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Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti 总被引:15,自引:0,他引:15
Although large human populations have been safely immunized against tuberculosis with two live vaccines, Mycobacterium bovis BCG or Mycobacterium microti, the vole bacillus, the molecular basis for the avirulence of these vaccine strains remains unknown. Comparative genomics has identified a series of chromosomal deletions common to both virulent and avirulent species but only a single locus, RD1, that has been deleted from M. bovis BCG and M. microti. Restoration of RD1, by gene knock-in, resulted in a marked change in colonial morphology towards that of virulent tubercle bacilli. Three RD1-encoded proteins were localized in the cell wall, and two of them, the immunodominant T-cell antigens ESAT-6 and CFP-10, were also found in culture supernatants. The BCG::RD1 and M. microti::RD1 knock-ins grew more vigorously than controls in immunodeficient mice, inducing extensive splenomegaly and granuloma formation. Increased persistence and partial reversal of attenuation were observed when immunocompetent mice were infected with the BCG::RD1 knock-in, whereas BCG controls were cleared. Knocking-in five other RD loci did not affect the virulence of BCG. This study describes a genetic lesion that contributes to safety and opens new avenues for vaccine development. 相似文献
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Roxane Simeone Fadel Sayes Okryul Song Matthias I. Gr?schel Priscille Brodin Roland Brosch Laleh Majlessi 《PLoS pathogens》2015,11(2)
Mycobacterium tuberculosis (Mtb) uses efficient
strategies to evade the eradication by professional phagocytes, involving—as
recently confirmed—escape from phagosomal confinement. While
Mtb determinants, such as the ESX-1 type VII secretion system,
that contribute to this phenomenon are known, the host cell factors governing this
important biological process are yet unexplored. Using a newly developed
flow-cytometric approach for Mtb, we show that macrophages
expressing the phagosomal bivalent cation transporter Nramp-1, are much less
susceptible to phagosomal rupture. Together with results from the use of the
phagosome acidification inhibitor bafilomycin, we demonstrate that restriction of
phagosomal acidification is a prerequisite for mycobacterial phagosomal rupture and
cytosolic contact. Using different in vivo approaches including an
enrichment and screen for tracking rare infected phagocytes carrying the CD45.1
hematopoietic allelic marker, we here provide first and unique evidence of M.
tuberculosis-mediated phagosomal rupture in mouse spleen and lungs and in
numerous phagocyte types. Our results, linking the ability of restriction of
phagosome acidification to cytosolic access, provide an important conceptual advance
for our knowledge on host processes targeted by Mtb evasion
strategies. 相似文献
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Brodin P de Jonge MI Majlessi L Leclerc C Nilges M Cole ST Brosch R 《The Journal of biological chemistry》2005,280(40):33953-33959
Proteins of the 6-kDa early secreted antigenic target (ESAT-6) secretion system-1 of Mycobacterium tuberculosis are not only strongly involved in the anti-mycobacterial Th1-host immune response but are also key players for virulence. In this study, protein engineering together with bioinformatic, immunological, and virulence analyses allowed us to pinpoint regions of the ESAT-6 molecule that are critical for its biological activity in M. tuberculosis. Mutation of the Trp-Xaa-Gly motif, conserved in a wide variety of ESAT-6-like proteins, abolished complex formation with the partner protein CFP-10, induction of specific T-cell responses, and virulence. Replacement of conserved Leu residues interfered with secretion, coiled-coil formation, and virulence, whereas certain mutations at the extreme C terminus did not affect secretion but caused attenuation, possibly because of altered ESAT-6 targeting or trafficking. In contrast, the mutation of several residues on the outer surface of the four-helical bundle structure of the ESAT-6.CFP-10 complex showed much less effect. Construction of recombinant BCG expressing ESAT-6 with a C-terminal hexahistidine tag allowed us to co-purify ESAT-6 and CFP-10, experimentally confirming their strong interaction both in and outside of the mycobacterial cell. The strain induced potent, antigen-specific T-cell responses and intermediate in vivo growth in mice, suggesting that it remained immunogenic and biologically active despite the tag. Together with previous NMR data, the results of this study have allowed a biologically relevant model of the ESAT-6.CFP-10 complex to be constructed that is critical for understanding the structure-function relationship in tuberculosis pathogenesis. 相似文献
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Inactivation of Rv2525c, a substrate of the twin arginine translocation (Tat) system of Mycobacterium tuberculosis, increases beta-lactam susceptibility and virulence 下载免费PDF全文
Saint-Joanis B Demangel C Jackson M Brodin P Marsollier L Boshoff H Cole ST 《Journal of bacteriology》2006,188(18):6669-6679
The twin arginine translocation (Tat) system is used by many bacteria to export fully folded proteins containing cofactors. Here, we show genetically that this system is essential for Mycobacterium tuberculosis, as the tatAC operon and tatB genes could be inactivated only in partially diploid strains. Using comparative genomics, the rv2525c gene of M. tuberculosis was identified as encoding a histidine-rich protein, with a twin arginine signal peptide, and orthologous genes were shown to be present in several but not all actinobacterial species. Conservation of this gene by Mycobacterium leprae, which has undergone reductive evolution, suggested an important role for rv2525c. An rv2525c knockout mutant was constructed, and biochemical analysis indicated that the mature Rv2525c protein is secreted. Upon exposure to antituberculous drugs, rv2525c expression is significantly up-regulated together with those of other genes involved in cell wall biogenesis. Phenotypic comparison of the mutant with the parental strain revealed an increase in susceptibility to some beta-lactam antibiotics and, despite slower growth in vitro, enhanced virulence in both cellular and murine models of tuberculosis. The Tat system thus contributes in multiple ways to survival of the tubercle bacillus. 相似文献
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Carralot JP Ogier A Boese A Genovesio A Brodin P Sommer P Dorval T 《Bioinformatics (Oxford, England)》2012,28(2):261-268
MOTIVATION: High-throughput screening (HTS) is an important method in drug discovery in which the activities of a large number of candidate chemicals or genetic materials are rapidly evaluated. Data are usually obtained by measurements on samples in microwell plates and are often subjected to artefacts that can bias the result selection. We report here a novel edge effect correction algorithm suitable for RNA interference (RNAi) screening, because its normalization does not rely on the entire dataset and takes into account the specificities of such a screening process. The proposed method is able to estimate the edge effects for each assay plate individually using the data from a single control column based on diffusion model, and thus targeting a specific but recurrent well-known HTS artefact. This method was first developed and validated using control plates and was then applied to the correction of experimental data generated during a genome-wide siRNA screen aimed at studying HIV-host interactions. The proposed algorithm was able to correct the edge effect biasing the control data and thus improve assay quality and, consequently, the hit-selection step. 相似文献
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Rachel E. Butler Priscille Brodin Jichan Jang Mi-Seon Jang Brian D. Robertson Brigitte Gicquel Graham R. Stewart 《PloS one》2012,7(10)