Characterization of Novel Mycobacterium tuberculosis and Mycobacterium smegmatis Mutants Hypersusceptible to β-Lactam Antibiotics

Abstract

Our laboratory previously constructed mutants of Mycobacterium tuberculosis and Mycobacterium smegmatis with deletions in the genes for their major ␤-lactamases, BlaC and BlaS, respectively, and showed that the mutants have increased susceptibilities to most ␤-lactam antibiotics, particularly the penicillins. However, there is still a basal level of resistance in the mutants to certain penicillins, and the susceptibilities of the mutants to some cephalosporin-based ␤-lactams are essentially the same as those of the wild types. We hypothesized that characterizing additional mutants (derived from ␤-lactamase deletion mutants) that are hypersusceptible to ␤-lactam antibiotics might reveal novel genes involved with other mechanisms of ␤-lactam resistance, peptidoglycan assembly, and cell envelope physiology. We report here the isolation and characterization of nine ␤-lactam antibiotic-hypersusceptible transposon mutants, two of which have insertions in genes known to be involved with peptidoglycan biosynthesis (ponA2 and dapB); the other seven mutants have insertions which affect novel genes. These genes can be classified into three groups: those involved with peptidoglycan biosynthesis, cell division, and other cell envelope processes. Two of the peptidoglycan-biosynthetic genes (ponA2 and pbpX) may encode ␤-lactam antibiotic-resistant enzymes proposed to be involved with the synthesis of the unusual diaminopimelyl linkages within the mycobacterial peptidoglycan.The mycobacteria consist of a diverse group of organisms representing saprophytic bacteria as well as important human pathogens. Organisms such as Mycobacterium chelonae, Mycobacterium smegmatis, and Mycobacterium fortuitum are environmental bacteria and rarely cause disease. On the other hand, Mycobacterium tuberculosis, the causative agent of the disease tuberculosis, has successfully infected one-third of the world's population and is responsible for over 2 million deaths annually (15). Interest in mycobacterial pathogenesis and physiology has been stimulated by the emergence of multidrugresistant strains of M. tuberculosis as well as the observation that the immunocompromised (e.g., those with AIDS) are at a higher risk for M. tuberculosis infection (20,21).The biosynthesis of the mycobacterial cell envelope is an area of considerable research interest, as several of the antibiotics used to treat mycobacterial infections target the synthesis pathways of several envelope components. The main feature of the mycobacterial cell envelope is a single, covalently linked structure composed of peptidoglycan, arabinogalactan, and mycolic acids (the mAGP complex) (5). The peptidoglycan is the innermost layer of the complex and is attached to the arabinogalactan layer via a rhamnose-N-acetylglucosamine linker. The arabinose residues are in turn esterified to mycolic acids, which are long-chain (60 to 90 carbon) ␣-alkyl, ␤-hydroxy fatty acids oriented perpendicularly to the cell membrane (36). Extractable lipids in association with the mycolic acids serve to form an...

Mycobacterium tuberculosis Rv2224c modulates innate immune responses

“…Interestingly, M. smegmatis expA mutants showed increased susceptibility to lysozyme and displayed elongated cell morphology with swollen termini. The authors speculated that expA and expB (the Rv2223c homolog) might be directly involved in hydrolytic breakdown of cell wall components or indirectly involved by regulating proteins involved in autolysis or cell wall remodeling (24). Rv2224c is classified as an ␣/␤ hydrolase in a family with both esterase and amidase members (25).…”

Listeria monocytogenes Is Resistant to Lysozyme through the Regulation, Not the Acquisition, of Cell Wall-Modifying Enzymes

“…pbpX encodes a ␤-lactamase domain; however, it has been reported that in Mycobacterium smegmatis, PbpX does not contribute to ␤-lactam antibiotic resistance. Rather, it was proposed to function as a D,D-carboxypeptidase; however, this has never been confirmed biochemically (38). DegU is an orphan response regulator that regulates flagellar and chemotaxis genes in L. monocytogenes and is severely attenuated during the infection of mice (39)(40)(41)(42)(43).…”

Bacterial Growth and Cell Division: a Mycobacterial Perspective

“…A mycobacterial AmpG homologue has not yet been identified and characterized, but some transport of the PG material back into the cytosol would be needed. It should be noted that mycobacteria do have ␤-lactamases, enzymes associated with cell wall monitoring and remodeling (136,137,271,434).…”