Ethambutol (EMB), a frontline antituberculous drug, targets the mycobacterial cell wall, a unique structure among prokaryotes which consists of an outer layer of mycolic acids covalently bound to peptidoglycan via the arabinogalactan. EMB inhibits the polymerization of cell wall arabinan, and results in the accumulation of the lipid carrier decaprenol phosphoarabinose, which suggests that the drug interferes with the transfer of arabinose to the cell wall acceptor. Unfortunately, resistance to EMB has been described in up to 4% of clinical isolates of Mycobacterium tuberculosis and is prevalent among isolates from patients with multidrug-resistant tuberculosis. We used resistance to EMB as a tool to identify genes participating in the biosynthesis of the mycobacterial cell wall. This approach led to the identification of the embCAB gene cluster, recently proposed to encode for mycobacterial arabinosyl transferases. Resistance to EMB results from an accumulation of genetic events determining overexpression of the Emb protein(s), structural mutation in EmbB, or both. Further characterization of these proteins might provide information on targets for new chemotherapeutic agents and might help development of diagnostic strategies for the detection of resistant M. tuberculosis.

In this paper we describe identification and characterization of Mycobacterium leprae ESAT-6 (L-ESAT-6), the homologue of M. tuberculosis ESAT-6 (T-ESAT-6). T-ESAT-6 is expressed by all pathogenic strains belonging to the M. tuberculosis complex but is absent from virtually all other mycobacterial species, and it is a promising antigen for immunodiagnosis of tuberculosis (TB). Therefore, we analyzed whether L-ESAT-6 is a similarly powerful tool for the study of leprosy by examining T-cell responses against L-ESAT-6 in leprosy patients, TB patients, and exposed or nonexposed healthy controls from areas where leprosy and TB are endemic and areas where they are not endemic. L-ESAT-6 was recognized by T cells from leprosy patients, TB patients, individuals who had contact with TB patients, and healthy individuals from an area where TB and leprosy are endemic but not by T cells from individuals who were not exposed to M. tuberculosis and M. leprae. Moreover, leprosy patients who were not responsive to M. leprae failed to respond to L-ESAT-6. A very similar pattern was obtained with T-ESAT-6. These results show that L-ESAT-6 is a potent M. leprae antigen that stimulates T-celldependent gamma interferon production in a large proportion of individuals exposed to M. leprae. Moreover, our results suggest that there is significant cross-reactivity between T-ESAT-6 and L-ESAT-6, which has implications for the use of ESAT-6 as tool for diagnosis of leprosy and TB in areas where both diseases are endemic.Tuberculosis (TB) and leprosy are major public health problems in the developing world. One-third of the world's population is infected with Mycobacterium tuberculosis, and around 2 million individuals suffer from leprosy (9). Many studies have shown that early culture filtrate proteins of M. tuberculosis can be dominant target antigens for CD4 ϩ Th1 cells both in animal models of TB (1, 2, 5, 12, 13) and in humans. One of these secreted antigens, called T-ESAT-6, is a 10-kDa protein which is present in M. tuberculosis and virulent M. bovis but not in M. bovis BCG, and this protein could not be detected in M. leprae, M. avium, M. scrofulaceum, M. intracellulare, M. fortuitum, and M. xenopi (3, 11). Indeed, when the T-ESAT-6 protein was used, TB-infected cattle could be distinguished from cattle sensitized by environmental mycobacteria (15), and in humans T-ESAT-6 and T-ESAT-6-derived peptides were shown to be very efficiently and specifically recognized by individuals exposed to M. tuberculosis (4,14,16,21). The gene for ESAT-6 (Rv3875) is in a region of the M. tuberculosis genome, designated RD1, that is indeed absent from M. bovis BCG and most nontuberculous mycobacteria (NTM).The existing diagnostic skin test reagents for leprosy, lepromin and leprosin, are prepared from whole autoclaved M. leprae and from the soluble fraction of M. leprae, respectively, and contain many mycobacterial antigens shared with other species (20). A more specific skin test reagent for leprosy would be a highly desirable diagnostic tool. Becaus...

Early detection of Mycobacterium leprae infection is considered an important component of strategies aiming at reducing transmission of infection, but currently available diagnostic tools often lack sufficient sensitivity and specificity to reach this goal. Recent comparative genomics have revealed the presence of 165 M. leprae genes with no homologue in M. tuberculosis. We selected 17 of these genes for further study. All 17 genes were found to be expressed at the mRNA level in M. leprae from infected mice and from a multibacillary leprosy patient. Additional comparative genomic analyses of all currently available mycobacterial genome databases confirmed 12 candidate genes to be unique to M. leprae, whereas 5 genes had homologues in mycobacteria other than M. tuberculosis. Evaluation of the immunogenicity of all 17 recombinant proteins in PBMC from 127 Brazilians showed that five antigens (ML0576, ML1989, ML1990, ML2283, and ML2567) induced significant gamma interferon levels in paucibacillary leprosy patients, reactional leprosy patients, and exposed healthy controls but not in most multibacillary leprosy patients, tuberculosis patients, or endemic controls. Importantly, among exposed healthy controls 71% had no detectable immunoglobulin M antibodies to the M. leprae-specific PGL-I but responded to one or more M. leprae antigen(s). Collectively, the M. leprae proteins identified are expressed at the transcriptome level and can efficiently activate T cells of M. leprae-exposed individuals. These proteins may provide new tools to develop tests for specific diagnosis of M. leprae infection and may enhance our understanding of leprosy and its transmission.

CD8+ T cells have the potential to attack and eradicate cancer cells. The efficacy of therapeutic vaccines against cancer, however, lacks defined immune correlates of tumor eradication after (therapeutic) vaccination based on features of Ag-specific T cell responses. In this study, we examined CD8+ T cell responses elicited by various peptide and TLR agonist-based vaccine formulations in nontumor settings and show that the formation of CD62L−KLRG1+ effector-memory CD8+ T cells producing the effector cytokines IFN-γ and TNF predicts the degree of therapeutic efficacy of these vaccines against established s.c. tumors. Thus, characteristics of vaccine-induced CD8+ T cell responses instill a predictive determinant for the efficacy of vaccines during tumor therapy.

Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) (Rv3874) is considered a promising antigen for the immunodiagnosis of tuberculosis (TB) together with early secreted antigens of M. tuberculosis (ESAT-6). Both ESAT-6 and CFP-10 are encoded by the RD1 region that is deleted from all tested M. bovis bacille Calmette-Guérin (BCG) strains but present in M. leprae, M. tuberculosis, M. bovis, M. kansasii, M. africanum and M. marinum. In this study, the homologue of CFP-10 in M. leprae (ML0050) is identified and characterized. Interferon-g production in response to this homologue by T cells from leprosy patients, TB patients and unexposed controls shows that CFP-10 of M. leprae is a potent antigen that crossreacts with CFP-10 of M. tuberculosis at the T-cell level. This crossreactivity has implications for the use of CFP-10 of these mycobacterial species as diagnostic tool in areas endemic for both the diseases.