Characterisation of the ATP-binding cassette transporters of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a ubiquitous, Gram-negative, rod-shaped, environmental bacterium. However, it is also a clinically significant, opportunistic human pathogen, responsible for life-threatening infections in immunocompromised persons, including those with cancer and significant burn wounds. Furthermore, P. aeruginosa infections are the primary cause of morbidity and mortality in individuals with the genetic disease cystic fibrosis. The success of P. aeruginosa as both an environmental organism and a human pathogen can be attributed to its 6.3 Mbp genome, which encodes an array of mechanisms that enable adaptation, persistence and propagation in diverse environments. Membrane transporters are critical to the ability of P. aeruginosa to adapt and respond to its environment. Of these proteins, the ATP-binding cassette (ABC) transporters hold a prominent role in cellular processes, facilitating the active translocation of molecules across the inner membrane. The conserved structural organisation of ABC transporters has enabled their classification based upon the vector of transport, as either efflux or import transporters. Bioinformatic analyses reveal that P. aeruginosa PAO1 is predicted to encode 289 proteins associated with ABC transporter functionality, with a subset shown to contribute to in vivo virulence. This study presents the phenotypic characterisation of five putative ABC export proteins in P. aeruginosa PAO1, assessing their contribution to antibiotic efflux. Deletion mutants of each identified ABC exporter were assessed for changes in their antibiotic resistance profile. Transcriptional analysis of the ABC transporter genes in response to antibiotic treatment was also performed to detect drug-stimulated expression. These analyses revealed the PA0860, PA1113, PA1876, PA3228 and PA5231 ABC efflux proteins to have a negligible contribution to antibiotic resistance, with bioinformatic analyses subsequently utilized to propose alternative roles. ABC importers, also known as ABC permeases, typically feature one, but sometimes multiple, solute-binding protein (SBP) component(s) that deliver the cargo molecule to the ABC transporter for cytoplasmic translocation. ABC permeases are central to the uptake of many essential nutrients in prokaryotes, including transition metal ions. Herein, this study characterises the acquisition and role of two crucial transition metal ions, molybdenum and zinc, in P. aeruginosa. Acquisition of either metal ion was shown to occur primarily via ABC permeases, molybdenum via ModABC, and zinc via ZnuABC. Deletion of the modA SBP gene abrogated molybdenum accumulation in P. aeruginosa and abolished the capacity for anaerobic growth or nitrate reduction. Unexpectedly, conditions that permitted nitrate reduction were shown to inhibit biofilm formation and alter membrane fatty acid composition. By contrast, although deletion of the zinc-specific SBP gene, znuA, reduced cellular zinc accumulation, the mutant strain did not exhibit a phenotype corresponding with zinc depletion. Transcriptional analyses revealed that P. aeruginosa encodes a number of additional, previously unidentified, putative mechanisms that enable it to adapt to cellular zinc deficiency, including the use of several uncharacterized zinc acquisition systems. Collectively, this study represents a significant advance in our understanding of P. aeruginosa ABC transporters and offers detailed insight into their cellular functions. Furthermore, this work highlights the remarkable adaptability of the bacterium, which enables its survival in diverse environmental and host niches.