E-Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2021

Investigating the molecular basis for zinc homeostasis in A. baumannii (#201)

Saleh Alquethamy 1 , Felise Adams 2 , Katherine Ganio 1 , Ian Paulsen 3 , Tim Stinear 1 , Bart Eijkelkamp 2 , Christopher McDevitt 1
  1. Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
  2. College of Science and Engineering, Flinders University, Bedford Park, South Australia, Australia
  3. Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia

Acinetobacter baumannii is a Gram-negative nosocomial pathogen associated with significant disease in immunocompromised individuals. Due to the prevalence of carbapenem resistant A. baumannii clinical isolates, the World Health Organisation has called for new antimicrobial therapies to combat this pathogen. Host control of infection is multifactorial, and changes in the elemental composition of niches infected by A. baumannii is an important, but poorly understood aspect. Zinc is an essential metal for all forms of life, but can mediate significant toxicity in excess. A. baumannii harbours an extensive repertoire of metal ion homeostasis mechanisms that contribute to achieving Zn sufficiency, whilst avoiding potentially lethal intoxication. Here, we investigated this repertoire of pathways to identify those that contribute to Zn tolerance. Our analyses of a transposon mutant library of A. baumannii strains identified a role for the resistance nodulation cell-division (RND) transporter CzcCBA, and the cation diffusion facilitator (CDF) transporter CzcD, in zinc resistance. The mutant strains czcA::T26 and czcD::T26 showed a perturbed growth phenotype in the presence of Zn, relative to the wild-type strain, and an increase in cellular Zn accumulation. Although Zn intoxication at the host-pathogen interface is a major antimicrobial factor, Zn withholding also contributes to preventing infection of other niches by attempting to starve A. baumannii of essential Zn. Recruitment of Zn by A. baumannii is mediated, at least in part, by ZnuA, a periplasmic solute binding protein that has high affinity for the metal. However, the mechanistic basis by which ZnuA contributes to A. baumannii Zn import remains to be fully defined. Here, we investigated ZnuA by site-directed mutagenesis of three putative Zn-coordinating residues, His41, His109, and His170. By analysing the ZnuA variant proteins, using a combination of quantitative metal binding assays, thermostability studies, and metal affinity determination, we determined that all three residues contribute to ZnuA-mediated Zn recruitment. Collectively, these analyses expand our understanding of the Zn homeostatic mechanisms of A. baumannii.