Essential Gene Knockdowns Reveal Genetic Vulnerabilities and Antibiotic Sensitivities in Acinetobacter baumannii

Abstract

Abstract The emergence of multidrug-resistant Gram-negative bacteria underscores the need to define genetic vulnerabilities that can be therapeutically exploited. The Gram-negative pathogen,Acinetobacter baumannii, is considered an urgent threat due to its propensity to evade antibiotic treatments. Essential cellular processes are the target of existing antibiotics and a likely source of new vulnerabilities. AlthoughA. baumanniiessential genes have been identified by transposon sequencing (Tn-seq), they have not been prioritized by sensitivity to knockdown or antibiotics. Here, we take a systems biology approach to comprehensively characterizeA. baumanniiessential genes using CRISPR interference (CRISPRi). We show that certain essential genes and pathways are acutely sensitive to knockdown, providing a set of vulnerable targets for future therapeutic investigation. Screening our CRISPRi library against last-resort antibiotics uncovered genes and pathways that modulate beta-lactam sensitivity, an unexpected link between NADH dehydrogenase activity and growth inhibition by polymyxins, and anticorrelated phenotypes that underpin synergy between polymyxins and rifamycins. Our study demonstrates the power of systematic genetic approaches to identify vulnerabilities in Gram-negative pathogens and uncovers antibiotic-essential gene interactions that better inform combination therapies. Importance Acinetobacter baumanniiis a hospital-acquired pathogen that is resistant to many common antibiotic treatments. To combat resistantA. baumanniiinfections, we need to identify promising therapeutic targets and effective antibiotic combinations. In this study, we comprehensively characterize the genes and pathways that are critical forA. baumanniiviability. We show that genes involved in aerobic metabolism are central toA. baumanniiphysiology and may represent appealing drug targets. We also find antibiotic-gene interactions that may impact the efficacy of carbapenems, rifamycins, and polymyxins, providing a new window into how these antibiotics function in mono- and combination therapies. Our studies offer a useful approach for characterizing interactions between drugs and essential genes in pathogens to inform future therapies.