Differential impact of cell wall antibiotics on the Rod complex and aPBPs in Bacillus subtilis: Insights into the peptidoglycan elongation machineries

Abstract

Bacterial cell wall (CW), primarily composed of the biopolymer peptidoglycan, serve as essential protective barriers against external stresses and the internal turgor pressure. The peptidoglycan (PG) biosynthetic pathway encompasses sequential enzymatic reactions in the cytoplasm and in the membrane that involve critical enzymes susceptible to antibiotic targeting. Virtually each step of the pathway is the target of a known antibiotic. Antibiotic-induced inhibition of PG assembly typically weakens the sacculus, often leading to cell lysis. However, the cascade of events that follow inhibition of a specific enzyme of the pathway, and how these culminate in cell death remain largely unknown. Here, we investigated the effects on growing Bacillus subtilis cells of two categories of CW antibiotics: inhibitors of the synthesis of soluble PG precursors in the cytoplasm (fosfomycin and D-cycloserine) and inhibitors of the polymerisation and crosslinking reactions at the outer leaflet of the membrane, which incorporate newly externalised precursors into the existing network (vancomycin and penicillin). In B. subtilis, the latter reactions are catalysed along the sidewalls by the Rod complex, thought to primarily build the sacculus, and by class A penicillin-binding proteins (aPBPs), thought to add to repair it. Our findings reveal that the two antibiotic groups lead to growth arrest, sacculus thinning, and eventual cell lysis. However, while the impact of vancomycin and penicillin G is rapid, lacking morphological deformation, fosfomycin and D-cycloserine induce cell widening and bulging before lysis. During shortage of PG precursors, dysregulated PG hydrolytic activity contributes to elevated cell lysis but is not responsible of bulging. Instead, dispersed PG synthesis by aPBPs persists while the activity of the Rod system is rapidly arrested, resulting in cell rounding. We propose that this facilitates the redirection of the limited PG precursors to sites of CW repair, thereby preserving cell integrity and allowing for prolonged growth during antibiotic challenge.