Biophysical Society Thematic Meeting | Stockholm 2022

Physical and Quantitative Approaches to Overcome Antibiotic Resistance

Poster Abstracts

22-POS Board 22 MUTATIONS IN THE RND TRANSPORTER ADEJ OF ACINETOBACTER BAUMANNII REDUCING THE ACTIVITY OF ADEIJK EFFLUX PUMP

Aysegül Saral Sariyer 2 ; Emrah Sariyer 3 ; Inga V. Leus 1 ; Helen I. Zgurskaya 1 ; 1 University of Oklahoma, Department of Chemistry and Biochemistry, Norman, OK, USA 2 Artvin Coruh University, Department of Nutrition and Dietetics, Artvin, Turkey 3 Artvin Coruh University, Vocational School of Health Services, Artvin, Turkey

Acinetobacter baumannii is a gram-negative and opportunistic pathogen that causes infections in the blood, urinary tract, and lungs. The World Health Organization identified A. baumannii as one of the global priority list of antibiotic resistant bacteria. Although A. baumannii utilizes several different antibiotic resistance mechanisms, one of the most effective is antibiotic efflux mediated by RND type efflux pumps such as AdeABC, AdeIJK and AdeFGH. In this study, the critical residues in the inner membrane transporter AdeJ were identified and the effect of substitutions in these residues on the activity of AdeIJK complex was analyzed experimentally and in silico. Substitutions G721I, R701A, N81A, E675A, F618A, F136A and A134I were introduced into AdeJ expressed from a plasmid carrying the AdeIJK operon under an arabinose inducible promoter. Plasmids producing the mutated pump were electroporated into the efflux deficient A. baumannii Ab 3-pore strain and minimal inhibitory concentrations (MIC) of antibiotics novobiocin and chloramphenicol were determined. Cells producing five AdeJ mutants R701A, N81A, E675A, F618A, A134I became by 4-8 fold more susceptible to chloramphenicol, whereas cells carrying mutant with the F136A substitution were more susceptible to novobiocin. Mutations of R701A in the proximal binding site of AdeJ and N81A, F136A and E675A mutations in the distal binding site of AdeJ were analyzed using molecular dynamics simulations during 200 ns. The mobility and loop dynamics of AdeJ, hydrogen bonding of chloramphenicol with the residues and the path the antibiotic followed in the pocket were examined using trajectory files. E675A, F136A and N81A had a higher deviation than AdeJ-WT but R701A had lesser. In addition, calculations of the binding affinity of chloramphenicol to the wild type and mutant AdeJ variants using the MMGBSA method showed that the binding affinity increased in mutants. The mutations changed the hydrogen bonding profile of AdeJ.

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