Author: Sylvia Sapula
Sapula, Sylvia, 2015 Structural and functional assessment of the Staphylococcus aureus multidrug transporter QacC, Flinders University, School of Biological Sciences
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Antimicrobial efflux is a common bacterial resistance mechanism performed by membrane transport proteins. Some of these transporters can extrude a number of structurally dissimilar toxic compounds including antibiotics and biocides. Amongst the six currently characterised superfamilies and families of membrane transporters, is the small multidrug resistance (SMR) family. Members of this family typically range in size from ~100 to 150 amino acids and are composed of four α-helical transmembrane segments (TMS). Due to their small size their functional unit is thought to be multimeric. Within the SMR family, the Staphylococcus aureus QacC multidrug efflux pump was one of the first identified and characterised. Composed of 107 amino acids, QacC extrudes quaternary ammonium compounds (QACs) and dyes such as ethidium. Extensive analysis of both the structure and function of the QacC multidrug transporter was carried out using Cys-scanning mutagenesis, whereby residues are selectively changed to a Cys residue and then challenged with various sulphydryl reagents to evaluate exposure and accessibility. In this study, 103 QacC residues were consecutively replaced with Cys and their influence on protein topology examined by the accessibility of these residues to fluorescein-5-maleimide, a membrane-impermeable sulphydryl-specific reagent. The relative position of each residue was then mapped as either being within the TMS, when shown to be non-accessible, or part of a connecting loop when accessible. These results were compared to models of QacC generated by computer modelling, which allowed for the formation of a final topological map of QacC. As SMR proteins like QacC are believed to function as dimers, with monomers displaying an antiparallel arrangement within the membrane, whole cell accessibility studies were also carried out to determine whether this monomeric arrangement occurs in QacC. In addition to structural analysis, the ability of each QacC mutant to extrude the fluorescent substrate ethidium was analysed as was the resistance profile to a select group of compounds. Finally, topological assessment coupled with functional analysis was used to evaluate conserved SMR family motifs found in TMS1 and TMS4, and to assess the distribution of positively charged residues within QacC which may be contributing the orientation of the monomers with the membrane. Of the 103 residues examined, 14 were identified to be significant for protein function and/or expression. Analysis of the conserved motifs identified residues involved in substrate binding, such as Glu14 and those possibly involved in dimerisation, such as Ser90 and Gly97. Assessment of topology using whole cells has revealed that QacC orientation of monomers within the membrane takes on an antiparallel arrangement with the same reactive residues found on both side of the membrane. Finally, computer analysis and accessibility studies allowed for the formulation of a topological map of QacC, mapping each residue to the TMS or loop region. The data obtained will shed more light into the topology of transport proteins which will further our understanding of the evolution of membrane transporters. Furthermore, the essential residues identified in this study will help us gain a better understanding into the transport mechanism of multidrug transporters and SMR proteins in particular.
Keywords: QacC, multidrug efflux pumps, Staphylococcus aureus, bacterial resistance mechanims
Subject: Biological Sciences thesis
Thesis type: Doctor of Philosophy
School: School of Biological Sciences
Supervisor: Professor Melissa Brown