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 -diaminopimelic acids [-DAPs]) and 4-3 cross-links (between d-Ala and -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, and, both individually and in combination, from mc155. 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, , 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. The glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. The dd-CPases generate tetrapeptides by acting on the pentapeptides, and ld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of two dd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, including ) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology of dd-CPases might help us design antimycobacterial therapeutics in the future.

Amino Acid-Dependent Alterations in Cell Wall and Cell Morphology of Deinococcus indicus DR1

“…Changes in PG cross-linking lead to alteration in curvature and helicity of Helicobacter pylori (Sycuro et al, 2010). Alteration in the ratio of tetra–tri and tri–tri cross-links also lead to changes in surface glycopeptidolipids, colony morphology, and biofilm formation in Mycobacterium smegmatis (Pandey et al, 2018). Growth medium-dependent glycine incorporation in PG is also observed in Caulobacter crescentus (Takacs et al, 2013).…”

Antibiotics and resistance: the two-sided coin of the mycobacterial cell wall

“…In mycobacteria, however, 80% of the crosslinks are the non-classical 3 → 3 linkage ( Kumar et al, 2012 , Lavollay et al, 2008 ). These cross-links are formed by the combined activities of the D,D-carboxypeptidases of the monofunctional PBPs, which remove the terminal D-Ala from one sidechain ( Pandey et al, 2018 ), followed by the activity of the L,D-transpeptidases (Ldts), which cleaves the next D-Ala and forms a 3 → 3 crosslink between two m -DAP residues of nearby sidechains ( Laponogov et al, 2009 ). There are two Ldts encoded in the Mtb genome, termed Ldt Mt1 (LdtA; Rv0116c) and Ldt Mt2 (LdtB; Rv2518c) ( Gupta et al, 2010 ), which are structurally unrelated to the PBPs and contain an active-site cysteine instead of serine ( Biarrotte-Sorin et al, 2006 , Mainardi et al, 2005 ).…”

MSMEG_2432 of Mycobacterium smegmatis mc2155 is a dual function enzyme that exhibits DD-carboxypeptidase and β-lactamase activities

“…PBP5 of E. coli helps the cells to recover from morphological deformities in septuple PBP mutant E. coli where L182 in its omega loop plays an imperative role in its activity [16,19]. A previous study from our laboratory showed that the deletion of MSMEG_2433 and MSMEG_2432 changed the cell shape and length along with the nature of PG cross-link formation in M. smegmatis [15]. The dual functionality (β-lactamases and dd-CPase) of MSMEG_2433 has also been reported whose activity is affected by E104A substitution (E75A in gene devoid of signal sequence) [10].…”

“…dd-CPases catalyze the cleavage of terminal d-Ala 5 from the muramyl-pentapeptide (l-Ala 1 -d-iso-Glu 2 -meso-DAP 3 -d-Ala 4 -d-Ala 5 ) to generate tetrapeptides, the substrates of l-dtranspeptidases, which in turn is responsible for forming the 3-3 crosslinks between the adjacent peptides [14]. dd-CPases in mycobacteria are also responsible for preventing the unwanted crosslink formation (4-3 crosslinks in mycobacteria) as the deletion of dd-CPases shifts the 3-3 to 4-3 crosslink formation [15], though the dd-CPases are non-essential for bacterial survival [16]. Biochemically, dd-CPases contain SXXK, (S/Y)XN, and (K/H)(T/S)G motifs where Serine of SXXK plays a major role in the substrate binding [17,18].…”