PBP1A and LdtJ support cell envelope homeostasis and impact selection of Colistin-resistance in Acinetobacter baumannii

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-resource antibiotic. Although lipid A is essential in most Gram-negatives, 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 {Delta}mrcA mutants. Here, we identify a 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 affected growth, viability, morphology, and OM homeostasis. PG analyses revealed that the {Delta}ldtJ {Delta}mrcA mutants displays reduced overall cross-linkage and shorter glycan chains, producing a weakened sacculus. Co-immunoprecipitation demonstrated that PBP1A associates with LdtJ, supporting their coordinated activity at sites of PG synthesis. Notably, {Delta}ldtJ {Delta}mrcA mutants exhibited the highest recovery frequency of colistin-resistant, LOS-deficient variants compared with wild type or single mutants. Together, our findings demonstrate that coupling between 4-3 and 3-3 transpeptidation is critical for envelope stability in A. baumannii and highlight how disrupting this coordination favors the emergence of colistin resistance. This work identifies a conserved PG remodeling vulnerability that directly links PG integrity to the evolution of antibiotic resistance, offering a new conceptual framework for destabilizing the A. baumannii envelope. ImportanceThe global rise of multidrug-resistant Acinetobacter baumannii represents an urgent clinical threat, largely driven by its extraordinary capacity to withstand cell envelope damages and escape last-resort antibiotics such as colistin. Although peptidoglycan synthesis and remodeling are known to influence outer membrane stability, how these processes are coordinated in A. baumannii has remained unclear. Here, we identify a crucial interplay between two critical envelope biogenesis and remodeling activities, 4-3 and 3-3 transpeptidations, mediated by the bifunctional PBP1A and the LD-transpeptidase LdtJ, respectively. Disrupting this coordination weakens the peptidoglycan, destabilizes the outer membrane, and increases the emergence frequency of colistin-resistant, LOS-deficient variants. These findings highlight a previously unrecognized vulnerability in the envelope homeostasis of A. baumannii, suggesting that simultaneously targeting DD and LD-transpeptidation could potentiate therapeutic strategies aimed at limiting antibiotic resistance development.