The G132S Mutation Enhances the Resistance of Mycobacterium tuberculosis β-Lactamase against Sulbactam.
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ABSTRACT:
SUBMITTER: van Alen I
PROVIDER: S-EPMC8383266 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:beta-Lactam antibiotics are extremely effective in disrupting the synthesis of the bacterial cell wall in both gram-positive and gram-negative bacteria. However, they are ineffective against Mycobacterium tuberculosis, due to the production of a beta-lactamase enzyme encoded on the chromosome of M. tuberculosis that degrades these antibiotics. Indeed, recent studies have demonstrated that deletion of the blaC gene, the only gene encoding a beta-lactamase in M. tuberculosis, or inhibition of the encoded enzyme resulted in significantly increased sensitivity to beta-lactam antibiotics. In this paper we present a biochemical and structural characterization of M. tuberculosis BlaC. Recombinant BlaC shows a broad range of specificity with almost equal penicillinase and cepholothinase activity. While clavulanate is a mechanism-based inhibitor to class A beta-lactamase with high potency (typically K(i) < 0.1 microM), it is a relatively poor inhibitor of the M. tuberculosis BlaC (K(i) = 2.4 microM). The crystal structure of the enzyme, determined at a resolution of 1.7 A, shows that the overall fold of the M. tuberculosis enzyme is similar to other class A beta-lactamases. There are, however, several distinct features of the active site, such as the amino acid substitutions N132G, R164A, R244A, and R276E, that explain the broad specificity of the enzyme, relatively low penicillinase activity, and resistance to clavulanate.
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Project description:Capreomycin, an important drug for the treatment of multidrug-resistant tuberculosis, is a macrocyclic peptide antibiotic produced by Saccharothrix mutabolis subspecies capreolus. The basis of resistance to this drug was investigated by isolating and characterizing capreomycin-resistant strains of Mycobacterium smegmatis and Mycobacterium tuberculosis. Colonies resistant to capreomycin were recovered from a library of transposon-mutagenized M. smegmatis. The transposon insertion site of one mutant was mapped to an open reading frame in the unfinished M. smegmatis genome corresponding to the tlyA gene (Rv1694) in the M. tuberculosis H37Rv genome. In M. smegmatis spontaneous capreomycin-resistant mutants, the tlyA gene was disrupted by one of three different naturally occurring insertion elements. Genomic DNAs from pools of transposon mutants of M. tuberculosis H37Rv were screened by PCR by using primers to the tlyA gene and the transposon to detect mutants with an insertion in the tlyA gene. One capreomycin-resistant mutant was recovered that contained the transposon inserted at base 644 of the tlyA gene. Complementation with the wild-type tlyA gene restored susceptibility to capreomycin in the M. smegmatis and M. tuberculosis tlyA transposon mutants. Mutations were found in the tlyA genes of 28 spontaneous capreomycin-resistant mutants generated from three different M. tuberculosis strains and in the tlyA genes of capreomycin-resistant clinical isolates. In in vitro transcription-translation assays, ribosomes from tlyA mutant but not tlyA(+) strains resist capreomycin inhibition of transcription-translation. Therefore, TlyA appears to affect the ribosome, and mutation of tlyA confers capreomycin resistance.
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