OXA-23 beta-Lactamase Overexpression in Acinetobacter baumannii Drives Physiological Changes Resulting in New Genetic Vulnerabilities

beta-Lactamase expression is the major mechanism of resistance to penicillins, cephalosporins, and carbapenems in the multidrug-resistant (MDR) bacterium Acinetobacter baumannii. In fact, stable high-level expression of at least one beta-lactamase has been rapidly increasing and reported to occur in up to 98.5% of modern A. baumannii isolates recovered in the clinic. Moreover, the OXA-51 beta-lactamase is universally present in the A. baumannii chromosome, suggesting it may have a cellular function beyond antibiotic resistance. However, the consequences associated with OXA beta-lactamase overexpression on A. baumannii physiology are not well understood. Using peptidoglycan composition analysis, we show that overexpressing the OXA-23 beta-lactamase in A. baumannii drives significant collateral changes with alterations consistent with increased amidase activity. Consequently, we predicted that these changes create new cellular vulnerabilities. As proof of principle, a small screen of random transposon insertions revealed three genes, where mutations resulted in a greater than 19-fold loss of viability when OXA-23 was overexpressed. The identified genes remained conditionally essential even when a catalytically inactive OXA-23 beta-lactamase was overexpressed. In addition, we demonstrated a synergistic lethal relationship between OXA-23 overexpression and a CRISPR interference (CRISPRi) knockdown of the essential peptidoglycan synthesis enzyme MurA. Last, OXA-23 overexpression sensitized cells to two inhibitors of peptidoglycan synthesis, D-cycloserine and fosfomycin. Our results highlight the impact of OXA-23 hyperexpression on peptidoglycan integrity and reveal new genetic vulnerabilities, which may represent novel targets for antimicrobial agents specific to MDR A. baumannii and other OXA beta-lactamase-overexpressing Enterobacteriaceae, while having no impact on the normal flora. IMPORTANCE Acinetobacter baumannii has become a serious pathogen in both hospital and community settings. The beta-lactam class of antibiotics is a primary treatment option for A. baumannii infections, and expression of beta-lactamases is the most frequent mechanism of resistance in this bacterium. New approaches to treating multi drug-resistant A. baumannii strains are needed. In this study, we demonstrate that overexpressing the OXA-23 beta-lactamase leads to significant collateral changes, where peptidoglycan structure is altered. We have identified genes that become selectively essential in OXA-23-expressing strains and confirmed the relationship between altered peptidoglycan and OXA-23 expression by demonstrating that OXA23 overexpression sensitizes cells to genetic and chemical inhibition of peptidoglycan synthesis. This work paves the way for the identification of new antimicrobial targets, where inhibitors would selectively kill beta-lactamase-expressing strains.