão de resposta imune, induzindo a secreção de citocinas de padrão Th1 criando um ambiente favorável à erradicação do bacilo. Os resultados demonstram ainda que a via de administração e a formulação na qual a vacina é administrada exerce fundamental influência no padrão e duração da resposta imune desencadeada. O conjunto de resultados hoje disponíveis mostra que uma vacina de DNA contra a tuberculose contribuirá de maneira significativa no controle desta doença. Abstract in english The DNA vaccines currently under pre-clinical and clinical development may prove to be important tools in combating infectious diseases, such as tuberculosis, for which no safe and effective form of prevention has yet been developed. In recent years, several studies have aimed to develop a DNA vacci [...] ne encoding mycobacterial proteins such as antigen 85 (Ag85) and the 65-kDa mycobacterial heat shock protein (hsp65). The latter is protective against virulent infection with Mycobacterium tuberculosis (including multidrug-resistant strains). The hsp65 DNA vaccine, currently under clinical evaluation in Brazil for cancer therapy, is able to induce the secretion of Th1 cytokines, such as gamma-interferon, associated with disease control. Furthermore, this vaccine stimulates cytotoxic CD8 and CD4 T-cell clones that can be characterized as memory cells, which are responsible for effective and long-lasting immunity against tuberculosis. When used as a therapeutic agent in inoculated mice, the hsp65 DNA vaccine promotes changes in the immunity profile, triggering the secretion of Th1 cytokines and establishing a favorable environment for the elimination of bacilli. The results also demonstrate that the route of administration, as well as the formulation in which the vaccine is administered, fundamentally influence the pattern and duration of the immune response induced. Taking all currently available data into account, we can conclude that a DNA vaccine against tuberculosis could contribute significantly to the control of the disease.

José Maciel, Rodrigues Júnior; Karla de Melo, Lima; Arlete Aparecida Martins Coelho, Castelo; Vânia Luiza Deperon Bonato, Martins; Sandra Aparecida dos, Santos; Lucia Helena, Faccioli; Célio Lopes, Silva.

317

É possível uma vacina gênica auxiliar no controle da tuberculose? Could a DNA vaccine be useful in the control of tuberculosis?  

Directory of Open Access Journals (Sweden)

Full Text Available Vacinas de DNA, ainda em fase de experimentação e testes clínicos, podem se tornar uma importante ferramenta de combate a doenças infecciosas para as quais, até hoje, não existe prevenção segura e eficaz, como a tuberculose. Nos últimos anos vários estudos têm sido dedicados ao desenvolvimento de vacinas de DNA que codificam proteínas de micobactérias, entre as quais destacam-se as que codificam o antígeno 85 (Ag 85 e a proteína de choque térmico de 65 kDa (hsp65. Estes dois antígenos foram os mais estudados apresentando resultados bastante satisfatórios em ensaios pré-clínicos e com grande volume de dados registrados na literatura. Além de proteger contra infecção experimental por Mycobacterium tuberculosis virulenta, a vacina DNA-hsp65 também apresenta atividade terapêutica, ou seja, é capaz de curar os animais previamente infectados, inclusive aqueles com bacilos resistentes a múltiplas drogas. Esta vacina, hoje em avaliação clínica no Brasil também para o tratamento de câncer, é capaz de induzir a produção de citocinas de padrão Th1 tal como IFN- interferon-gama, associadas ao controle da doença. Além disso, a vacina de DNA-hsp65 é capaz de estimular clones de células CD8 citotóxicos e CD4 que podem ser caracterizados como células de memória sendo responsáveis por conferir imunidade duradoura contra a infecção. Quando utilizada na terapia da infecção, a vacina de DNA-hsp65 faz com que haja uma mudança no padrão de resposta imune, induzindo a secreção de citocinas de padrão Th1 criando um ambiente favorável à erradicação do bacilo. Os resultados demonstram ainda que a via de administração e a formulação na qual a vacina é administrada exerce fundamental influência no padrão e duração da resposta imune desencadeada. O conjunto de resultados hoje disponíveis mostra que uma vacina de DNA contra a tuberculose contribuirá de maneira significativa no controle desta doença.The DNA vaccines currently under pre-clinical and clinical development may prove to be important tools in combating infectious diseases, such as tuberculosis, for which no safe and effective form of prevention has yet been developed. In recent years, several studies have aimed to develop a DNA vaccine encoding mycobacterial proteins such as antigen 85 (Ag85 and the 65-kDa mycobacterial heat shock protein (hsp65. The latter is protective against virulent infection with Mycobacterium tuberculosis (including multidrug-resistant strains. The hsp65 DNA vaccine, currently under clinical evaluation in Brazil for cancer therapy, is able to induce the secretion of Th1 cytokines, such as gamma-interferon, associated with disease control. Furthermore, this vaccine stimulates cytotoxic CD8 and CD4 T-cell clones that can be characterized as memory cells, which are responsible for effective and long-lasting immunity against tuberculosis. When used as a therapeutic agent in inoculated mice, the hsp65 DNA vaccine promotes changes in the immunity profile, triggering the secretion of Th1 cytokines and establishing a favorable environment for the elimination of bacilli. The results also demonstrate that the route of administration, as well as the formulation in which the vaccine is administered, fundamentally influence the pattern and duration of the immune response induced. Taking all currently available data into account, we can conclude that a DNA vaccine against tuberculosis could contribute significantly to the control of the disease.

José Maciel Rodrigues Júnior

2004-08-01

318

Isolation of acid-inducible genes of Mycobacterium tuberculosis with the use of recombinase-based in vivo expression technology.  

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A better understanding of mycobacterial gene regulation under certain stress conditions (e.g., low pH) may provide insight into mechanisms of adaptation during infection. To identify mycobacterial promoters induced at low pH, we adapted the recombinase-based in vivo expression technology (RIVET) promoter trap system for use with mycobacteria. Our results show that the TnpR recombinase of transposon gammadelta is active in Mycobacterium smegmatis and Mycobacterium tuberculosis. We developed a method to perform sequential double selection with mycobacteria by using RIVET, with a kanamycin preselection and a sucrose postselection. A library of M. tuberculosis DNA inserted upstream of tnpR was created, and using the double selection, we identified two promoters which are upregulated at low pH. The promoter regions drive the expression of a gene encoding a putative lipase, lipF (Rv3487c), as well as a PE-PGRS gene, Rv0834c, in a pH-dependent manner in both M. smegmatis and M. tuberculosis. The acid inducibility of lipF and Rv0834c was independent of the stress response sigma factor, SigF, as acid induction of the two genes in an M. tuberculosis sigF mutant strain was similar to that in the wild-type strain. No induction of lipF or Rv0834c was observed during infection of J774 murine macrophages, an observation which is in agreement with previous reports on the failure of phagosomes containing M. tuberculosis to acidify. PMID:12595455

Saviola, Beatrice; Woolwine, Samuel C; Bishai, William R

2003-03-01

319

Patients with inhibitory and neutralizing auto-antibodies to interferon-? resemble the sporadic adult-onset phenotype of Mendelian Susceptibility to Mycobacterial Disease (MSMD) lacking Bacille Calmette-Guerin (BCG)-induced diseases.  

Science.gov (United States)

To recognize patients with inhibitory and neutralizing auto-antibodies to interferon-? (AutoAbs-IFN-?) presenting with the sporadic phenotype of Mendelian Susceptibility to Mycobacterial Disease (MSMD) mainly characterized by recurrent intracellular mycobacterium or/and salmonella infections, we comprehensively investigated IL12/23-IFN-? signaling, candidate genetic sequencings or/and protein expressions of IL12RB1, IFNRG1, IL12p40, IFNRG2, STAT1, IKKA, NEMO, CYBB and IRF8 in four patients. Their serum was further titrated to detect AutoAbs-IFN-?, for which the biological activity was assessed in Jurkat T cells. The patients mainly presented with recurrent non-tuberculous mycobacterium osteomyelitis and lymphadenopathy (Mycobacterium abscessus, chelonae and avium intracellular complex), and salmonella sepsis (S. enterica serogroup B, C2 and D). Additionally, Penicillium marneffei, varicella-zoster virus, and herpes simplex virus infections occurred. Inhibitory and neutralizing IFN-? downstream signaling was elucidated in Jurkat cell lines as decreased MHC class I and phosphorylated STAT1 expression. Together with 24 patients from the PubMed search, the majority of the AutoAbs-IFN-? patients were Asian (25/28). The most common involvement was lymph nodes (in 22/28), lungs (19/28) and bones (12/28). Mycobacterium avium complex (in 14) and chelonae (7) were the most common pathogens from 40 isolations. In contrast to those with the mild form of MSMD phenotype, AutoAbs-IFN-? patients, in the absence of BCG-induced diseases, had a more persistent course and poor response to IFN-? treatment. In conclusion, AutoAbs-IFN-? patients may have a sporadic adult-onset MSMD phenotype in Asian regions endemic for mycobacterial infections. PMID:23083630

Lee, Wen-I; Huang, Jing-Long; Wu, Ting-Shu; Lee, Ming-Hsun; Chen, I-Jung; Yu, Kuang-Hiu; Liu, Chien-Ying; Yang, Chih-Hsun; Hsieh, Meng-Ying; Lin, Yi-Ling; Shih, Ying-Fan; Jaing, Tang-Her; Huang, Shih-Chiang; Kuo, Tseng-Tong; Ku, Cheng-Lung

2013-05-01

320

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-p [...] erform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

Letícia Muraro, Wildner; Maria Luiza, Bazzo; Susie Coutinho, Liedke; Christiane Lourenço, Nogueira; Gabriela, Segat; Simone Gonçalves, Senna; Aline Daiane, Schlindwein; Jaquelline Germano de, Oliveira; Darcita B, Rovaris; Claudio A, Bonjardim; Erna G, Kroon; Paulo CP, Ferreira.

 
 
 
 
321

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-p [...] erform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

Wildner, Letícia Muraro; Bazzo, Maria Luiza; Liedke, Susie Coutinho; Nogueira, Christiane Lourenço; Segat, Gabriela; Senna, Simone Gonçalves; Schlindwein, Aline Daiane; Oliveira, Jaquelline Germano de; Rovaris, Darcita B; Bonjardim, Claudio A; Kroon, Erna G; Ferreira, Paulo CP.

2014-05-07

322

Endocrine genes  

Energy Technology Data Exchange (ETDEWEB)

This book contains 13 chapters. Some of the titles are: Gene Transfer and Expression of Mammalian Cell Receptors; Mapping Endocrine Genes with Sorted Human Chromosomes; Structure, Function, Hormonal Regulation of Steroidogenic Enzyme Genes; Molecular Analysis of Steroid Hormone Action Using the Human Metallothionein Genes as a Model.

Lau, Y.F.

1988-01-01

323

Efficient and simple generation of unmarked gene deletions in Mycobacterium smegmatis.  

Science.gov (United States)

Genetic research in molecular laboratories relies heavily on directed mutagenesis and gene deletion techniques. In mycobacteria, however, genetic analysis is often hindered by difficulties in the preparation of deletion mutants. Indeed, in comparison to the allelic exchange systems available for the study of other common model organisms, such as Saccharomyces cerevisiae and Escherichia coli, mycobacterial gene disruption systems suffer from low mutant isolation success rates, mostly due to inefficient homologous recombination and a high degree of non-specific recombination. Here, we present a gene deletion system that combines efficient homologous recombination with advanced screening of mutants. This novel methodology allows for gene disruption in three consecutive steps. The first step relies on the use of phage Che9c recombineering proteins for directed insertion into the chromosome of a linear DNA fragment that encodes GFP and confers hygromycin resistance. In the second step, GFP positive and hygromycin resistant colonies are selected, and in the last step, the gfp-hyg cassette is excised from the chromosome, thus resulting in the formation of an unmarked deletion. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the prcSBA operon of Mycobacterium smegmatis. PMID:24100088

Shenkerman, Yael; Elharar, Yifat; Vishkautzan, Marina; Gur, Eyal

2014-01-01

324

Gene cooption in Mycobacteria and search for virulence attributes: Comparative proteomic analyses of Mycobacterium tuberculosis, Mycobacterium indicus pranii and other mycobacteria.  

Science.gov (United States)

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a leading infectious disease taking one human life every 15s globally. Mycobacterium undergoes reductive evolution; the ancestors have bigger genome size and rich in metabolic pathways. Mycobacterium indicus pranii (MIP) is placed much above Mycobacterium tuberculosis (M.tb) in evolutionary scale and is a non-pathogenic, saprophytic mycobacterium. Our in silico comparative proteomic analyses of virulence factors of M.tb and their homologs in 12 different Mycobacterial species, including MIP, point toward gene cooption as an important mechanism in evolution of mycobacteria. We propose that adaptive changes in niche factors of non-pathogenic mycobacterium, together with novel gene acquisitions, are key players in the evolution of pathogenicity. Antigenic analyses between M.tb and MIP highlighted the importance of PE/PPE family in host immunomodulation, further supporting the likely potential of MIP as an effective vaccine against TB. PMID:24951307

Singh, Yadvir; Kohli, Sakshi; Sowpati, Divya Tej; Rahman, Syed Asad; Tyagi, Anil K; Hasnain, Seyed E

2014-07-01

325

Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA? †  

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Mycobacterium tuberculosis EsxA and EsxB proteins are founding members of the WXG100 (WXG) protein family, characterized by their small size (?100 amino acids) and conserved WXG amino acid motif. M. tuberculosis contains 11 tandem pairs of WXG genes; each gene pair is thought to be coexpressed to form a heterodimer. The precise role of these proteins in the biology of M. tuberculosis is unknown, but several of the heterodimers are secreted, which is important for virulence. However, WXG pro...

Callahan, Brian; Nguyen, Kiet; Collins, Alissa; Valdes, Kayla; Caplow, Michael; Crossman, David K.; Steyn, Adrie J. C.; Eisele, Leslie; Derbyshire, Keith M.

2010-01-01

326

Ulcera lingual como signo único de infección recurrente por micobacteria en un paciente con VIH/SIDA / Lingual ulcer as the only sign of recurrent mycobacterial infection in an HIV/AIDS-infected patient  

Scientific Electronic Library Online (English)

Full Text Available SciELO Spain | Language: Spanish Abstract in spanish Se describe un paciente con VIH/SIDA en el que se identificó una infección por micobacteria en la mucosa bucal, probablemente tuberculosis, en un centro de referencia para VIH/SIDA de la Ciudad de México. El propósito del presente informe es describir los hallazgos clínicos e histológicos en un paci [...] ente con VIH/SIDA, quien después de haber sido tratado exitosamente para tuberculosis ganglionar 4 años antes, presentó una úlcera lingual como único signo que sugirió recurrencia de infección por micobacteria, probablemente tuberculosis. Hombre de 39 años de edad, atendido desde 1991 en el Instituto Nacional de Ciencias Médicas y Nutrición "Salvador Zubirán", por el diagnóstico de infección con VIH. En 1999, el paciente presentó tuberculosis ganglionar, recibiendo tratamiento antifímico con involución de las adenopatías y desaparición de los síntomas sistémicos. En mayo del 2003 acudió a consulta por presentar una úlcera superficial en lengua, dolorosa, de 4 meses de evolución, de 0.7 cm. de diámetro, bien circunscrita, crateriforme, con bordes ligeramente elevados, irregulares e indurados. El estudio histopatológico mostró inflamación granulomatosa crónica con células gigantes multinucleadas sugestivas de infección por micobacteria, lo cual hizo pensar en recurrencia de tuberculosis, por lo que se indicó rifampicina, pirazinamida, etambutol y estreptomicina. En junio del 2003 el paciente inició TARAA, que incluyó dos ITRAN y un ITRNN. La lesión lingual evolucionó favorablemente, con cicatrización parcial a la primera semana y remisión total a los 45 días del inicio del tratamiento antifímico; a los 7 meses de seguimiento permanece sin lesión. El presente caso tiene la particularidad de que la úlcera lingual fue la única manifestación de infección por micobacteria, sugestiva de tuberculosis, en un paciente con VIH/SIDA, que pudo ocurrir como resultado de la recurrencia del episodio previo de TB ganglionar. Abstract in english The report describes an HIV/AIDS patient seen at a referral center in Mexico City, in whom a mycobacterial infection in the oral mucosa, probably tuberculosis (TB) was identified. The purpose is to describe the clinical and histological findings in an HIV-infected patient, who after being treated su [...] ccessfully for tuberculous lymphangitis 4 years ago, presented with a lingual ulcer as the only suggestive sign of recurrence of mycobacterial infection, probably M. tuberculosis. A 39-year-old man seen inthe HIV clinic of the Instituto Nacional de Ciencias Médicas y Nutrición "Salvador Zubirán" in Mexico City since 1991 for HIV infection. In 1999 the patient developed tuberculous lymphangitis; he was managed with a 4-drug regimen for 12 months, with improvement of local and systemic symptoms. In May of 2003, the patient presented a painful superficial lingual ulcer, 0.7 cm in diameter, well circumscribed, crateriform with slightly elevated, irregular and indurated borders, of 4 months duration. The histopathological examination showed chronic granulomatous inflammation with giant multinucleated cells, suggestive of mycobacterial infection, and recurrence of TB was considered. Rifampin, isoniazide, pyrazinamide, ethambutol and streptomycin were administered. The lingual lesion improved with partial healing at the first week and total remission at 45 days after the beginning of the antituberculous treatment. In June, 2003, the patient began highly active antiretroviral therapy (HAART) that included two NRTIs and one NNRTI. At 7 months of follow-up, the patient remains free of lingual lesions. The particularity of the present case is that the lingual ulcer was the only sign of infection by mycobacteria, suggestive of TB, in an HIV/AIDS patient that probably represented a recurrence of a previous episode.

Velia, Ramírez Amador; Gabriela, Anaya Saavedra; Imelda, González Ramírez; Juan Luis, Mosqueda Gómez; Lilly, Esquivel Pedraza; Edgardo, Reyes Gutiérrez; Juan, Sierra Madero.

327

Gene expression  

International Nuclear Information System (INIS)

We prepared probes for isolating functional pieces of the metallothionein locus. The probes enabled a variety of experiments, eventually revealing two mechanisms for metallothionein gene expression, the order of the DNA coding units at the locus, and the location of the gene site in its chromosome. Once the switch regulating metallothionein synthesis was located, it could be joined by recombinant DNA methods to other, unrelated genes, then reintroduced into cells by gene-transfer techniques. The expression of these recombinant genes could then be induced by exposing the cells to Zn2+ or Cd2+. We would thus take advantage of the clearly defined switching properties of the metallothionein gene to manipulate the expression of other, perhaps normally constitutive, genes. Already, despite an incomplete understanding of how the regulatory switch of the metallothionein locus operates, such experiments have been performed successfully

1983-01-01

328

Piperidinols That Show Anti-Tubercular Activity as Inhibitors of Arylamine N-Acetyltransferase: An Essential Enzyme for Mycobacterial Survival Inside Macrophages  

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Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of tuberculosis (TB). Cholesterol plays a critical role in the persistence of M. tuberculosis within the macrophage during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the cholesterol sterol-ring degradation and is e...

Abuhammad, Areej; Fullam, Elizabeth; Lowe, Edward D.; Staunton, David; Kawamura, Akane; Westwood, Isaac M.; Bhakta, Sanjib; Garner, Alun Christopher; Wilson, David L.; Seden, Peter T.; Davies, Stephen G.; Russell, Angela J.; Garman, Elspeth F.; Sim, Edith

2012-01-01

329

Clinical and laboratory features of Mycobacterium porcinum.  

Science.gov (United States)

Recent molecular studies have shown Mycobacterium porcinum, recovered from cases of lymphadenitis in swine, to have complete 16S rDNA sequence identity and >70% DNA-DNA homology with human isolates within the M. fortuitum third biovariant complex. We identified 67 clinical and two environmental isolates of the M. fortuitum third biovariant sorbitol-negative group, of which 48 (70%) had the same PCR restriction enzyme analysis (PRA) profile as the hsp65 gene of M. porcinum (ATCC 33776(T)) and were studied in more detail. Most U.S. patient isolates were from Texas (44%), Florida (19%), or other southern coastal states (15%). Clinical infections included wound infections (62%), central catheter infections and/or bacteremia (16%), and possible pneumonitis (18%). Sequencing of the 16S rRNA gene (1,463 bp) showed 100% identity with M. porcinum ATCC 33776(T). Sequencing of 441 bp of the hsp65 gene showed four sequevars that differed by 2 to 3 bp from the porcine strains. Clinical isolates were positive for arylsulfatase activity at 3 days, nitrate, iron uptake, D-mannitol, i-myo-inositol, and catalase at 68 degrees C. They were negative for L-rhamnose and D-glucitol (sorbitol). Clinical isolates were susceptible to ciprofloxacin, sulfamethoxazole, and linezolid and susceptible or intermediate to cefoxitin, clarithromycin, imipenem, and amikacin. M. porcinum ATCC 33776(T) gave similar results except for being nitrate negative. These studies showed almost complete phenotypic and molecular identity between clinical isolates of the M. fortuitum third biovariant D-sorbitol-negative group and porcine strains of M. porcinum and confirmed that they belong to the same species. Identification of M. porcinum presently requires hsp65 gene PRA or 16S rRNA or hsp65 gene sequencing. PMID:15583300

Wallace, Richard J; Brown-Elliott, Barbara A; Wilson, Rebecca W; Mann, Linda; Hall, Leslie; Zhang, Yansheng; Jost, Kenneth C; Brown, June M; Kabani, Amin; Schinsky, Mark F; Steigerwalt, Arnold G; Crist, Christopher J; Roberts, Glenn D; Blacklock, Zeta; Tsukamura, Michio; Silcox, Vella; Turenne, Christine

2004-12-01

330

Gene Modifications  

Science.gov (United States)

This animation shows how a gene is constructed to eventually produce a protein in a Bt corn plant. This is the fifth of a series of seven animations that detail the process of crop genetic engineering. To begin at the beginning, see Overview of Crop Genetic Engineering. (To return to the animation previous to this, go to Gene Regions. To go to the next animation, go to Gene Gun.)

331

Identification of Mycobacteria from Solid and Liquid Media by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in the Clinical Laboratory  

Science.gov (United States)

Mycobacteria cause significant morbidity in humans. Rapid and accurate mycobacterial identification is important for improvement of patient outcomes. However, identification may be challenging due to the slow and fastidious growth of mycobacteria. Several diagnostic methods, such as biochemical, sequencing, and probe methods, are used for mycobacterial identification. We compared the matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) Biotyper system (Bruker Daltonics) to 16S rRNA/hsp65 sequencing and/or DNA probes (Gen-Probe) for mycobacterial identification. One hundred seventy-eight mycobacterial isolates grown on solid and/or broth medium were included in the study. MALDI-TOF MS identified 93.8% of the mycobacteria isolates accurately to the species level and 98.3% to the genus level, independent of the type of medium used for isolation. The identification of mycobacteria directly from cultures using MALDI-TOF MS allows for precise identification in an hour compared to traditional biochemical and phenotypic methods that can take weeks or probes and sequencing that may take a few hours. Identification by MALDI-TOF MS potentially reduces the turnaround time and cost, thereby saving resources within the health care system.

Kamboj, Kamal; Pancholi, Preeti

2013-01-01

332

Development of a Real-Time qPCR Method for Detection and Enumeration of Mycobacterium spp. in Surface Water ? †  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A real-time quantitative PCR method was developed for the detection and enumeration of Mycobacterium spp. from environmental samples and was compared to two other methods already described. The results showed that our method, targeting 16S rRNA, was more specific than the two previously published real-time quantitative PCR methods targeting another 16S rRNA locus and the hsp65 gene (100% versus 44% and 91%, respectively).

2010-01-01

333

An Outbreak of Keratitis Caused by Mycobacterium immunogenum  

Digital Repository Infrastructure Vision for European Research (DRIVER)

From 8 October to 12 November 2003, 36 patients underwent surgical correction of myopia in a São Paulo, Brazil, clinic. Five patients had clinical signs of infectious keratitis, and a Mycobacterium species with previously unreported patterns determined by PCR restriction enzyme analysis of the hsp65 gene and PCR restriction enzyme analysis of the 16S-23S rRNA internal transcribed spacer (ITS) was isolated from corneal scrapings from four of these patients. Subsequent evaluation by phenotypic...

2006-01-01

334

Gene therapy.  

Science.gov (United States)

Applications of gene therapy have been evaluated in virtually every oral tissue, and many of these have proved successful at least in animal models. While gene therapy will not be used routinely in the next decade, practitioners of oral medicine should be aware of the potential of this novel type of treatment that doubtless will benefit many patients with oral diseases. PMID:24372817

Baum, B J

2014-03-01

335

A Mycobacterium tuberculosis gene cluster encoding proteins of a phosphate transporter homologous to the Escherichia coli Pst system.  

Science.gov (United States)

We report the cloning and sequencing of three M. tuberculosis genes encoding proteins homologous to E. coli PstA, PstC and PstB. They are tentatively called pstA-2, pstC-1 and pstB. They encode proteins of 302, 336 and 275 amino acids, respectively. In E. coli, PstB is the ATP binding component and PstA/PstC are the two hydrophobic subunits of a phosphate permease belonging to the family of ABC (ATP-binding cassette) transporters. In mycobacteria, PstS-1, the phosphate binding subunit (Andersen and Hansen, 1989), is encoded by a gene directly surrounded by pstB, pstC-1 and pstA-2 within a potential operon (pstB, pstS-1, pstC-1, pstA-2). Phosphate uptake by whole, suspension grown, M. bovis BCG cells was measured and could be inhibited by a monoclonal antibody directed against the PstS-1 subunit, suggesting that these genes encode subunits of a functional mycobacterial phosphate permease. PMID:8918249

Braibant, M; Lefèvre, P; de Wit, L; Peirs, P; Ooms, J; Huygen, K; Andersen, A B; Content, J

1996-10-17

336

STRESS AND ATHEROSCLEROSIS: MAY HSP60 BE THE MOLECULAR LINK?  

Directory of Open Access Journals (Sweden)

Full Text Available In last decades, incidence of cardiovascular diseases is increased. Among them, atherosclerosis is one of the most commons. It is a disorder of in- flammation and innate immunity following lipid accumulation. From a bio- logic perspective, the process of adhesion and transmigration of immune cells (monocytes and macrophages across the endothelium is a crucial step for atherogenesis and mature plaque rupture. Moreover, there is a relationship between inflammation, infection, autoimmunity and athero- sclerosis. Inflammation has received increasing attention in recent years as a cause of atherosclerosis and cardiovascular diseases. Autoimmune diseases are characterized by enhanced atherosclerosis. Humoral immune responses to mycobacterial Hsp65, as well as to human Hsp60 and oxLDL, have been established in a number of human autoimmune diseases and are considered to be significantly associated also with atherosclerosis.

Luana Lipari

2009-01-01

337

???: GeneMark  

Full Text Available GeneMark GeneMark.hmm Webgenemark Webgenemark.hmm GenMark???????????????????? 5'?????? ?????????????????? ?????|1000 ????/????|141150000 the Georgia Institute of Technology |http://opal.biology.gatech.edu/GeneMark/ GeneMark.

338

???: PubGene  

Full Text Available 01000 01200 ???????????? | ???? PubGene WWW PubGene Inc.|http://www.pubgene.com/ ???? ??????????????? ?????????????????? ???????? ???????????????????????????? ??????????????????? ???????

339

The changing pattern of nontuberculous mycobacterial disease  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Nontuberculous mycobacteria are human opportunistic pathogens whose source of infection is the environment. These include both slow-growing (eg, Mycobacterium kansasii and Mycobacterium avium) and rapid-growing (eg, Mycobacterium abscessus and Mycobacterium fortuitum) species. Transmission is through ingestion or inhalation of water, particulate matter or aerosols, or through trauma. The historic presentation of pulmonary disease in older individuals with predisposing lung conditions and in c...

Falkinham, Joseph O.

2003-01-01

340

Mycobacterial cross contamination during radiometric culturing.  

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Pseudobacteremias in blood cultures performed on the BACTEC radiometric blood culture system have been reported. We report three cases of cross contamination with Mycobacterium avium that occurred when mycobacteria were cultured with the BACTEC 460 TB system. Malfunction of the needle sterilization heating block was demonstrated.

Vannier, A. M.; Tarrand, J. J.; Murray, P. R.

1988-01-01

 
 
 
 
341

A highly conserved mycobacterial cholesterol catabolic pathway.  

Science.gov (United States)

Degradation of the cholesterol side-chain in Mycobacterium tuberculosis is initiated by two cytochromes P450, CYP125A1 and CYP142A1, that sequentially oxidize C26 to the alcohol, aldehyde and acid metabolites. Here we report characterization of the homologous enzymes CYP125A3 and CYP142A2 from Mycobacterium smegmatis mc(2) 155. Heterologously expressed, purified CYP125A3 and CYP142A2 bound cholesterol, 4-cholesten-3-one, and antifungal azole drugs. CYP125A3 or CYP142A2 reconstituted with spinach ferredoxin and ferredoxin reductase efficiently hydroxylated 4-cholesten-3-one to the C-26 alcohol and subsequently to the acid. The X-ray structures of both substrate-free CYP125A3 and CYP142A2 and of cholest-4-en-3-one-bound CYP142A2 reveal significant differences in the substrate binding sites compared with the homologous M.?tuberculosis proteins. Deletion only of cyp125A3 causes a reduction of both the alcohol and acid metabolites and a strong induction of cyp142 at the mRNA and protein levels, indicating that CYP142A2 serves as a functionally redundant back up enzyme for CYP125A3. In contrast to M.?tuberculosis, the M.?smegmatis??cyp125?cyp142 double mutant retains its ability to grow on cholesterol albeit with a diminished capacity, indicating an additional level of redundancy within its genome. PMID:23489718

García-Fernández, Esther; Frank, Daniel J; Galán, Beatriz; Kells, Petrea M; Podust, Larissa M; García, José L; Ortiz de Montellano, Paul R

2013-08-01

342

Assessment of mycobacterial viability by RNA amplification.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We investigated whether the presence of intact RNA is a valuable indicator of viability of mycobacteria with Mycobacterium smegmatis. M. smegmatis was exposed to various concentrations of rifampin and ofloxacin suspended in broth for different periods of time. The NASBA nucleic acid amplification system was used because of its rapid, sensitive, and specific detection of 16S rRNA. During drug exposure, the viability of the mycobacteria, expressed by the number of CFU, was compared with the pre...

Vliet, G. M.; Schepers, P.; Schukkink, R. A.; Gemen, B.; Klatser, P. R.

1994-01-01

343

Antigen stimulation of peripheral blood mononuclear cells from Mycobacterium bovis infected cattle yields evidence for a novel gene expression program  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Bovine tuberculosis (BTB caused by Mycobacterium bovis continues to cause substantial losses to global agriculture and has significant repercussions for human health. The advent of high throughput genomics has facilitated large scale gene expression analyses that present a novel opportunity for revealing the molecular mechanisms underlying mycobacterial infection. Using this approach, we have previously shown that innate immune genes in peripheral blood mononuclear cells (PBMC from BTB-infected animals are repressed in vivo in the absence of exogenous antigen stimulation. In the present study, we hypothesized that the PBMC from BTB-infected cattle would display a distinct gene expression program resulting from exposure to M. bovis. A functional genomics approach was used to examine the immune response of BTB-infected (n = 6 and healthy control (n = 6 cattle to stimulation with bovine tuberculin (purified protein derivative – PPD-b in vitro. PBMC were harvested before, and at 3 h and 12 h post in vitro stimulation with bovine tuberculin. Gene expression changes were catalogued within each group using a reference hybridization design and a targeted immunospecific cDNA microarray platform (BOTL-5 with 4,800 spot features representing 1,391 genes. Results 250 gene spot features were significantly differentially expressed in BTB-infected animals at 3 h post-stimulation contrasting with only 88 gene spot features in the non-infected control animals (P ? 0.05. At 12 h post-stimulation, 56 and 80 gene spot features were differentially expressed in both groups respectively. The results provided evidence of a proinflammatory gene expression profile in PBMC from BTB-infected animals in response to antigen stimulation. Furthermore, a common panel of eighteen genes, including transcription factors were significantly expressed in opposite directions in both groups. Real-time quantitative reverse transcription PCR (qRT-PCR demonstrated that many innate immune genes, including components of the TLR pathway and cytokines were differentially expressed in BTB-infected (n = 8 versus control animals (n = 8 after stimulation with bovine tuberculin. Conclusion The PBMC from BTB-infected animals exhibit different transcriptional profiles compared with PBMC from healthy control animals in response to M. bovis antigen stimulation, providing evidence of a novel gene expression program due to M. bovis exposure.

Zhao Yingdong

2008-09-01

344

Treatment of bladder carcinomas using recombinant BCG DNA vaccines and electroporative gene immunotherapy.  

Science.gov (United States)

Intravesical immunotherapy with live Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the treatment of choice for superficial bladder cancers. Nevertheless, a significant proportion of patients do not respond to this therapy, and adverse effects are common. Here, we report the cloning of recombinant mycobacterial DNA vaccines and demonstrate the ability of multicomponent and multisubunit DNA vaccines to enhance Th1-polarized cytokine-mediated responses as well as effector cell responses. Splenocytes from immunized groups of mice were restimulated in vitro and examined for cytotoxicity against murine bladder tumur (MBT-2) cells. We used four combined recombinant BCG DNA vaccines (poly-rBCG) for electroporative gene immunotherapy (EPGIT) in vivo, and found that tumor growth was significantly inhibited and mouse survival was prolonged. Increased immune cell infiltration and induction of apoptosis were noted after treatment with poly-rBCG alone, with the murine interleukin-12 (mIL-12) vaccine alone, and-most significantly-with the poly-rBCG+mIL-12 vaccine combination. Electroporation of poly-rBCG+mIL-12 resulted in complete tumor eradication in seven of eight mice (PBCG is highly effective in the treatment of bladder cancer. This approach presents new possibilities for the treatment of bladder cancer using recombinant BCG DNA vaccines. PMID:14973549

Lee, Chi-Feng; Chang, Sun-Yran; Hsieh, Dar-Shih; Yu, Dah-Shyong

2004-03-01

345

Identification of polymorphisms and sequence variants in the human homologue of the mouse natural resistance-associated macrophage protein gene  

Energy Technology Data Exchange (ETDEWEB)

The most common mycobacterial disease in humans is tuberculosis, and there is evidence for genetic factors in susceptibility to tuberculosis. In the mouse, the Bcg gene controls macrophage priming for activation and is a major gene for susceptibility to infection with mycobacteria. A candidate gene for Bcg was identified by positional cloning and was designated {open_quotes}natural resistance-associated macrophage protein gene{close_quotes} (Nramp1), and the human homologue (NRAMP1) has recently been cloned. Here we report (1) the physical mapping NRAMP1 close to VIL in chromosome region 2q35 by PCR analysis of somatic cell hybrids and YAC cloning and (2) the identification of nine sequence variants in NRAMP1. Of the four variants in the coding region, there were two missense mutations and two silent substitutions. The missense mutations were a conservative alanine-to-valine substitution at codon 318 in exon9 and an aspartic acid-to-asparagine substitution at codon 543 in the predicted cytoplasmic tail of the NRAMP1 protein. A microsatellite was located in the immediate 5{prime} region of the gene, three variants were in introns, and one variant was located in the 3{prime} UTR. The allele frequencies of each of the nine variants were determined in DNA samples of 60 Caucasians and 20 Asians. In addition, we have physically linked two highly polymorphic microsatellite markers, D2S104 and D2S173, to NRAMP1 on a 1.5-Mb YAC contig. These molecular markers will be useful to assess the role of NRAMP1 in susceptibility to tuberculosis and other macrophage-mediated diseases. 40 refs., 3 figs., 2 tabs.

Liu, Jing; Fujiwara, T.M.; Buu, N.T.; Sanchez, F.O.; Cellier, M.; Paradis, A.J.; Frappier, D.; Skamene, E.; Gros, P.; Morgan, K. [McGill Univ., Montreal (Canada)

1995-04-01

346

Comparative genomic analysis of sixty mycobacteriophage genomes: Genome clustering, gene acquisition and gene size  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Mycobacteriophages are viruses that infect mycobacterial hosts. Expansion of a collection of sequenced phage genomes to a total of sixty – all infecting a common bacterial host – provides further insight into their diversity and evolution. Of the sixty phage genomes, 55 can be grouped into nine clusters according to their nucleotide sequence similarities, five of which can be further divided into subclusters; five genomes do not cluster with other phages. The sequence diversity between ge...

Hatfull, Graham F.; Jacobs-sera, Deborah; Lawrence, Jeffrey G.; Pope, Welkin H.; Russell, Daniel A.; Ko, Ching-chung; Weber, Rebecca J.; Patel, Manisha C.; Germane, Katherine L.; Edgar, Robert H.; Hoyte, Natasha N.; Bowman, Charles A.; Tantoco, Anthony T.; Paladin, Elizabeth C.; Myers, Marlana S.

2010-01-01

347

Genes and Hearing Loss  

Science.gov (United States)

... mutation may only have dystopia canthorum. How Do Genes Work? Genes are a road map for the synthesis of ... lung, etc. Every child inherits half of its genes from one parent and half from the other ...

348

???: GeneMark.hmm  

Full Text Available 05000 05300 05301 ?????? | ?????????????? | Ab initio? GeneMark.hmm WWW the Georgia Institute of Technology |http://opal.biology.gatech.edu/GeneMark/ GenMark? ????????????? ??????5'?????? ?????????????????? GeneMark.hmm: n

349

Gene conversion in steroid 21-hydroxylase genes.  

Science.gov (United States)

The steroid 21-hydroxylase gene, CYP21B, encodes cytochrome P450c21, which mediates 21-hydroxylation. The gene is located about 30 kb downstream from pseudogene CYP21A. The CYP21A gene is homologous to the CYP21B gene but contains some mutations, including a C----T change which leads a termination codon, TAG, in the eighth exon. We found the same change in a mutant CYP21B gene isolated from a patient with 21-hydroxylase deficiency. Furthermore, a reciprocal change--i.e., a T----C change in the eighth exon of the CYP21A gene--was observed in the Japanese population and was associated with the two HLA haplotypes, HLA-B44-DRw13 and HLA-Bw46-DRw8. These changes may be considered the result of gene conversion-like events. PMID:1971153

Urabe, K; Kimura, A; Harada, F; Iwanaga, T; Sasazuki, T

1990-06-01

350

Essential Bacillus subtilis genes  

Digital Repository Infrastructure Vision for European Research (DRIVER)

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among ?4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the syn...

Kobayashi, Kazuo; Ehrlich, Stanislav Dusko; Deuerling, Elke

2003-01-01

351

Mycobacterium Bovis Ornithine Carbamoyltransferase, MB1684, Induces Proinflammatory Cytokine Gene Expression by Activating NF-?B in Macrophages.  

Science.gov (United States)

Mycobacterium bovis is the etiological factor of bovine tuberculosis (BTB), posing a significant problem to domestic cattle. The bacterium is also zoonotic, affecting human health worldwide. Macrophage evasion of the bacterium involves mycobacterial molecules such as MB1684 (ornithine carbamoyltransferase). In this study, we confirmed a concentration-dependent decrease in proliferation of Ana-1 macrophages when treated with rMB1684 when compared with mycobacterium bovis purified protein derivative of tuberculosis (MbPPD) or phosphate buffer solution incubation groups. We examined the activation of nuclear factor-kappa B (NF-?B) upon exposure to MB1684, and its role in MB1684-induced upregulation of interferon (IFN)-? and proinflammatory cytokines (interleukin [IL]-1?, IL-6, and tumor necrosis factor-?) in Ana-1 macrophages. The levels of proinflammatory cytokines and IFN-? were significantly high in MB1684-treated Ana-1 macrophages. The treatment led to an increase in NF-?B activation and a high expression of the just mentioned proinflammatory cytokines. NF-?B inhibition significantly abrogated MB1684-induced upregulation of proinflammatory cytokine mRNA expression, which suggests that MB1684-induced activation of NF-?B in turn stimulates gene expression of IFN-? and proinflammatory cytokines in Ana-1 macrophages. The experiment was repeated in bone marrow macrophages, a more in-vivo-like model system, and similar results validated our conclusion. Further, we identified the possibility of the application of MB1684 antigen for the detection of BTB in cattle serum. PMID:24568683

Zhao, Wei; Zhou, Xiangmei; Lu, Yun; Peng, Yun; Lin, Zhu; Lin, Jingjun; Yang, Lifeng; Yin, Xiaomin; Zhao, Deming

2014-05-01

352

???: GeneCluster  

Full Text Available GeneCluster ?????????k?????SOM????????????????? ?????|100000 000 > ????|120000000 > ????????????? |122000000 MIT|http://www.broad.mit.edu/cancer/software/ genecluster2/gc2.html GeneCluster 2.0 : an advanced toolset for bioarray analysis.

353

???: GeneFisher2  

Full Text Available 08000 08300 ?????PCR?????? | ??????? GeneFisher2 WWW Univ. Bielefeld|http://bi or the detection of postulated genes. Giegerich R, Meyer F, Schleiermacher C. Proc Int Conf Intell Syst Mol

354

Understanding Cancer Series: Gene Testing  

Science.gov (United States)

... Disease Inheritance Is Complex Gene Tests - Three Common Methods Genetic Tests Serve Many Purposes Microarray Analysis Human Genome Project Studying Genes Searching Disease Families Disease-Linked Genes ...

355

Nocardia altamirensis sp. nov., isolated from Altamira Cave, Cantabria, Spain  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A novel actinomycete strain, OFN S17T, was isolated from a sample collected from Altamira Cave, Cantabria, Spain. This strain was identified by using a polyphasic taxonomic approach. The 16S rRNA, hsp65 and sod gene sequences of the strain were determined and compared with those of representative Nocardia species. The results showed that strain OFN S17T should be assigned to the genus Nocardia. Phylogenetic analysis indicated that strain OFN S17T was most closely related to the type strain of...

2008-01-01

356

Clinical and Laboratory Features of Mycobacterium porcinum†  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recent molecular studies have shown Mycobacterium porcinum, recovered from cases of lymphadenitis in swine, to have complete 16S rDNA sequence identity and >70% DNA-DNA homology with human isolates within the M. fortuitum third biovariant complex. We identified 67 clinical and two environmental isolates of the M. fortuitum third biovariant sorbitol-negative group, of which 48 (70%) had the same PCR restriction enzyme analysis (PRA) profile as the hsp65 gene of M. porcinum (ATCC 33776T) and we...

2004-01-01

357

Discovering genes underlying QTL  

International Nuclear Information System (INIS)

A map-based approach has allowed scientists to discover few genes at a time. In addition, the reproductive barrier between cultivated rice and wild relatives has prevented us from utilizing the germ plasm by a map-based approach. Most genetic traits important to agriculture or human diseases are manifested as observable, quantitative phenotypes called Quantitative Trait Loci (QTL). In many instances, the complexity of the phenotype/genotype interaction and the general lack of clearly identifiable gene products render the direct molecular cloning approach ineffective, thus additional strategies like genome mapping are required to identify the QTL in question. Genome mapping requires no prior knowledge of the gene function, but utilizes statistical methods to identify the most likely gene location. To completely characterize genes of interest, the initially mapped region of a gene location will have to be narrowed down to a size that is suitable for cloning and sequencing. Strategies for gene identification within the critical region have to be applied after the sequencing of a potentially large clone or set of clones that contains this gene(s). Tremendous success of positional cloning has been shown for cloning many genes responsible for human diseases, including cystic fibrosis and muscular dystrophy as well as plant disease resistance genes. Genome and QTL mapping, positional cloning: the pre-genomics era, comparative approaches to gene identification, and positional cloning: the genomics era are discussed in the report. (M. Suetake)

2002-02-01

358

Cloning Human Chromosome 17 Genes: Candidate Genes for BRCA1.  

Science.gov (United States)

Our research interest is focused on the identification of genes from chromosome 17. The isolation of genes transcribed from chromosome 17 will provide candidates for the proposed sporadic breast cancer genes and genes for other human disorders.The ability...

C. Lee

1998-01-01

359

Down-weighting overlapping genes improves gene set analysis  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Abstract Background The identification of gene sets that are significantly impacted in a given condition based on microarray data is a crucial step in current life science research. Most gene set analysis methods treat genes equally, regardless how specific they are to a given gene set. Results In this work we propose a new gene set analysis method that computes a gene set score as the mean of absolute values of weighted moderated gene t-scores. The gene weights...

Tarca Adi; Draghici Sorin; Bhatti Gaurav; Romero Roberto

2012-01-01

360

The synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE liposomes and DOTAP/DOPE lipoplexes.  

Science.gov (United States)

We present a comparative study of the physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-complexed DNA in EPC/DOTAP/DOPE (50/25/25% molar) liposomes and DOTAP/DOPE (50/50% molar) lipoplexes. The study aims to correlate the biological behavior and structural properties of the lipid carriers. We used DNA-hsp65, whose naked action as a gene vaccine against tuberculosis has already been demonstrated. Additionally, surface-complexed DNA-hsp65 in EPC/DOTAP/DOPE (50/25/25% molar) liposomes was effective as a single-dose tuberculosis vaccine. The results obtained showed that the EPC inclusion stabilized the DOTAP/DOPE structure, producing higher melting temperature and lower zeta potential despite a close mean hydrodynamic diameter. Resemblances in morphologies were identified in both structures, although a higher fraction of loaded DNA was not electrostatically bound in EPC/DOTAP/DOPE. EPC also induced a striking reduction in cytotoxicity, similar to naked DNA-hsp65. The proper immune response lead to a polarized antibody production of the IgG2a isotype, even for the cytotoxic DOTAP/DOPE. However, the antibody production was detected at 15 and 30 days for DOTAP/DOPE and EPC/DOTAP/DOPE, respectively. Therefore, the in vivo antibody production neither correlates with the in vitro cytotoxicity, nor with the structural stability alone. The synergistic effect of the structural stability and DNA electrostatic binding upon the surface of structures account for the immunological effects. By adjusting the composition to generate proper packing and cationic lipid/DNA interaction, we allow for the optimization of liposome formulations for required immunization or gene therapy. In a specific manner, our results contribute to studies on the tuberculosis therapy and vaccination. PMID:19540734

de la Torre, Lucimara Gaziola; Rosada, Rogério Silva; Trombone, Ana Paula Fávaro; Frantz, Fabiani Gai; Coelho-Castelo, Arlete A M; Silva, Celio Lopes; Santana, Maria Helena Andrade

2009-10-15

 
 
 
 
361

Gene expression in fungi  

Directory of Open Access Journals (Sweden)

Full Text Available This contribution is based on the four presentations made at the Special Interest Group (SIG meeting titled Gene Expression in Fungi held during IMC9 in Edinburgh. This overview is independent from other articles published or that will be published by each speaker. In the SIG meeting, basic principles of in vivo animal models for virulence studies were discussed. Infection associated genes of Candida albicans and fungal adaptation to the host was summarized. Azole susceptibility was evaluated as a combined result of several changes in expression of pertinent genes. Gene transfer in fungi, resulting in fungal evolution and gene adaptation to environmental factors, was reported.

A. Kalkanci

2011-06-01

362

Evaluation of GeneXpert MTB/RIF for diagnosis of tuberculous meningitis.  

Science.gov (United States)

Tuberculous meningitis (TBM) is the most severe form of tuberculosis. Microbiological confirmation is rare, and treatment is often delayed, increasing mortality and morbidity. The GeneXpert MTB/RIF test was evaluated in a large cohort of patients with suspected tuberculous meningitis. Three hundred seventy-nine patients presenting with suspected tuberculous meningitis to the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam, between 17 April 2011 and 31 December 2012 were included in the study. Cerebrospinal fluid samples were tested by Ziehl-Neelsen smear, mycobacterial growth indicator tube (MGIT) culture, and Xpert MTB/RIF. Rifampin (RIF) resistance results by Xpert were confirmed by an MTBDR-Plus line probe assay and all positive cultures were tested by phenotypic MGIT drug susceptibility testing. Overall, 182/379 included patients (48.0%) were diagnosed with tuberculous meningitis. Sensitivities of Xpert, smear, and MGIT culture among patients diagnosed with TBM were 59.3% (108/182 [95% confidence interval {CI}, 51.8 to 66.5%]), 78.6% (143/182 [95% CI, 71.9 to 84.3%]) and 66.5% (121/182 [95% CI, 59.1 to 73.3%]), respectively. There was one false-positive Xpert MTB/RIF test (99.5% specificity). Four cases of RIF resistance (4/109; 3.7%) were identified by Xpert, of which 3 were confirmed to be multidrug-resistant (MDR) TBM and one was culture negative. Xpert MTB/RIF is a rapid and specific test for the diagnosis of tuberculous meningitis. The addition of a vortexing step to sample processing increased sensitivity for confirmed TBM by 20% (P = 0.04). Meticulous examination of a smear from a large volume of cerebrospinal fluid (CSF) remains the most sensitive technique but is not practical in most laboratories. The Xpert MTB/RIF represents a significant advance in the early diagnosis of this devastating condition. PMID:24197880

Nhu, Nguyen Thi Quynh; Heemskerk, Dorothee; Thu, Do Dang Anh; Chau, Tran Thi Hong; Mai, Nguyen Thi Hoang; Nghia, Ho Dang Trung; Loc, Pham Phu; Ha, Dang Thi Minh; Merson, Laura; Thinh, Tran Thi Van; Day, Jeremy; Chau, Nguyen van Vinh; Wolbers, Marcel; Farrar, Jeremy; Caws, Maxine

2014-01-01

363

Identification of Mycobacterium Species by Multiple-Fluorescence PCR–Single-Strand Conformation Polymorphism Analysis of the 16S rRNA Gene  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Identification of mycobacteria to the species level by growth-based methodologies is a process that has been fraught with difficulties due to the long generation times of mycobacteria. There is an increasing incidence of unusual nontuberculous mycobacterial infections, especially in patients with concomitant immunocompromised states, which has led to the discovery of new mycobacterial species and the recognition of the pathogenicity of organisms that were once considered nonpathogens. Therefo...

2001-01-01

364

Entrez Gene: gene-centered information at NCBI  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Entrez Gene (www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene) is NCBI's database for gene-specific information. It does not include all known or predicted genes; instead Entrez Gene focuses on the genomes that have been completely sequenced, that have an active research community to contribute gene-specific information, or that are scheduled for intense sequence analysis. The content of Entrez Gene represents the result of curation and automated integration of data from NCBI's Reference Sequen...

2005-01-01

365

Physiological function of mycobacterial mtFabD, an essential malonyl-CoA:AcpM transacylase of type 2 fatty acid synthase FASII, in yeast mct1Delta cells.  

Science.gov (United States)

Mycobacterium tuberculosis mtFabD is an essential malonyl-CoA:AcpM transacylase and is important for vital protein-protein interactions within type 2 fatty acid synthase FASII. mtFabD contacts KasA, KasB, FabH, InhA, and possibly also HadAB, HadBC, and FabG1/MabA. Disruption of mtFabD's interactions during FASII has been proposed for drug development. Here, the gene for a mitochondrially targeted mtFabD was ectopically expressed in Saccharomyces cerevisiae mct1Delta mutant cells lacking the corresponding mitochondrial malonyl-CoA transferase Mct1p, allowing the mutants to recover their abilities to respire on glycerol and synthesize lipoic acid. Hence, mtFabD could physiologically function in an environment lacking holo-AcpM or other native interaction partners. PMID:19859569

Gurvitz, Aner

2009-01-01

366

???: GeneSplicer  

Full Text Available GeneSplicer ?????|100000000 > ??/????|140000000 > ??????????/????|14100 ???|400000000 > [Arabidopsis_Workflow_All] ???? ????????(???????)|410000000 > [Arabid ??|400000000 > [ArabidopsisChr1_Workflow_All] ???? ???1?????????(?????)|420000000 > [A

367

???: GenePublisher  

Full Text Available 01000 01200 01203 01204 01205 01208 05000 05800 07000 07100 ???????????? | ???? vices/GenePublisher t?? ?????? k???? ?? ???????? ??????? ?????????? ?????????t??????????k??????? ??????????????????????????

368

Gene Conversion and Evolution of Gene Families: An Overview  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The importance of gene conversion for the evolution of gene families is reviewed. Four problems concerning gene conversion, i.e., concerted evolution, generation of useful variation, deleterious effects, and relation to neofunctionalization, are discussed by surveying reported examples of evolving gene families. Emphasis is given toward understanding interactive effects of gene conversion and natural selection.

Tomoko Ohta

2010-01-01

369

DNA repair genes  

International Nuclear Information System (INIS)

Fission yeast S. pombe is assumed to be a good model for cloning of human DNA repair genes, because human gene is normally expressed in S. pombe and has a very similar protein sequence to yeast protein. We have tried to elucidate the DNA repair mechanisms of S. pombe as a model system for those of mammals. (J.P.N.)

1995-12-01

370

TIGR Drosophila Gene Index  

Science.gov (United States)

The Institute for Genomic Resources (TIGR) placed online the Drosophila Gene Index (version 1.1) in 1999. The index is searchable by nucleotide or protein sequence, identifier (TC, ET, EST, GB), tissue, or gene product name. Note that this resource is available free of charge "only to researchers at non-profit institutions using it for non-commercial purposes."

371

TIGR Tomato Gene Index  

Science.gov (United States)

The Institute for Genomic Resources (TIGR) placed online the Tomato Gene Index (version 1.2) in 1999. The index is searchable by nucleotide or protein sequence, identifier (TC, ET, EST, GB), tissue, or gene product name. Note that this resource is available free of charge "only to researchers at non-profit institutions using it for non-commercial purposes."

372

A review on microcephaly genes  

Directory of Open Access Journals (Sweden)

Full Text Available This review aims to summarize the recent findings regarding microcephaly genes. We have discussed the molecular genetics studies of microcephaly genes including a comprehensive appraisal of the seven mapped loci (MCPH1–MCPH7, their corresponding genes and protein products of the genes, their likely role in normal brain development and the details of the mutations reported in these genes.

Irshad S.

2012-06-01

373

Correlations Between Gene Expression and Gene Conservation in Fission Yeast  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Genes can be expressed at a wide range of levels, and they show different degrees of cross-species conservation. We compared gene expression levels to gene conservation by integrating microarray data from fission yeast (Schizosaccharomyces pombe) with lists of “core” genes (present in worm and budding and fission yeasts), “yeast-specific” genes (present in budding and fission yeasts, but not in worm), and “pombe-specific” genes (present in fission yeast only). Whereas a disproport...

Mata, Juan; Ba?hler, Ju?rg

2003-01-01

374

GeneCards Version 3: the human gene integrator  

Digital Repository Infrastructure Vision for European Research (DRIVER)

GeneCards (www.genecards.org) is a comprehensive, authoritative compendium of annotative information about human genes, widely used for nearly 15 years. Its gene-centric content is automatically mined and integrated from over 80 digital sources, resulting in a web-based deep-linked card for each of >73 000 human gene entries, encompassing the following categories: protein coding, pseudogene, RNA gene, genetic locus, cluster and uncategorized. We now introduce GeneCards Version 3, featuring a ...

Safran, Marilyn; Dalah, Irina; Alexander, Justin; Rosen, Naomi; Iny Stein, Tsippi; Shmoish, Michael; Nativ, Noam; Bahir, Iris; Doniger, Tirza; Krug, Hagit; Sirota-madi, Alexandra; Olender, Tsviya; Golan, Yaron; Stelzer, Gil; Harel, Arye

2010-01-01

375

Gene-gene interaction filtering with ensemble of filters  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Abstract Background Complex diseases are commonly caused by multiple genes and their interactions with each other. Genome-wide association (GWA) studies provide us the opportunity to capture those disease associated genes and gene-gene interactions through panels of SNP markers. However, a proper filtering procedure is critical to reduce the search space prior to the computationally intensive gene-gene interaction identification step. In this study, we show that two commonly ...

Yang Pengyi; Wk, Ho Joshua; Yang Yee; Zhou Bing B

2011-01-01

376

Cloning Human Chromosome 17 Genes:.  

Science.gov (United States)

Our research interest is focused on the development of new strategies to identify genes from chromosome 17. The isolation of genes transcribed from chromosome 17 will provide candidates for the proposed sporadic breast and ovarian cancer genes. We have re...

C. C. Lee

1995-01-01

377

Gene amplification in carcinogenesis  

Directory of Open Access Journals (Sweden)

Full Text Available Gene amplification increases the number of genes in a genome and can give rise to karyotype abnormalities called double minutes (DM and homogeneously staining regions (HSR, both of which have been widely observed in human tumors but are also known to play a major role during embryonic development due to the fact that they are responsible for the programmed increase of gene expression. The etiology of gene amplification during carcinogenesis is not yet completely understood but can be considered a result of genetic instability. Gene amplification leads to an increase in protein expression and provides a selective advantage during cell growth. Oncogenes such as CCND1, c-MET, c-MYC, ERBB2, EGFR and MDM2 are amplified in human tumors and can be associated with increased expression of their respective proteins or not. In general, gene amplification is associated with more aggressive tumors, metastases, resistance to chemotherapy and a decrease in the period during which the patient stays free of the disease. This review discusses the major role of gene amplification in the progression of carcinomas, formation of genetic markers and as possible therapeutic targets for the development of drugs for the treatment of some types of tumors.

Lucimari Bizari

2006-01-01

378

Both Corynebacterium diphtheriae DtxR(E175K) and Mycobacterium tuberculosis IdeR(D177K) Are Dominant Positive Repressors of IdeR-Regulated Genes in M. tuberculosis  

Science.gov (United States)

The diphtheria toxin repressor (DtxR) is an important iron-dependent transcriptional regulator of known virulence genes in Corynebacterium diphtheriae. The mycobacterial iron-dependent repressor (IdeR) is phylogenetically closely related to DtxR, with high amino acid similarity in the DNA binding and metal ion binding site domains. We have previously shown that an iron-insensitive, dominant-positive dtxR(E175K) mutant allele from Corynebacterium diphtheriae can be expressed in Mycobacterium tuberculosis and results in an attenuated phenotype in mice (Y. C. Manabe, B. J. Saviola, L. Sun, J. R. Murphy, and W. R. Bishai, Proc. Natl. Acad. Sci. USA 96:12844-12848, 1999). In this paper, we report the M. tuberculosis IdeR(D177K) strain that has the cognate point mutation. We tested four known and predicted IdeR-regulated gene promoters (mbtI, Rv2123, Rv3402c, and Rv1519) using a promoterless green fluorescent protein (GFP) construct. GFP expression from these promoters was abrogated under low-iron conditions in the presence of both IdeR(D177K) and DtxR(E175K), a result confirmed by reverse transcription-PCR. The IdeR regulon can be constitutively repressed in the presence of an integrated copy of ideR containing this point mutation. These data also suggest that mutant IdeR(D177K) has a mechanism similar to that of DtxR(E175K); iron insensitivity occurs as a result of SH3-like domain binding interactions that stabilize the intermediate form of the repressor after ancillary metal ion binding. This construct can be used to elucidate further the IdeR regulon and its virulence genes and to differentiate these from genes regulated by SirR, which does not have this domain.

Manabe, Yukari C.; Hatem, Christine L.; Kesavan, Anup K.; Durack, Justin; Murphy, John R.

2005-01-01

379

Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of Rond?nia, Brazil  

Science.gov (United States)

The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB). We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance.

de Lima, Cleoni Alves Mendes; Gomes, Harrison Magdinier; Oelemann, Maranibia Aparecida Cardoso; Ramos, Jesus Pais; Caldas, Paulo Cezar; Campos, Carlos Eduardo Dias; Pereira, Marcia Aparecida da Silva; Montes, Fatima Fandinho Onofre; de Oliveira, Maria do Socorro Calixto; Suffys, Philip Noel; Moura, Maria Manuela da Fonseca

2013-01-01

380

Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of Rondônia, Brazil.  

Science.gov (United States)

The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB). We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance. PMID:23827995

Lima, Cleoni Alves Mendes de; Gomes, Harrison Magdinier; Oelemann, Maraníbia Aparecida Cardoso; Ramos, Jesus Pais; Caldas, Paulo Cezar; Campos, Carlos Eduardo Dias; Pereira, Márcia Aparecida da Silva; Montes, Fátima Fandinho Onofre; Oliveira, Maria do Socorro Calixto de; Suffys, Philip Noel; Moura, Maria Manuela da Fonseca

2013-06-01

 
 
 
 
381

Rapid identification and classification of Mycobacterium spp. using whole-cell protein barcodes with matrix assisted laser desorption ionization time of flight mass spectrometry in comparison with multigene phylogenetic analysis.  

Science.gov (United States)

The need of quick diagnostics and increasing number of bacterial species isolated necessitate development of a rapid and effective phenotypic identification method. Mass spectrometry (MS) profiling of whole cell proteins has potential to satisfy the requirements. The genus Mycobacterium contains more than 154 species that are taxonomically very close and require use of multiple genes including 16S rDNA for phylogenetic identification and classification. Six strains of five Mycobacterium species were selected as model bacteria in the present study because of their 16S rDNA similarity (98.4-99.8%) and the high similarity of the concatenated 16S rDNA, rpoB and hsp65 gene sequences (95.9-99.9%), requiring high identification resolution. The classification of the six strains by MALDI TOF MS protein barcodes was consistent with, but at much higher resolution than, that of the multi-locus sequence analysis of using 16S rDNA, rpoB and hsp65. The species were well differentiated using MALDI TOF MS and MALDI BioTyper™ software after quick preparation of whole-cell proteins. Several proteins were selected as diagnostic markers for species confirmation. An integration of MALDI TOF MS, MALDI BioTyper™ software and diagnostic protein fragments provides a robust phenotypic approach for bacterial identification and classification. PMID:22284888