Disruption of a DNA Repair Protein Promotes Antibiotic Resistance in Acinetobacter Baumannii

About This Grant

Project Abstract The emergence of antibiotic resistance in pathogenic bacteria poses an eminent threat to the efficacy of our current antibiotic arsenal. Acinetobacter baumannii (Ab) is the top priority pathogen by the WHO and CDC in the fight against antibiotic resistance. Minocycline (MIN), a tetracycline class antibiotic, is one of the most effective antibiotics for treating Ab infections in patients. Unfortunately, MIN resistance is spreading internationally and has begun to emerge in the United States. The greater utility of MIN over other tetracycline class antibiotics lies in structural differences, which render common tetracycline efflux pumps ineffective. While efflux pumps are correlated with MIN resistant Ab, clinical data suggests alternative mechanisms of MIN resistance. To explore the genetic basis for MIN resistance in Ab we employed a machine learning model to predict genetic resistance correlates from clinical isolates. Mutations in ruvB, a DNA repair protein, were strongly correlated with MIN resistant clinical strains of Ab. Mutations in ruvB have not previously been associated with MIN resistance. Consistent with the prediction, tn26 insertion in the Ab strain AB5075 ruvB gene, and deletion of ruvB in strain ATCC 19606, increased MIN minimum inhibitory concentrations to a level that exceeds the MIN resistance breakpoint. RuvB complexes with RuvA and RuvC to resolve Holliday junctions during recombination. However, only ruvB mutants showed the resistance phenotype; neither ruvA nor ruvC mutants were MIN resistant, suggesting loss of the complex’s activity was not the basis for resistance. We observed ruvB mutants produced increased biomass during planktonic growth relative to the other two ruv mutants. Upon examination, the ruvB::tn26 mutant had a 451% increase in biomass and 360% thicker biofilms relative to wildtype. Prior studies in Escherichia coli, although not examined in relation to antibiotic resistance or relevance to disease, demonstrated RuvA stabilizes extracellular DNA (eDNA) to promote biofilm formation. Since RuvB binds RuvA to initiate formation of the RuvABC complex, we hypothesize ruvB disruption promotes increased RuvA eDNA binding and stabilization leading to enhanced biofilm formation and MIN resistance in Ab. Our preliminary experiments indicate ruvB mutant cultures have higher DNA content compared to wild type, supporting our hypothesis. Within this proposal we outline a detailed strategy to understand how ruvB mutations 1) mechanistically lead to MIN resistance, and 2) impact Ab disease progression and MIN resistance in vivo. The findings from this proposal will alter paradigms for how MIN resistance evolves in Ab and paves the way for future studies focused on understanding the correlations between ruvB mutations and infectious disease progression within patients. The results will also help inform future novel counter-resistance therapeutic strategies which will seek to preserve the clinical utility of essential drugs, like MIN, for use against the most dangerous pathogens.