Two DD-carboxypeptidases from Mycobacterium smegmatis affect cell surface properties through regulation of peptidoglycan cross-linking and glycopeptidolipids

Abstract

During the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between two meso-diaminopimelic acids [meso- DAPs]) and 4-3 cross-links (between D-Ala and meso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. The DD-carboxypeptidases (DD-CPases) act on pentapeptides to generate tetrapeptides that are used by LD-transpeptidases as substrates to form 3-3 cross-links. Therefore, DD-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology of DD-CPases in mycobacteria is relatively unexplored. In this study, we deleted two DD-CPase genes, msmeg_2433 and msmeg_2432, both individually and in combination, from Mycobacterium smegmatis mc2155. Though the single DD-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant, viz., a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to β-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of the DD-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.