Isoniazid is one of the most effective antituberculosis drugs, yet its precise mechanism of action is still controversial. Using specialized linkage transduction, a single point mutation allele (S94A) within the putative target gene inhA was transferred in Mycobacterium tuberculosis. The inhA(S94A) allele was sufficient to confer clinically relevant levels of resistance to isoniazid killing and inhibition of mycolic acid biosynthesis. This resistance correlated with the decreased binding of the INH-NAD inhibitor to InhA, as shown by enzymatic and X-ray crystallographic analyses, and establishes InhA as the primary target of isoniazid action in M. tuberculosis.

Multi-drug tolerance is a key phenotypic property that complicates the sterilization of mammals infected with Mycobacterium tuberculosis. Previous studies have established that iniBAC, an operon that confers multi-drug tolerance to M. bovis BCG through an associated pump-like activity, is induced by the antibiotics isoniazid (INH) and ethambutol (EMB). An improved understanding of the functional role of antibiotic-induced genes and the regulation of drug tolerance may be gained by studying the factors that regulate antibiotic-mediated gene expression. An M. smegmatis strain containing a lacZ gene fused to the promoter of M. tuberculosis iniBAC (PiniBAC) was subjected to transposon mutagenesis. Mutants with constitutive expression and increased EMB-mediated induction of PiniBAC::lacZ mapped to the lsr2 gene (MSMEG6065), a small basic protein of unknown function that is highly conserved among mycobacteria. These mutants had a marked change in colony morphology and generated a new polar lipid. Complementation with multi-copy M. tuberculosis lsr2 (Rv3597c) returned PiniBAC expression to baseline, reversed the observed morphological and lipid changes, and repressed PiniBAC induction by EMB to below that of the control M. smegmatis strain. Microarray analysis of an lsr2 knockout confirmed upregulation of M. smegmatis iniA and demonstrated upregulation of genes involved in cell wall and metabolic functions. Fully 121 of 584 genes induced by EMB treatment in wild-type M. smegmatis were upregulated (“hyperinduced”) to even higher levels by EMB in the M. smegmatis lsr2 knockout. The most highly upregulated genes and gene clusters had adenine-thymine (AT)–rich 5-prime untranslated regions. In M. tuberculosis, overexpression of lsr2 repressed INH-mediated induction of all three iniBAC genes, as well as another annotated pump, efpA. The low molecular weight and basic properties of Lsr2 (pI 10.69) suggested that it was a histone-like protein, although it did not exhibit sequence homology with other proteins in this class. Consistent with other histone-like proteins, Lsr2 bound DNA with a preference for circular DNA, forming large oligomers, inhibited DNase I activity, and introduced a modest degree of supercoiling into relaxed plasmids. Lsr2 also inhibited in vitro transcription and topoisomerase I activity. Lsr2 represents a novel class of histone-like proteins that inhibit a wide variety of DNA-interacting enzymes. Lsr2 appears to regulate several important pathways in mycobacteria by preferentially binding to AT-rich sequences, including genes induced by antibiotics and those associated with inducible multi-drug tolerance. An improved understanding of the role of lsr2 may provide important insights into the mechanisms of action of antibiotics and the way that mycobacteria adapt to stresses such as antibiotic treatment.

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SummaryLittle is known about the intracellular events that occur following the initial inhibition of Mycobacterium tuberculosis by the first-line antituberculosis drugs isoniazid (INH) and ethambutol (EMB). Understanding these pathways should provide significant insights into the adaptive strategies M. tuberculosis undertakes to survive antibiotics. We have discovered that the M. tuberculosis iniA gene (

We report a new class of thiophene (TP) compounds that kill Mycobacterium tuberculosis (Mtb) by the novel mechanism of Pks13 inhibition. An F79S mutation near the catalytic Ser-55 site in Pks13 conferred TP-resistance in Mtb. Over-expression of wild-type pks13 resulted in TP-resistance and over-expression of the F79S pks13 mutant conferred high-level resistance. In vitro, TP inhibited fatty acyl-AMP loading onto Pks13. TP inhibited mycolic acid biosynthesis in wild-type Mtb, but to a much lesser extent in TP-resistant Mtb. TP treatment was bactericidal and equivalent to the first-line drug isoniazid, but it was less likely to permit emergent resistance. Combined isoniazid and TP treatment exhibited sterilizing activity. Computational-docking identified a possible TP-binding groove within the Pks13 ACP domain. This study confirms that Mtb Pks13 is required for mycolic acid biosynthesis, validates it as a druggable target and demonstrates the therapeutic potential of simultaneously inhibiting multiple targets in the same biosynthetic pathway.

SummarySpontaneous mutants of Mycobacterium tuberculosis that were resistant to the anti-tuberculosis drugs ethionamide and isoniazid were isolated and found to map to mshA, a gene encoding the first enzyme involved in the biosynthesis of mycothiol, a major low-molecular-weight thiol in M. tuberculosis. Seven independent missense or frameshift mutations within mshA were identified and characterized. Precise null deletion mutations of the mshA gene were generated by specialized transduction in three different strains of M. tuberculosis. The mshA deletion mutants were defective in mycothiol biosynthesis, were only ethionamide-resistant and required catalase to grow. Biochemical studies suggested that the mechanism of ethionamide resistance in mshA mutants was likely due to a defect in ethionamide activation. In vivo, a mycothiol-deficient strain grew normally in immunodeficient mice, but was slightly defective for growth in immunocompetent mice. Mutations in mshA demonstrate the nonessentiality of mycothiol for growth in vitro and in vivo, and provide a novel mechanism of ethionamide resistance in M. tuberculosis.

Vaccination against and diagnosis of tuberculosis are still insufficient. Proteins secreted by Mycobacterium tuberculosis induce strong immune responses in tuberculosis and constitute prime candidates for development of novel vaccines against tuberculosis as well as for immunodiagnostic assays. We investigated the role of the secreted proteins MPT63, MPT64 and ESAT6 from M. tuberculosis in healthy individuals and tuberculosis patients. None of the secreted proteins stimulated peripheral blood mononuclear cells from healthy donors. In contrast, CD4+ T cells from many tuberculosis patients were stimulated in an MHC class II-restricted fashion by ESAT6, but not by MPT63 or MPT64. T cell reactivities of tuberculosis patients were focused on the N-terminal region of ESAT6. The ESAT6 T cell epitopes were presented by different HLA-DR phenotypes. Cell cultures responding to either ESAT6 or synthetic peptides thereof showed mRNA transcripts for macrophage inflammatory protein (MIP)-1alpha, monocyte chemotactic protein (MCP)-1 or IL-8 and production of IFN-gamma and MIP-1alpha. Our results suggest that the secreted M. tuberculosis proteins MPT63, MPT64 or ESAT6 do not stimulate unprimed T cells, and that ESAT6 may be a potential candidate antigen for detection of clinical disease.

Mycobacterium tuberculosis has evolved a number of strategies to survive within the hostile environment of host phagocytes. Reactive nitrogen and oxygen intermediates (RNI and ROI) are among the most effective antimycobacterial molecules generated by the host during infection. Lsr2 is a M. tuberculosis protein with histonelike features, including the ability to regulate a variety of transcriptional responses in mycobacteria. Here we demonstrate that Lsr2 protects mycobacteria against ROI in vitro and during macrophage infection. Furthermore, using macrophages derived from NOS ؊/؊ and Phox ؊/؊ mice, we demonstrate that Lsr2 is important in protecting against ROI but not RNI. The protection provided by Lsr2 protein is not the result of its ability to either bind iron or scavenge hydroxyl radicals. Instead, electron microscopy and DNAbinding studies suggest that Lsr2 shields DNA from reactive intermediates by binding bacterial DNA and physically protecting it. Thus, Lsr2 appears to be a unique protein with both histone-like properties and protective features that may be central to M. tuberculosis pathogenesis. In addition, evidence indicates that lsr2 is an essential gene in M. tuberculosis. Because of its essentiality, Lsr2 may represent an excellent candidate as a drug target.Mycobacterium tuberculosis ͉ ROI ͉ DPS ͉ DNA