Class A Penicillin-Binding Protein-Mediated Cell Wall Synthesis Promotes Structural Integrity during Peptidoglycan Endopeptidase Insufficiency in Vibrio cholerae

Abstract

The bacterial cell wall is composed primarily of peptidoglycan (PG), a poly-aminosugar that is essential to sustain cell shape, growth, and structural integrity. PG is synthesized by class A/B penicillin-binding proteins (a/bPBPs) and shape, elongation, division, and sporulation (SEDS) proteins like RodA (as part of the Rod system cell elongation machinery) and degraded by “autolytic” enzymes to accommodate growth processes. It is thought that autolysins (particularly endopeptidases [EPs]) are required for PG synthesis and incorporation by creating gaps that are patched and paved by PG synthases, but the exact relationship between autolysins and PG synthesis remains incompletely understood. Here, we have probed the consequences of EP depletion for PG synthesis in the diarrheal pathogen Vibrio cholerae. We found that EP depletion resulted in severe morphological and division defects, but these cells continued to increase in mass and aberrantly incorporated new cell wall material. Mass increase proceeded in the presence of Rod system inhibitors, but cells lysed upon inhibition of aPBPs, suggesting that aPBPs are required for structural integrity under these conditions. The Rod system, although not essential for the observed mass increase, remained functional even after prolonged EP depletion. Last, heterologous expression of an EP from Neisseria gonorrhoeae fully complemented growth and morphology of an EP-insufficient V. cholerae, highlighting the possibility that the PG synthases may not necessarily function via direct interaction with EPs. Overall, our findings suggest that during EP insufficiency in V. cholerae, aPBPs become essential for structural integrity while the Rod system is unable to promote proper cell expansion. IMPORTANCE Synthesis and turnover of the bacterial cell wall must be tightly coordinated to avoid structural integrity failure and cell death. Details of this coordination are poorly understood, particularly if and how cell wall turnover enzymes are required for the activity of the different cell wall synthesis machines, the aPBPs and the Rod system. Our results suggest that in Vibrio cholerae, one class of turnover enzymes, the endopeptidases, are necessary for proper cell elongation and division. aPBPs become essential for maintaining structural integrity during EP insufficiency, while the Rod system remains active but contributes little to cell expansion under these conditions. Our results suggest that aPBPs are more versatile than the Rod system in their ability to recognize cell wall gaps formed by autolysins other than the major endopeptidases, adding to our understanding of the coordination between autolysins and cell wall synthases. A detailed understanding of autolysin biology may promote the development of antibiotics that target these essential turnover processes.

Identification of the potential active site of the septal peptidoglycan polymerase FtsW

“…For decades, aPBPs were thought to be the primary PG synthases in bacteria [13]. However, in the last few years accumulating evidence indicates that SEDS-bPBP complexes are responsible for synthesizing PG during bacterial cell growth and division [5][6][7][8][9][10][11], while aPBPs appear to be largely involved in repair and maintenance of the PG layer [7,[14][15][16][17][18]. Nonetheless, both sets of PG synthases are essential for the survival of most bacteria having a cell wall, although some manage to survive without aPBPs under certain circumstances [5,[19][20][21][22].…”

Cell wall damage reveals spatial flexibility in peptidoglycan synthesis and a non-redundant role for RodA in mycobacteria

“…In laterally-growing, rod-shaped bacteria, the emerging narrative is that RodA lays the template for elongation and aPBPs fill in the gaps for maintenance and repair (10,(16)(17)(18)(19)21). Unlike the organisms in which this model has been tested, pole-growing bacteria like members of the Mycobacteriales and Hyphomicrobiales lack the cytoskeletal protein MreB and either lack or do not require RodA for viability or shape (28,29).…”

“…Loss of damage-induced sidewall shift supports this type of active role for RodA in mycobacteria. However if true, this would be in contrast to the repair function of aPBPs, rather than RodA, in other organisms (16)(17)(18)(19)21). Thus a second model to explain the lysis phenotype of ∆rodA is that RodA builds a cell wall with an architecture that is inherently more resistant to damage or that is more amenable to repair.…”

“…This essential pathway contributes to elongation and rod-shape homeostasis by directed motion around the cell (10)(11)(12)(13). A second, non-essential, pathway utilizes bifunctional, class A PBP (aPBPs) that perform both transglycosylation and transpeptidation (9,14,15), move diffusively, and are thought to maintain and repair the cell wall (16)(17)(18)(19)(20)(21). Despite a growing body of evidence suggesting that aPBPs are important for stress response while the Rod complex contributes to normal growth, there are also reports that Rod complex components can sense and respond to stress (22)(23)(24).…”

Lytic transglycosylases mitigate periplasmic crowding by degrading soluble cell wall turnover products

“…Endopeptidases (EPs), for example, cleave the peptide crosslinks and are particularly integral to the “space-making” autolytic function that permits sacculus expansion during cell elongation; without EP activity, PG synthesis results in a thicker cell wall or integrity failure and lysis. 17, 18 Another major class of autolysins, the lytic transglycosylases (LTGs), cleave the glycosidic linkages between disaccharide subunits within PG strands. Their biochemistry has been exquisitely well-studied and the diversity of their structures and mechanisms of action well characterized.…”