Interplay between peptidoglycan biosynthesis and remodeling and outer membrane homeostasis in Acinetobacter baumannii

The exceptional capacity of the Gram-negative superbug Acinetobacter baumannii to persist under environmental pressures and rapidly develop antibiotic resistance is closely tied to the intrinsic adaptability of its cell envelope, whose structural and regulatory components operate in intimate continuous coordination. In this pathogen, the peptidoglycan (PG) layer and the outer membrane (OM) constantly interact with each another, such that alterations in one compartment often reshape the stability, composition, and stress tolerance of the other. Understanding this reciprocal dependence is essential for explaining the clinical success of A. baumannii in recent years, particularly its growing ability to withstand colistin, one of the last available treatment options. In this work, we investigated different envelope systems whose interactions collectively sustain A. baumannii's adaptability. We first showed that PBP1A, a bifunctional PG synthase with both transglycosylase and D,D-transpeptidase activities, and the L,Dtranspeptidase LdtJ form a functional pair whose coordinated action ensures proper PG assembly and envelope robustness. Their combined inactivation disrupts cell wall architecture, compromises OM stability and integrity, and markedly increases the emergence of colistin-resistant, lipooligosaccharide (LOS)-deficient variants. These findings illustrate how perturbing PG biosynthesis can generate envelope states permissive to extreme OM remodeling, enabling survival even in the absence of LOS. We also examined a second route to colistin resistance based on the loss of OM lipid asymmetry through simultaneous inactivation of both the Mla phospholipid transport system and the OM phospholipase PldA. This condition promoted stable LOS-deficient, colistin-resistant variants even in strains otherwise unable to tolerate the loss of LOS. When PG synthesis was additionally weakened, through inactivation of PBP1A, this permissive state was further amplified, highlighting that PG architecture and OM lipid balance jointly determine the capacity of the envelope to adapt under colistin exposure. This interdependence between the PG structure and OM homeostasis was further underscored by our characterization of A. baumannii DD-carboxypeptidases and DDendopeptidases. Each enzyme contributed uniquely to PG synthesis, maturation and remodeling, cell shape, and mechanical integrity, revealing a set of specialized, nonredundant PG hydrolases in A. baumannii. The effect of DD-endopeptidases on envelope stability became even clearer when considering their tight regulation. The periplasmic protease CtpA and its adaptor partner LbcA control the levels of key endopeptidases and modulate PG turnover and cell separation by coordinating with PBP7, directly influencing cell division, OM integrity and LOS abundance. Together, these findings depicted a cell envelope in which PG synthesis, PG hydrolysis, its proteolytic control, and OM lipid homeostasis form tightly interconnected processes that collectively sustained viability under stress in A. baumannii. By showing how modifications in one component propagated across the envelope and facilitated colistin resistance, this work identified structural and regulatory vulnerabilities that may guide future strategies to counteract multidrug-resistant A. baumannii.