Structural and functional analyses of the QacA multidrug resistance efflux protein from Staphylococcus aureus

Author: Abolfazl Dashtbani-Roozbehani

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Dashtbani-Roozbehani, Abolfazl, 2021 Structural and functional analyses of the QacA multidrug resistance efflux protein from Staphylococcus aureus, Flinders University, College of Science and Engineering

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The increasing emergence of multidrug resistant bacteria is a major health threat worldwide. Among the most serious human pathogens is Staphylococcus aureus, which is responsible for burgeoning multidrug resistant hospital- or community-acquired infections with significant morbidity and mortality globally. S. aureus utilises a number of mechanisms to circumvent the effects of antimicrobials. One of these is the export of antimicrobial agents through the activity of membrane-embedded multidrug efflux pump proteins. QacA is one such protein that mediates resistance to a diverse array of cationic and lipophilic antimicrobial compounds, including many commonly used antiseptics and disinfectants such as benzalkonium and chlorhexidine.

The proton motive force-driven efflux pump QacA belongs to the major facilitator superfamily of transporter proteins. QacA comprises 514 amino acids which are organised into 14 transmembrane-spanning (TMS) regions. Being plasmid-borne allows QacA to rapidly spread among staphylococci in clinical and natural environments and challenge our modern lifestyle by causing not just biocide resistance but also cross-resistance with antibiotics. Thus, a detailed understanding of the structural and functional features of QacA is a required step in finding a strategy to overcome its efflux activity and thus breaking QacA-mediated resistance. The main aim of this thesis was to analyse QacA and the architectural features of its substrate-binding pockets by performing a blend of microbiological, biochemical and bioinformatic analyses.

To interact specifically with each antimicrobial substrate QacA employs distinct subsets of amino acid residues. TMS 12 of QacA is postulated to be involved in interactions with substrates due to being one of the helices that directly contribute to forming the QacA internal cavity, which is purported to be the central location of substrate-binding and transport. To further understand the importance of TMS 12 in the structure and function of QacA, 38 amino acid residues in the putative TMS 12 and its flanking regions were replaced individually with cysteine using site-directed mutagenesis. These mutants were subsequently analysed to determine the impact of the introduced mutation on protein expression and QacA activity (Chapter 3).

Minimum inhibitory concentration analysis for representative monovalent and bivalent cationic antimicrobial substrates and fluorimetric transport assays identified a number of functionally important amino acid residues. Moreover, accessibility analysis of cysteine-substituted mutants with fluorescein maleimide found that TMS 12 is amphipathic and identified its termini which allowed for refinement of the QacA topology model.

The roles of the identified functionally important residues in QacA substrate-binding and transport pathway were further examined by a suite of biochemical assays (Chapter 4). DAPI fluorescent transport assays, together with results obtained from the effects of substrate preincubation on maleimide reactivity and maleimide pre-treatment on QacA-mediated DAPI efflux, demonstrated that the residues G361 and S387 directly partake in the DAPI transport pathway and perhaps that for other bivalent cationic substrates. Moreover, the solvent accessible residue, S387 at the cytoplasmic end of TMS 12, was found to be directly involved in or located in close vicinity to the substrate-binding site and translocation pathway of chlorhexidine and DAPI. Importantly, the conserved residue G379 in TMS 12 appeared to be structurally important in QacA for transport of bivalent substrates, commensurate with the role of glycine residues in helical flexibility and inter-helical interactions.

A high resolution structure of QacA still remains unresolved. Therefore, a QacA model structure was generated based on homology modelling with the available high resolution structure of a 14 TMS major facilitator superfamily (MFS) protein as a template (Chapter 5). The residues important for QacA mediated resistance to benzalkonium and chlorhexidine were overlaid onto the 3D QacA model. This identified regions lining the QacA central cavity as potentially critical interaction sites for such biocides. Molecular docking analysis revealed the putative substrate-binding pockets for the six antimicrobials used in the functional analyses.

Overall, the work described in thesis makes an original contribution to basic and applied understanding of the structure-function of QacA and provides new insights into the architecture of its drug binding pockets. More generally, the findings can be used in complementing the high-resolution structural data when they are available and also for rational development of efflux pump inhibitor.

Keywords: antimicrobial resistance, bacterial multidrug efflux pumps, staphylococci, Staphylococcus aureus, QacA protein, structure, function, inhibition, inhibitor

Subject: Microbiology & Infectious Diseases thesis

Thesis type: Doctor of Philosophy
Completed: 2021
School: College of Science and Engineering
Supervisor: Professor Melissa Brown