PBP1A and LdtJ transpeptidase activities are crucial for cell envelope homeostasis and counteract the selection of colistin resistance in Acinetobacter baumannii

ABSTRACT The multilayered cell envelope of Acinetobacter baumannii is an essential structure that maintains cellular integrity and protects the bacterial cell against external stresses and antibiotics. It consists of an inner membrane, a thin peptidoglycan (PG) layer, and an asymmetric outer membrane (OM) enriched in lipooligosaccharide (LOS), whose lipid A moiety is the target of colistin, a last-resort antibiotic. Although lipid A is essential in most Gram-negative bacteria, A. baumannii can survive without LOS through envelope remodeling, particularly in strains producing low levels of the bifunctional penicillin-binding protein PBP1A (encoded by mrcA ) or in Δ mrcA mutants. Here, we identify a genetic and functional interplay between the LD-transpeptidase LdtJ, which generates 3-3 cross-links, and PBP1A, which catalyzes 4-3 transpeptidation during PG synthesis. We show that simultaneous inactivation of both enzymes severely affects growth, viable counts, morphology, and OM homeostasis. PG analyses revealed that the Δ ldtJ Δ mrcA mutants display reduced overall cross-linking and shorter glycan chains, consistent with a weakened sacculus. Co-immunoprecipitation demonstrated that PBP1A associates with LdtJ, suggesting that the two proteins may act in close proximity during PG synthesis. Notably, Δ ldtJ Δ mrcA mutants exhibited the highest recovery frequency of colistin-resistant, LOS-deficient variants compared with wild type or single mutants. Together, our findings indicate that 4-3 and 3-3 transpeptidase activities play complementary roles in PG synthesis and remodeling, contributing to envelope stability in A. baumannii , and that disruption of these activities favors the emergence of colistin resistance. This work further highlights the importance of PG remodeling in maintaining envelope homeostasis and suggests that proper PG cross-linking acts as a physiological constraint to limit the emergence of LOS-deficient colistin-resistant variants in A. baumannii , providing new insight into mechanisms that contribute to envelope destabilization. IMPORTANCE The global rise of multidrug-resistant Acinetobacter baumannii represents an urgent clinical threat, driven by its capacity to withstand cell envelope damage and escape last-resort antibiotics such as colistin. Although peptidoglycan synthesis and remodeling are known to influence outer membrane stability, how these processes interact in A. baumannii remains unclear. Here, we identify genetic and functional interactions between two major peptidoglycan biosynthetic and remodeling activities, 4-3 and 3-3 transpeptidation, mediated by the bifunctional PBP1A and the LD-transpeptidase LdtJ, respectively. Disrupting these activities weakens the peptidoglycan, destabilizes the outer membrane, and increases the emergence of lipooligosaccharide-deficient, colistin-resistant variants. These findings highlight the importance of proper peptidoglycan cross-linking for envelope homeostasis in A. baumannii and provide insight into how disruptions in peptidoglycan architecture influence antibiotic resistance.