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

• Type of Material: Article
• Authors: Jennifer M Colquhoun, Emory University; Marjan Farokhyfar, Atlanta VA Medical Center; Anna R Hutcheson, Atlanta VA Medical Center; Alexander Anderson, University of Guelph; Christopher R Bethel, Louis Stokes Cleveland Department of Veterans Affairs; Robert A Bonomo, Louis Stokes Cleveland Department of Veterans Affairs; Anthony J Clarke, Wilfrid Laurier University; Philip Rather, Emory University
• Language: English
• Date: 2021-11-01
• Publisher: AMER SOC MICROBIOLOGY
• Publication Version: Final Published Version
• Copyright Statement: © 2021 Colquhoun et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1128/mBio.03137-21
• Title of Journal or Parent Work: MBIO
• Volume: 12
• Issue: 6
• Start Page: e0313721
• End Page: e0313721
• Grant/Funding Information: This work was supported by NIH R01 AI72219 to P.N.R. and R.A.B. In addition, P.N.R. is supported by grant R21AI115183 and Department of Veterans Affairs awards I01BX001725 and IK6BX004470. R.A.B. is supported by grants R01AI100560 and R01AI063517 and Department of Veterans Affairs award 1I01BX001974. A.J.C. is funded by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (RGPIN3215-11). A.A. holds an NSERC PGS-D scholarship. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the Department of Veterans Affairs.
• Keywords: EXPRESSION; Acinetobacter; CELL-DIVISION; RESISTANCE; beta-lactamases; peptidoglycan; Microbiology; VIRULENCE; Science & Technology; CLONING; Life Sciences & Biomedicine
• Research Categories: Chemistry, Biochemistry; Health Sciences, Immunology; Biology, Microbiology; Health Sciences, Pharmacology; Biology, Molecular

Abstract

b-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 b-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 b-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 b-lactamase overexpression on A. baumannii physiology are not well understood. Using peptidoglycan composition analysis, we show that overexpressing the OXA-23 b-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 b-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 b-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 b-lactam class of antibiotics is a primary treatment option for A. baumannii infections, and expression of b-lactamases is the most frequent mechanism of resistance in this bacterium. New approaches to treating multidrug-resistant A. baumannii strains are needed. In this study, we demonstrate that overexpressing the OXA-23 b-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 OXA-23 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 b-lactamase-expressing strains.