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ABSTRACTThe 23-membered Esx protein family is involved in the host-pathogen interactions of Mycobacterium tuberculosis. These secreted proteins are among the most immunodominant antigens recognized by the human immune system and have thus been used to develop vaccines and immunodiagnostic tests for tuberculosis (TB). Gene pairs for 10 Esx proteins are contained in the ESX-1 to ESX-5 loci, encoding type VII secretion systems. A subset of Esx proteins can be further classified into the Mtb9.9, QILSS, and TB10.4 subfamilies. To survey genetic diversity in the Esx family and its potential for antigenic variation, we sequenced all esxgenes from 108 clinical isolates of M. tuberculosisfrom different clades by using a targeted approach. A total of 109 unique single nucleotide polymorphisms (SNPs) were observed, and 59 of these were nonsynonymous. Some of the resultant amino acid substitutions affect known Esx epitopes, including two in the EsxB (CFP-10) and EsxH (TB10.4) antigens. Assessment of the SNP distribution across the Esx proteins revealed high genetic variability, especially in the Mtb9.9 and QILSS subfamilies, and more conservation in the ESX-1 to ESX-4 loci. Comparison of the DNA sequences of variable esxgenes provided clear evidence for recombination events between different genes in the same strain, some of which are predicted to truncate the corresponding protein. Many of these polymorphisms escape detection by ultrahigh-throughput sequencing using short sequence reads, as such approaches cannot distinguish between closely related genes. The esxgene family is dynamic, and sequence changes likely lead to immune variation.

ABSTRACTTo further unravel the mechanisms responsible for attenuation of the tuberculosis vaccine Mycobacterium bovisBCG, comparative genomics was used to identify single nucleotide polymorphisms (SNPs) that differed between sequenced strains of Mycobacterium bovisand M. bovisBCG. SNPs were assayed in M. bovisisolates from France and the United Kingdom and from different BCG vaccines in order to identify those that arose during the attenuation process which gave rise to BCG. Informative data sets were obtained for 658 SNPs from 21 virulent M. bovisstrains and 13 BCG strains; these SNPs showed phylogenetic clustering that was consistent with the geographical origin of the strains and previous schemes for BCG genealogies. The data revealed a closer relationship between BCG Tice and BCG Pasteur than was previously appreciated, while we were able to position BCG Beijing within a grouping of BCG Denmark-derived strains. Only 186 SNPs were identified between virulent M. bovisstrains and all BCG strains, with 115 nonsynonymous SNPs affecting important functions such as global regulators, transcriptional factors, and central metabolism, which might impact on virulence. We therefore refine previous genealogies of BCG vaccines and define a minimal set of SNPs between virulent M. bovisstrains and the attenuated BCG strain that will underpin future functional analyses.

ABSTRACTThe dedicated secretion system ESX-1 of Mycobacterium tuberculosisencoded by the extended RD1 region (extRD1) assures export of the ESAT-6 protein and its partner, the 10-kDa culture filtrate protein CFP-10, and is missing from the vaccine strains M. bovisBCG and M. microti. Here, we systematically investigated the involvement of each individual ESX-1 gene in the secretion of both antigens, specific immunogenicity, and virulence. ESX-1-complemented BCG and M. microtistrains were more efficiently engulfed by bone-marrow-derived macrophages than controls, and this may account for the enhanced in vivo growth of ESX-1-carrying strains. Inactivation of gene pe35(Rv3872) impaired expression of CFP-10 and ESAT-6, suggesting a role in regulation. Genes Rv3868, Rv3869, Rv3870, Rv3871, and Rv3877 encoding an ATP-dependent chaperone and translocon were essential for secretion of ESAT-6 and CFP-10 in contrast to ppe68Rv3873 and Rv3876, whose inactivation did not impair secretion of ESAT-6. A strict correlation was found between ESAT-6 export and the generation of ESAT-6 specific T-cell responses in mice. Furthermore, ESAT-6 secretion and specific immunogenicity were almost always correlated with enhanced virulence in the SCID mouse model. Only loss of Rv3865 and part of Rv3866 did not affect ESAT-6 secretion or immunogenicity but led to attenuation. This suggests that Rv3865/66 represent a new virulence factor that is independent from ESAT-6 secretion. The present study has allowed us to identify new aspects of the extRD1 region of M. tuberculosisand to explore its role in the pathogenesis of tuberculosis.

ABSTRACTIn silico analysis reveals that most protective antigens expressed by the antituberculous vaccine Mycobacterium bovisBCG (BCG) are conserved in M. avium, supporting the hypothesis that exposure to environmental mycobacteria generates cross-reactive immune responses blocking BCG activity. We investigated the impact of sensitization with M. avium, M. scrofulaceum, or M. vaccaeon the protective efficacy of a recombinant BCG strain expressing RD1 antigens (BCG::RD1), using a mouse model of experimental tuberculosis (TB). No evidence that the RD1-encoded antigens ESAT-6, CFP-10, and PPE68 were expressed by these environmental strains could be demonstrated by Western blot analysis. Mice sensitized with each of these strains did not prime cellular immune responses cross-reacting with the immunodominant ESAT-6. Importantly, clearance of BCG::RD1 from the lungs and spleens of mice exposed to each of the environmental strains before vaccination was minimal compared to that of BCG. In mice sensitized with M. avium, increased persistence of BCG::RD1 correlated with stronger antimycobacterial gamma interferon responses and enhanced protection against aerosol infection with M. tuberculosis, compared to BCG. In contrast, animals exposed to M. scrofulaceumor M. vaccaeprior to vaccination with BCG or BCG::RD1 were better protected against TB than were the unsensitized controls. Our results suggest that the inhibitory effect of environmental mycobacteria on the protective efficacy of BCG depends critically on the extent of cross-recognition of antigens shared with the vaccine. In hosts sensitized with M. avium, potent immunogenicity of ESAT-6 and increased persistence of BCG::RD1 may allow this recombinant vaccine to overcome preexisting antimycobacterial responses.

ABSTRACTCulture filtrate protein 10 (CFP-10) from Mycobacterium tuberculosisis a well-characterized immunodominant 10-kDa protein antigen known to elicit a very potent early gamma interferon response in T cells from M. tuberculosis-infected mice and humans. The sequence of the Mycobacterium lepraehomologue of CFP-10 shows only 40% identity (60% homology) at the protein level with M. tuberculosisCFP-10 and thus has the potential for development as a T- or B-cell reactive antigen for specific diagnosis of leprosy. Antisera raised in mice or rabbits against recombinant M. lepraeand M. tuberculosisCFP-10 proteins reacted only with homologous peptides from arrays of overlapping synthetic peptides, indicating that there was no detectable cross-reactivity at the antibody level. Sera from leprosy and tuberculosis patients were also specific for the homologous protein or peptides and showed distinct patterns of recognition for either M. lepraeor M. tuberculosisCFP-10 peptides. At the cellular level, only 2 of 45 mouse T-cell hybridomas raised against either M. lepraeor M. tuberculosisCFP-10 displayed a cross-reactive response against the N-terminal heterologous CFP-10 peptide, the region that exhibits the highest level of identity in the two proteins; however, the majority of peptide epitopes recognized by mouse T-cell hybridomas specific for each protein did not cross-react with heterologous peptides. Coupled with the human serology data, these results raise the possibility that peptides that could be used to differentiate infections caused by these two related microorganisms could be developed. Immunohistochemical staining of sections of M. leprae-infected nude mouse footpads resulted in strongly positive staining in macrophages and dendritic cells, as well as weaker staining in extracellular areas, suggesting that M. lepraeCFP-10, like its homologue in M. tuberculosis, is a secreted protein.

ABSTRACTThe protective efficacy of Mycobacterium bovisBCG can be markedly augmented by stable integration of Mycobacterium tuberculosisgenomic region RD1. BCG complemented with RD1 (BCG::RD1) encodes nine additional proteins. Among them, 10-kDa culture filtrate protein (CFP-10) and ESAT-6 (6-kDa early secreted antigenic target) are low-molecular-weight proteins that induce potent Th1 responses. Using pools of synthetic peptides, we have examined the potential immunogenicity of four other RD1 products (PE35, PPE68, Rv3878, and Rv3879c). PPE68, the protein encoded by rv3873, was the only one to elicit gamma interferon (IFN-γ)-producing cells in C57BL/6 mice infected with M. tuberculosis. Anti-PPE68 T cells were predominantly raised against an epitope mapped in the N-terminal end of the protein. Importantly, inactivation of rv3873in BCG::RD1 did not modify CFP-10 and ESAT-6 secretion. Moreover, the generation of IFN-γ responses to these antigens following immunization with BCG::RD1 was independent of PPE68 expression. Taken together, these results show that PPE68 is an immunogenic product of the RD1 region, which does not interfere with the secretion and immunogenicity of CFP-10 and ESAT-6.

ABSTRACTMycobacterium microtiis a member of the Mycobacterium tuberculosiscomplex that causes tuberculosis in voles. Most strains of M. microtiare harmless for humans, and some have been successfully used as live tuberculosis vaccines. In an attempt to identify putative virulence factors of the tubercle bacilli, genes that are absent from the avirulent M. microtibut present in human pathogen M. tuberculosisor Mycobacterium boviswere searched for. A minimal set of 50 bacterial artificial chromosome (BAC) clones that covers almost all of the genome of M. microtiOV254 was constructed, and individual BACs were compared to the corresponding BACs from M. bovisAF2122/97 and M. tuberculosisH37Rv. Comparison of pulsed-field gel-separated DNA digests of BAC clones led to the identification of 10 regions of difference (RD) between M. microtiOV254 and M. tuberculosis. A 14-kb chromosomal region (RD1mic) that partly overlaps the RD1 deletion in the BCG vaccine strain was missing from the genomes of all nine tested M. microtistrains. This region covers 13 genes, Rv3864 to Rv3876, in M. tuberculosis, including those encoding the potent ESAT-6 and CFP-10 antigens. In contrast, RD5mic, a region that contains three phospholipase C genes (plcAto -C), was missing from only the vole isolates and was present in M. microtistrains isolated from humans. Apart from RD1micand RD5micother M. microti-specific deleted regions have been identified (MiD1 to MiD3). Deletion of MiD1 has removed parts of the direct repeat region in M. microtiand thus contributes to the characteristic spoligotype of M. microtistrains.

ABSTRACTThe usefulness of isoniazid (INH), a key component of short-course chemotherapy of tuberculosis, is threatened by the emergence of drug-resistant strains of Mycobacterium tuberculosiswith mutations in the katGgene. It is shown here that the most commonly occurring KatG mutation, where Ser 315 is replaced by Thr (S315T), is associated with clinically significant levels of INH resistance. In contrast to another resistant isolate, in which Pro replaces Thr 275, the S315T mutant produces active catalase-peroxidase and is virulent in the mouse model of the disease, indicating that a significant loss of bacterial fitness does not result from this frequent mutation. The implications of this finding for the transmission and reactivation of multidrug-resistant strains of M. tuberculosisare severe.

ABSTRACTMycobacterium tuberculosisH37Ra is an attenuated tubercle bacillus closely related to the virulent type strain M. tuberculosisH37Rv. Despite extensive study, the reason for the decreased virulence of M. tuberculosisH37Ra has not been determined. A genomic approach was therefore initiated to identify genetic differences between M. tuberculosisH37Rv andM. tuberculosisH37Ra as a means of pinpointing the attenuating mutation(s). Digestion with the rare-cutting restriction endonuclease DraI revealed two polymorphisms between the strains: a 480-kb fragment in M. tuberculosisH37Rv was replaced by two fragments of 220 and 260 kb in M. tuberculosisH37Ra, while there was a ∼7.9-kb DraI fragment in M. tuberculosisH37Ra that had no counterpart in M. tuberculosisH37Rv. As the M. tuberculosisinsertion sequence IS6110contains a single DraI restriction site, it was considered possible that these polymorphisms were the result of IS6110transposition events in M. tuberculosisH37Ra, events that may have inactivated virulence genes. The 7.9-kb polymorphism was found to be due to the presence of the previously described H37Rv RvD2 deletion in M. tuberculosisH37Ra, with sequence analysis suggesting an IS6110-mediated deletion mechanism for loss of RvD2. Three other IS6110-catalyzed deletions from theM. tuberculosisH37Rv chromosome (RvD3 to RvD5) were also identified, suggesting that this mechanism plays an important role in genome plasticity in the tubercle bacilli. Comparative mapping and sequencing revealed that the 480-kb polymorphism was due to an IS6110insertion in M. tuberculosisH37Ra nearoriC. Complementation of M. tuberculosisH37Ra with a 2.9-kb restriction fragment from M. tuberculosisH37Rv that encompassed the IS6110insertion did not increase the survival of recombinant M. tuberculosisH37Ra in mice. In conclusion, this study describes the presence and mechanisms of genomic variation between M. tuberculosisH37Ra and M. tuberculosisH37Rv, although the role that they play in the attenuation of M. tuberculosisH37Ra is unclear.

ABSTRACTDuring DNA sequence analysis of cosmid L373 from theMycobacterium lepraegenome, an open reading frame of 1.4 kb encoding a protein with some homology to the immunodominant 34-kDa protein of Mycobacterium paratuberculosis, but lacking significant serological activity, was detected. The DNA sequence predicted a signal peptide with a modified lipoprotein consensus sequence, but the protein proved to be devoid of lipid attachment.

ABSTRACTThe bacterial artificial chromosome (BAC) cloning system is capable of stably propagating large, complex DNA inserts in Escherichia coli. As part of the Mycobacterium tuberculosisH37Rv genome sequencing project, a BAC library was constructed in the pBeloBAC11 vector and used for genome mapping, confirmation of sequence assembly, and sequencing. The library contains about 5,000 BAC clones, with inserts ranging in size from 25 to 104 kb, representing theoretically a 70-fold coverage of the M. tuberculosisgenome (4.4 Mb). A total of 840 sequences from the T7 and SP6 termini of 420 BACs were determined and compared to those of a partial genomic database. These sequences showed excellent correlation between the estimated sizes and positions of the BAC clones and the sizes and positions of previously sequenced cosmids and the resulting contigs. Many BAC clones represent linking clones between sequenced cosmids, allowing full coverage of the H37Rv chromosome, and they are now being shotgun sequenced in the framework of the H37Rv sequencing project. Also, no chimeric, deleted, or rearranged BAC clones were detected, which was of major importance for the correct mapping and assembly of the H37Rv sequence. The minimal overlapping set contains 68 unique BAC clones and spans the whole H37Rv chromosome with the exception of a single gap of ∼150 kb. As a postgenomic application, the canonical BAC set was used in a comparative study to reveal chromosomal polymorphisms between M. tuberculosis, M. bovis, and M. bovisBCG Pasteur, and a novel 12.7-kb segment present in M. tuberculosisbut absent from M. bovisand M. bovisBCG was characterized. This region contains a set of genes whose products show low similarity to proteins involved in polysaccharide biosynthesis. The H37Rv BAC library therefore provides us with a powerful tool both for the generation and confirmation of sequence data as well as for comparative genomics and other postgenomic applications. It represents a major resource for present and future M. tuberculosisresearch projects.