Partial complementation of Sinorhizobium meliloti bacA mutant phenotypes by the Mycobacterium tuberculosis BacA protein.

The Sinorhizobium meliloti BacA ABC transporter protein plays an important role in its nodulating symbiosis with the legume alfalfa (Medicago sativa). The Mycobacterium tuberculosis BacA homolog was found to be important for the maintenance of chronic murine infections, yet its in vivo function is unknown. In the legume plant as well as in the mammalian host, bacteria encounter host antimicrobial peptides (AMPs). We found that the M. tuberculosis BacA protein was able to partially complement the symbiotic defect of an S. meliloti BacA-deficient mutant on alfalfa plants and to protect this mutant in vitro from the antimicrobial activity of a synthetic legume peptide, NCR247, and a recombinant human ß-defensin 2 (HBD2). This finding was also confirmed using an M. tuberculosis insertion mutant. Furthermore, M. tuberculosis BacA-mediated protection of the legume symbiont S. meliloti against legume defensins as well as HBD2 is dependent on its attached ATPase domain. In addition, we show that M. tuberculosis BacA mediates peptide uptake of the truncated bovine AMP, Bac7(1-16). This process required a functional ATPase domain. We therefore suggest that M. tuberculosis BacA is important for the transport of peptides across the cytoplasmic membrane and is part of a complete ABC transporter. Hence, BacA-mediated protection against host AMPs might be important for the maintenance of latent infections.

Effects of clofazimine on potassium uptake by a Trk-deletion mutant of Mycobacterium tuberculosis.

OBJECTIVES: This study was designed to investigate the effects of the membrane-active, anti-mycobacterial agent, clofazimine, on potassium (K+)-uptake by a mutant of Mycobacterium tuberculosis (MTB), in which the Trk system, the major K+ transporter of this microbial pathogen, had been selectively inactivated. METHODS: The ceoB and ceoC genes of MTB, which encode the TrkA proteins, CeoB and CeoC, were deleted by homologous recombination, and the double-knockout mutant and wild-type strains compared with respect to K+ uptake and growth in the presence and absence of clofazimine (0.015-2.5 mg/L) using radioassay procedures. RESULTS: Surprisingly, the magnitudes of K+ uptake and rate of growth of the ceoBC-knockout mutant were significantly (P < 0.05) greater than those of the wild-type strain, due, presumably, to induction of a back-up transporter. Exposure of both the wild-type strain and ceoBC-knockout mutant of MTB to clofazimine was accompanied by dose-related decreases in K+ uptake, as well as growth, which were of comparable magnitude for both strains. CONCLUSIONS: These observations demonstrate that the major K+ transporter of MTB, Trk, as well as an uncharacterized inducible back-up system, is equally sensitive to the inhibitory actions of clofazimine.

DNA metabolism in mycobacterium tuberculosis: implications for drug resistance and strain variability.

In this paper, we review the evidence supporting the notion that the genome of Mycobacterium tuberculosis sustains considerable damage as a result of exposure to nitrosative and oxidative stress. On these grounds, we propose a model in which stress-induced DNA damage in M. tuberculosis plays a role in the evolution of chromosomally encoded drug resistance mutations by altering the global mutation rate by mechanisms akin to SOS mutagenesis. Finally we review some of the factors determining the evolution of PE/PPE and MIRU (There are many abbreviations in this paper which are not defined, e.g. SOS, PE/PPE and MIRU. Please indicate whether these are well known and will be understood by readers or whether they should be defined at first mention) loci whose sequence characteristics are suggestive of their classification as heritable local mutators.

Expression of Mycobacterium smegmatis pyrazinamidase in Mycobacterium tuberculosis confers hypersensitivity to pyrazinamide and related amides.

A pyrazinamidase (PZase)-deficient pncA mutant of Mycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 microg/ml), in accordance with the well-established role of pncA in the PZA susceptibility of M. tuberculosis (A. Scorpio and Y. Zhang, Nat. Med. 2:662-667, 1996). Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809-5814, 1998) or the M. tuberculosis pncA gene into the pncA mutant complemented its PZase/nicotinamidase defect. In both pzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. The pzaA-complemented strain was hypersensitive to PZA (MIC, /=20 microg/ml) and was also sensitive to benzamide (MIC, 20 microg/ml), unlike the wild-type and pncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 microg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 microg/ml in all cases) and rendered Escherichia coli hypersensitive for growth at low pH.

Purification, gene cloning, targeted knockout, overexpression, and biochemical characterization of the major pyrazinamidase from Mycobacterium smegmatis.

The pyrazinamidase from Mycobacterium smegmatis was purified to homogeneity to yield a product of approximately 50 kDa. The deduced amino-terminal amino acid sequence of this polypeptide was used to design an oligonucleotide probe for screening a DNA library of M. smegmatis. An open reading frame, designated pzaA, which encodes a polypeptide of 49.3 kDa containing motifs conserved in several amidases was identified. Targeted knockout of the pzaA gene by homologous recombination yielded a mutant, pzaA::aph, with a more-than-threefold-reduced level of pyrazinamidase activity, suggesting that this gene encodes the major pyrazinamidase of M. smegmatis. Recombinant forms of the M. smegmatis PzaA and the Mycobacterium tuberculosis pyrazinamidase/nicotinamidase (PncA) were produced in Escherichia coli and were partially purified and compared in terms of their kinetics of nicotinamidase and pyrazinamidase activity. The comparable Km values obtained from this study suggested that the unique specificity of pyrazinamide (PZA) for M. tuberculosis was not based on an unusually high PZA-specific activity of the PncA protein. Overexpression of pzaA conferred PZA susceptibility on M. smegmatis by reducing the MIC of this drug to 150 micrograms/ml.