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As the number of multidrug-resistant tuberculosis (TB) cases continues to rise, so does the amplification of multidrug-resistant Mycobacterium tuberculosis strains during treatment (1,2). This amplification is generally assumed to result [...]

The 6-kDa early secretory antigenic target of Mycobacterium tuberculosis (ESAT-6) and the 1O-kDa culture filtrate antigen (CFP-1O), encoded in region of difference 1 (RD1) and secreted by the ESAT-6 system 1 (Esx-1) secretion system, are the most immunodominant and highly M. tuberculosis (MTB)-specific antigens. These attributes are responsible for their primary importance in tuberculosis (TB) immunodiagnosis and vaccine development. Rv361 5c [Esx-1 substrate protein C (EspC)], encoded outside RD1, is similar in size and sequence homology to CFP-1O and ESAT-6, suggesting it might be a target of cellular immunity in TB. Using ex vivo enzyme-linked immunospotand flow cytometry-based cytokine-secretion assay, we comprehensively assessed cellular immune responses to EspC in patients with active TB, latently infected persons, and uninfected bacillus CalmetteGuérin (BCG)-vaccinated controls. EspC was at least as immunodominant as ESAT-6 and CFP-1O in both active and latent TB infection. EspC contained broadly recognized CD4 and CD8 epitopes, inducing a predominantly CD4* T-cell response that comprised functional T-cell subsets secreting both IFN-y and lL-2 as well as functional T-cell subsets secreting only IFN-y. Surprisingly, T-cell responses to EspC were as highly specific (93%) for MTB infection as responses to ESAT-6 and CFP-1O, with only 2 of 27 BCG-vaccinated controls responding to each antigen. Using quantitative proteomics and metabolically labeled mutant and genetically complemented MTB strains, we identified the mechanism of the specificity of anti-EspC immunity as the Esx-1 dependence of EspC secretion. The high immunodominance of EspC, equivalent to that of ESAT-6 and CFP-1O, makes it a TB vaccine candidate, and its high specificity confers strong potential for T-cell-based immunodiagnosis. [ABSTRACT FROM AUTHOR]

Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette-Guérin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated. A single region (RD1), which is absent in all BCG substrains, was deleted from virulent M. bovis and Mycobacterium tuberculosis strains, and the resulting δRD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis δRD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the δRD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M. tuberculosis from BCG. A specific transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10 esat-6) operon of RD1, also caused loss of the cytolytic phenotype in both pneumocytes and macrophages. This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness. Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target). We ,conclude that the primary attenuating mechanism of bacillus Calmette-Guérin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) caused by bacteria of the Mycobacterium tuberculosis complex remains one of the most important infectious diseases of mankind. Rifampicin is a first line drug used in multi-drug treatment of TB, however, the necessary duration of treatment with these drugs is long and development of resistance is an increasing impediment to treatment programmes. As a result, there is a requirement for research and development of new TB drugs, which can form the basis of new drug combinations, either due to their own anti-mycobacterial activity or by augmenting the activity of existing drugs such as rifampicin. This study describes a TnSeq analysis to identify mutants with enhanced sensitivity to sub-minimum inhibitory concentrations (MIC) of rifampicin. The rifampicin-sensitive mutants were disrupted in genes of a variety of functions and the majority fitted into three thematic groups: firstly, genes that were involved in DNA/RNA metabolism, secondly, genes involved in sensing and regulating mycobacterial cellular systems, and thirdly, genes involved in the synthesis and maintenance of the cell wall. Selection at two concentrations of rifampicin (1/250 and 1/62 MIC) demonstrated a dose response for mutants with statistically significant sensitivity to rifampicin. The dataset reveals mechanisms of how mycobacteria are innately tolerant to and initiate an adaptive response to rifampicin; providing putative targets for the development of adjunctive therapies that potentiate the action of rifampicin.