SummaryUnlike many pathogens that are overtly harmful to their hosts, Mycobacterium tuberculosis can persist for years within humans in a clinically latent state. Latency is often linked to hypoxic conditions within the host. Among M. tuberculosis genes induced by hypoxia is a putative transcription factor, Rv3133c/ DosR. We performed targeted disruption of this locus followed by transcriptome analysis of wild-type and mutant bacilli. Nearly all the genes powerfully regulated by hypoxia require Rv3133c/DosR for their induction. Computer analysis identified a consensus motif, a variant of which is located upstream of nearly all M. tuberculosis genes rapidly induced by hypoxia. Further, Rv3133c/DosR binds to the two copies of this motif upstream of the hypoxic response gene alphacrystallin. Mutations within the binding sites abolish both Rv3133c/DosR binding as well as hypoxic induction of a downstream reporter gene. Also, mutation experiments with Rv3133c/DosR confirmed sequence-based predictions that the C-terminus is responsible for DNA binding and that the aspartate at position 54 is essential for function. Together, these results demonstrate that Rv3133c/DosR is a transcription factor of the two-component response regulator class, and that it is the primary mediator of a hypoxic signal within M. tuberculosis .

SummaryThe RD1 genomic region is present in virulent strains of Mycobacterium tuberculosis (MTB), missing from the vaccine strain M. bovis BCG, and its importance to virulence has been established experimentally. Based on in silico analysis, it has been suggested that RD1 may encode a novel secretion system, but the mechanism by which this region affects virulence is unknown. Here we examined mutants disrupted in five individual RD1 genes. Both in vitro and in vivo , each mutant displayed an attenuated phenotype very similar to a mutant missing the entire RD1 region. Genetic complementation of individual genes restored virulence. Attenuated mutants could multiply within THP-1 cells, but they were unable to spread to uninfected macrophages. We also examined export of two immunodominant RD1 proteins, CFP-10 and ESAT-6. Export of these proteins was greatly reduced or abolished in each attenuated mutant. Again, genetic complementation restored a wild-type phenotype. Our results indicate that RD1 genes work together to form a single virulence determinant, and argue that RD1 encodes a novel specialized secretion system that is required for pathogenesis of MTB.

The tuberculosis (TB) vaccine bacille Calmette-Guérin (BCG) is a live attenuated organism, but the mutation responsible for its attenuation has never been defined. Recent genetic studies identified a single DNA region of difference, RD1, which is absent in all BCG strains and present in all Mycobacterium tuberculosis (MTB) strains. The 9 open-reading frames predicted within this 9.5-kb region are of unknown function, although they include the TB-specific immunodominant antigens ESAT-6 and CFP-10. In this study, RD1 was deleted from MTB strain H37Rv, and virulence of H37Rv:DeltaRD1 was assessed after infections of the human macrophage-like cell line THP-1, human peripheral blood monocyte-derived macrophages, and C57BL/6 mice. In each of these systems, the H37Rv:DeltaRD1 strain was strikingly less virulent than MTB and was very similar to BCG controls. Therefore, it was concluded that genes within or controlled by RD1 are essential for MTB virulence and that loss of RD1 was important in BCG attenuation.

It is estimated that one-third of the world's population is infected with

Despite widespread use of the Bacillus Calmette-Guerin vaccine, tuberculosis, caused by infection with Mycobacterium tuberculosis, remains a leading cause of morbidity and mortality worldwide. As CD8+ T cells are critical to tuberculosis host defense and a phase 2b vaccine trial of modified vaccinia Ankara expressing Ag85a that failed to demonstrate efficacy, also failed to induce a CD8+ T cell response, an effective tuberculosis vaccine may need to induce CD8+ T cells. However, little is known about CD8, as compared to CD4, antigens in tuberculosis. Herein, we report the results of the first ever HLA allele independent genome-wide CD8 antigen discovery program. Using CD8+ T cells derived from humans with latent tuberculosis infection or tuberculosis and an interferon-γ ELISPOT assay, we screened a synthetic peptide library representing 10% of the Mycobacterium tuberculosis proteome, selected to be enriched for Mycobacterium tuberculosis antigens. We defined a set of immunodominant CD8 antigens including part or all of 74 Mycobacterium tuberculosis proteins, only 16 of which are previously known CD8 antigens. Immunogenicity was associated with the degree of expression of mRNA and protein. Immunodominant antigens were enriched in cell wall proteins with preferential recognition of Esx protein family members, and within proteins comprising the Mycobacterium tuberculosis secretome. A validation study of immunodominant antigens demonstrated that these antigens were strongly recognized in Mycobacterium tuberculosis-infected individuals from a tuberculosis endemic region in Africa. The tuberculosis vaccine field will likely benefit from this greatly increased known repertoire of CD8 immunodominant antigens and definition of properties of Mycobacterium tuberculosis proteins important for CD8 antigenicity.