A coat protein on phagosomes involved in the intracellular survival of mycobacteria.
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Mycobacteria are intracellular pathogens that can survive within macrophage phagosomes, thereby evading host defense strategies by largely unknown mechanisms. We have identified a WD repeat host protein that was recruited to and actively retained on phagosomes by living, but not dead, mycobacteria. This protein, termed TACO, represents a component of the phagosome coat that is normally released prior to phagosome fusion with or maturation into lysosomes. In macrophages lacking TACO, mycobacteria were readily transported to lysosomes followed by their degradation. Expression of TACO in nonmacrophages prevented lysosomal delivery of mycobacteria and prolonged their intracellular survival. Active retention of TACO on phagosomes by living mycobacteria thus represents a mechanism preventing cargo delivery to lysosomes, allowing mycobacteria to survive within macrophages. (+info)
Persistence and protective efficacy of a Mycobacterium tuberculosis auxotroph vaccine.
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New vaccines against tuberculosis are urgently required because of the impressive incidence of this disease worldwide and the highly variable protective efficacy of the current vaccine. The possibility of creating new live vaccines by the rational attenuation of strains from the Mycobacterium tuberculosis complex was investigated. Two auxotrophic mutants of M. tuberculosis and M. bovis BCG were constructed by disruption of one of their purine biosynthetic genes. These mutants appeared unable to multiply in vitro within mouse bone-marrow derived macrophages. They were also attenuated in vivo in the mouse and guinea pig animal models. In guinea pigs, the two mutants induced strong delayed-type hypersensitivity response to purified protein derivative. In a preliminary experiment, the two mutants were compared to the BCG vaccine for their protective efficacy in a challenge against aerosolized virulent M. tuberculosis in the guinea pig model. Both mutants conferred some level of protection. These experiments demonstrate that the rational attenuation of M. tuberculosis could lead to the design of new candidate live vaccines against tuberculosis. (+info)
Molecular characterization and human T-cell responses to a member of a novel Mycobacterium tuberculosis mtb39 gene family.
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We have used expression screening of a genomic Mycobacterium tuberculosis library with tuberculosis (TB) patient sera to identify novel genes that may be used diagnostically or in the development of a TB vaccine. Using this strategy, we have cloned a novel gene, termed mtb39a, that encodes a 39-kDa protein. Molecular characterization revealed that mtb39a is a member of a family of three highly related genes that are conserved among strains of M. tuberculosis and Mycobacterium bovis BCG but not in other mycobacterial species tested. Immunoblot analysis demonstrated the presence of Mtb39A in M. tuberculosis lysate but not in culture filtrate proteins (CFP), indicating that it is not a secreted antigen. This conclusion is strengthened by the observation that a human T-cell clone specific for purified recombinant Mtb39A protein recognized autologous dendritic cells infected with TB or pulsed with purified protein derivative (PPD) but did not respond to M. tuberculosis CFP. Purified recombinant Mtb39A elicited strong T-cell proliferative and gamma interferon responses in peripheral blood mononuclear cells from 9 of 12 PPD-positive individuals tested, and overlapping peptides were used to identify a minimum of 10 distinct T-cell epitopes. Additionally, mice immunized with mtb39a DNA have shown increased protection from M. tuberculosis challenge, as indicated by a reduction of bacterial load. The human T-cell responses and initial animal studies provide support for further evaluation of this antigen as a possible component of a subunit vaccine for M.tuberculosis. (+info)
Increased antimycobacterial immunity in interleukin-10-deficient mice.
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Macrophage effector functions are essential for clearing mycobacterial infections. Interleukin 10 (IL-10) negatively regulates macrophages and could be a factor inhibiting effective antimycobacterial immunity. We previously showed that transgenic mice which produce excess IL-10 from T cells are susceptible to infection, even though these mice continue to produce gamma interferon (IFN-gamma) at levels similar to those in controls. Here, we extend our genetic analysis of the functions of IL-10 in antimycobacterial immunity by testing the hypothesis that IL-10-deficient (IL-10(-/-)) mice should be more resistant to mycobacteria than control mice. Mycobacterium bovis bacillus Calmette-Guerin-infected IL-10(-/-) mice had significantly lower bacterial burdens than control mice early in the infection. Contrary to expectations, however, IL-10(-/-) mice did not have increased levels of IFN-gamma, either from T cells or in the plasma, suggesting that other mechanisms are responsible for the increased resistance. However, macrophages from IL-10(-/-) mice produced increased levels of inflammatory cytokines, including IFN-gamma, as well as nitric oxide and prostaglandins, which could account for increased antimycobacterial immunity. Our genetic analysis revealed that IL-10 is an inhibitor of early mycobacterial clearance. The data also suggest that IL-10 negatively regulates numerous macrophage functions as well as playing a role in down-regulating the general inflammatory response, especially in conditions where an infection must be controlled through macrophage activity. (+info)
Host defense against Mycobacterium avium does not have an absolute requirement for major histocompatibility complex class I-restricted T cells.
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The role of CD8(+) T cells was evaluated in a mouse model of disseminated Mycobacterium avium infection. C57BL/6J and C57BL/6Jbeta2-/- (beta2-/-) mice were infected intravenously, and the number of viable bacteria in each liver and spleen was determined. No significant difference between the number of bacteria in the two strains of mice was observed at 2, 4, 6, and 8 weeks after infection. Histopathological examination of granulomas from C57BL/6J and beta2-/- mice did not show any difference either in the number of organisms per granuloma or in the size of the granulomas. Investigation of the cytokine profile in the spleen demonstrated that the beta2-/- strain of mice produced a significantly lower amount of gamma interferon at 8 weeks after infection and significantly increased concentrations of tumor necrosis factor alpha compared with that from the wild-type mouse. Interleukin-12 and transforming growth factor beta1 levels did not differ between the two strains of mice at 2, 4, 6, and 8 weeks. Although previous work had found that host response against Mycobacterium tuberculosis involves major histocompatibility complex class I-restricted T cells, our results indicate that chronic deficiency of CD8(+) T cells does not lead to a different expression of the disease and that if CD8(+) T cells are involved in the host response, their function can be assumed by other immune cells. (+info)
Tuberculosis in the Caribbean: using spacer oligonucleotide typing to understand strain origin and transmission.
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We used direct repeat (DR)-based spacer oligonucleotide typing (spoligotyping) (in association with double-repetitive element polymerase chain reaction, IS6110-restriction fragment length polymorphism [RFLP], and sometimes DR-RFLP and polymorphic GC-rich sequence-RFLP) to detect epidemiologic links and transmission patterns of Mycobacterium tuberculosis on Martinique, Guadeloupe, and French Guiana. In more than a third of the 218 strains we typed from this region, clusters and isolates shared genetic identity, which suggests epidemiologic links. However, because of limited epidemiologic information, only 14.2% of the strains could be directly linked. When spoligotyping patterns shared by two or more isolates were pooled with 392 spoligotypes from other parts of the world, new matches were detected, which suggests imported transmission. Persisting foci of endemic disease and increased active transmission due to high population flux and HIV-coinfection may be linked to the recent reemergence of tuberculosis in the Caribbean. We also found that several distinct families of spoligotypes are overrepresented in this region. (+info)
Improved clearance of Mycobacterium avium upon disruption of the inducible nitric oxide synthase gene.
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Mice genetically deficient in the inducible NO synthase gene (iNOS-/-) were used to study the role played by NO during infection by Mycobacterium avium. iNOS-/- macrophages were equally able to restrict M. avium growth in vitro following stimulation by IFN-gamma and TNF-alpha as macrophages from wild-type mice. In vivo, the infection progressed at similar rates in wild-type and NO-deficient mice during the first 2 mo of infection, but the latter mice were subsequently more efficient in clearing the mycobacteria than the former. The increased resistance of iNOS-/- mice was associated with higher IFN-gamma levels in the serum and following in vitro restimulation of spleen cells with specific Ag, increased formation of granulomas and increased survival of CD4+ T cells. We show that NO is not involved in the antimycobacterial mechanisms of M. avium-infected macrophages and, furthermore, that it exacerbates the infection by causing the suppression of the immune response to the pathogen. (+info)
Mycobacterium tuberculosis CDC1551 induces a more vigorous host response in vivo and in vitro, but is not more virulent than other clinical isolates.
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Mycobacterium tuberculosis CDC1551, a clinical isolate reported to be hypervirulent and to grow faster than other isolates, was compared with two other clinical isolates (HN60 and HN878) and two laboratory strains (H37Rv and Erdman). The initial (1-14 days) growth of CDC1551, HN60, HN878, and H37Rv was similar in the lungs of aerosol-infected mice, but growth of Erdman was slower. Thereafter, the growth rate of CDC1551 decreased relative to the other strains which continued to grow at comparable rates up to day 21. In the lungs of CDC1551-infected mice, small well-organized granulomas with high levels of TNF-alpha, IL-6, IL-10, IL-12, and IFN-gamma mRNA were apparent sooner than in lungs of mice infected with the other strains. CDC1551-infected mice survived significantly longer. These findings were confirmed in vitro. The growth rates of H37Rv and CDC1551 in human monocytes were the same, but higher levels of TNF-alpha, IL-10, IL-6, and IL-12 were induced in monocytes after infection with CDC1551 or by exposure of monocytes to lipid fractions from CDC1551. CD14 expression on the surface of the monocytes was up-regulated to a greater extent by exposure to the lipids of CDC1551. Thus, CDC1551 is not more virulent than other M. tuberculosis isolates in terms of growth in vivo and in vitro, but it induces a more rapid and robust host response. (+info)