Mechanisms of intrinsic antibiotic resistance and envelope biosynthesis by two critical cell wall enzymes in Acinetobacter baumannii

Abstract

The opportunistic pathogen Acinetobacter baumannii is a major cause of hospital-acquired infections. Treatment options against A. baumannii infections are severely limited by its widespread resistance to existing drugs and the lack of new antibiotics. Gram-negative bacteria are shielded from environmental stress and antibiotic attack by their distinct cell envelope structure. A. baumannii has diverged from model organisms and may employ unique strategies in cell envelope biogenesis. The pathogen lacks several enzymes widely used by other bacteria for envelope construction, and instead produces a variety of poorly annotated proteins implicated in envelope assembly and critical to drug resistance. A clearer understanding of how these unknown proteins contribute to envelope integrity is a pivotal step towards developing more effective treatments.My thesis work first defines the function of a poorly understood protein, ElsL, that comprises an L,D-transpeptidases (LDTs)-family domain usually found in alternative cell wall crosslinking enzymes and provides A. baumannii with intrinsic resistance against β-lactam antibiotics and short-rod morphology. We reveal through genetic interaction and biochemical analyses that ElsL is not an LDT but is rather a novel class of cytoplasmic L,D-carboxypeptidase (LDC) key to cell wall recycling. ElsL has tetrapeptide-cleaving LDC activity in vitro. We found that ElsL deficient cells accumulate toxic, dead-end tetrapeptide recycling products, and rely on a separate cell wall LDT, which uses tetrapeptides, to avoid death. Blocking cell wall recycling relieves the toxic effects of lacking ElsL. We also demonstrate the multifaceted impact of ElsL deficiency on other envelope associated pathways, including cell division and outer membrane homeostasis. Work on ElsL reveals the distinct approaches to cell wall biogenesis in A. baumannii as well as vulnerabilities that can be leveraged in treatment. The biogenesis of the cell wall and the outer membrane are interdependent. How these two processes are orchestrated during bacterial growth and replication remains undefined. This dissertation identifies a cell wall hydrolase, PbpG, that supports A. baumannii outer membrane assembly. My work uncovered that PbpG is a critical determinant of resistance to bulky, hydrophobic drugs and detergents, indicating a compromised envelope barrier. We found that the barrier defect is associated with reduced production of lipooligosaccharide (LOS) and outer membrane proteins (OMPs). We also show that increasing flux into the LOS synthesis pathway by overexpressing LpxC is lethal in the absence of PbpG, suggesting that decreasing LOS is necessary for PbpG- viability. Finally, we found that PbpG inactivation requires a novel stress response pathway for survival, which we predict may act to downregulate LOS level. These findings illuminate the coordinated biogenesis of the cell wall and outer membrane previously underappreciated in A. baumannii.--Author's abstract