Genetic characterization of mycobacterial l,d-transpeptidases

Abstract

L,D-Transpeptidases (Ldts) catalyse the formation of 3-3 cross-links in peptidoglycans (PGs); however, the role of these enzymes in cell envelope physiology is not well understood. Mycobacterial PG contains a higher percentage of 3-3 cross-links (~30-80 %) than the PG in most other bacteria, suggesting that they are particularly important to mycobacterial cell wall biology. The genomes of Mycobacterium tuberculosis and Mycobacterium smegmatis encode multiple Ldt genes, but it is not clear if they are redundant. We compared the sequences of the Ldt proteins from 18 mycobacterial genomes and found that they can be grouped into six classes. We then constructed M. smegmatis strains lacking single or multiple Ldt genes to determine the physiological consequence of the loss of these enzymes. We report that of the single mutants, only one, DldtC (MSMEG_0929, class 5), displayed an increased susceptibility to imipenem -a carbapenem antibiotic that inhibits the Ldt enzymes. The invariant cysteine in the active site of LdtC was required for function, consistent with its role as an Ldt. A triple mutant missing ldtC and both of the class 2 genes displayed hypersusceptibility to antibiotics, lysozyme and D-methionine, and had an altered cellular morphology. These data demonstrated that the distinct classes of mycobacterial Ldts may reflect different, non-redundant functions and that the class 5 Ldt was peculiar in that its loss, alone and with the class 2 proteins, had the most profound effect on phenotype.

Loss of a Functionally and Structurally Distinct ld-Transpeptidase, LdtMt5, Compromises Cell Wall Integrity in Mycobacterium tuberculosis

“…2A). Proline-rich regions have been observed in other mycobacterial PG biosynthetic enzymes, including putative DD-transpeptidases PonA1, PonA2, and PonA3 and Ldt Mt4 , another paralog of Ldt Mt2 (41,42). Although these proline-rich regions are seemingly common among these M. tuberculosis cell wall biosynthetic enzymes, their role in M. tuberculosis physiology is still largely unknown.…”

“…We have previously demonstrated that M. tuberculosis lacking Ldt Mt2 is more susceptible to killing by ␤- lactams (8, 14). Sanders et al (42) have reported that LdtC (homologous to Ldt Mt5 in M. tuberculosis on the basis of sequence) is the primary LD-transpeptidase in Mycobacterium smegmatis. Strains lacking ldtC are hypersusceptible to imipenem, and ldt Mt5 from M tuberculosis fully complements this phenotype in an ldtC mutant, suggesting that these enzymes are equivalent (42).…”

“…Sanders et al (42) have reported that LdtC (homologous to Ldt Mt5 in M. tuberculosis on the basis of sequence) is the primary LD-transpeptidase in Mycobacterium smegmatis. Strains lacking ldtC are hypersusceptible to imipenem, and ldt Mt5 from M tuberculosis fully complements this phenotype in an ldtC mutant, suggesting that these enzymes are equivalent (42). We observed a modest enhancement in susceptibility of the ldt Mt5 ::Tn strain to select carbapenems (Table 5) presumably due to synthetic lethality as these ␤-lactams may inactivate other targets.…”

Targeting the cell wall ofMycobacterium tuberculosis: a molecular modeling investigation of the interaction of imipenem and meropenem withL,D-transpeptidase 2

“…The 3,3-transpeptidase has since then attracted the attention of several researchers. (De & McIntosh 2012;Dubee et al 2012;Erdemli et al 2012;Lecoq et al 2012;Both et al 2013;Cordillot et al 2013;Correale, Ruggiero, Capparelli, Pedone & Berisio 2013;Kim et al 2013;Lecoq et al 2013;Li et al 2013;Triboulet et al 2013;Sanders, Wright & Pavelka 2014;Schoonmaker et al 2014) We have determined the experimental binding free energies of imipenem and meropenem (see Figure 1) to ex-Ldt Mtb2 utilizing isothermal titration calorimetry (ITC) experiments. (Erdemli et al 2012) Figure 1.…”

Peptidoglycan synthesis in Mycobacterium tuberculosis is organized into networks with varying drug susceptibility

“…Recent work suggests that these agents might be far more efficacious against both dividing and nondividing bacteria (10). Although little is known about Mtb's five encoded Ldts (11), one, LdtB, is implicated in antibiotic tolerance (11)(12)(13), is required for normal virulence in a mouse model of TB (12), and is important for normal cell shape (13).…”