Dr Melissa Brown

Contact Details

Key Responsibilities

• Cluster leader - Molecular Biology
• Microbiology

Teaching

• Microbiology

Research

Multidrug resistance in bacteria

Bacteria exert resistance by a number of mechanisms, including antimicrobial inactivation, target modification and strategies that reduce antimicrobial concentration within the cell. Active extrusion of antimicrobial compounds from cells by membrane-bound efflux proteins is an effective mechanism of resistance and one of the major causes of failure of drug-based treatments of cancers and infectious diseases, which appears to be increasing in prevalence especially as the genes are frequently located on mobile genetic elements. Multidrug efflux systems present a serious threat to patient care and to successful therapy since the acquisition or upregulation of a single gene simultaneously renders the cell or organism resistant to compounds from different chemical classes. Research in my laboratory currently focuses on obtaining a detailed understanding of structure, function and regulation of these efflux systems in the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium

Neisseria gonorrhoea.

(1) The staphylococcal multidrug efflux protein QacA

The QacA protein from Staphylococcus aureus confers multidrug resistance by actively pumping antimicrobials out of the cell via a proton motive force-dependent mechanism. QacA mediates resistance to more than 30 different monovalent or bivalent cationic, lipophilic antimicrobial compounds, commonly used antiseptics and disinfectants, that belong to 12 distinct chemical families. The QacA membrane-bound protein is a member of the major facilitator superfamily of transport proteins, consisting of 14 a-helical transmembrane segments (TMS). The primary objective of this project is a detailed molecular characterisation of QacA, to: identify key domains and amino acid residues of QacA involved in the binding and transport of drugs; elucidate the salient architectural features of the QacA drug-binding pocket; elucidate a helix-packing model for the 3-D spatial arrangement of the 14 TMS in the QacA protein; and determine the 3-D crystallographic structure of QacA in its apo and drug-bound forms.

(2) The gonoccal multidrug efflux protein MtrD

In Neisseria gonorrhoeae and N. meningitidis the over-expression of the Mtr resistance proteins results in resistance to: bile salts; antibiotics such as penicillin, erythromycin, azithromycin, rifampicin, and macrolides; host-derived antimicrobial peptides; and nonionic detergents, such as the spermacide nonoxynol-9. Mtr is a multi-component system comprised of the MtrD inner membrane efflux protein, the MtrC periplasmic accessory protein, and the MtrE outer membrane channel; all components are required for direct export of substrates into the external medium. The MtrD drug-H⁺ antiporter, a member of the resistance-nodulation-cell division superfamily of transport proteins, is a large protein of 1067 amino acids. The availability of high-resolution crystal structures for an Escherichia coli MtrD homologue, AcrB in apo and substrate-bound forms, provides the framework to model the MtrD protein on and identify candidates for in-depth analyses. Thus, the goal of this project is a molecular characterisation of the gonococcal multidrug resistance protein, MtrD, the inner membrane protein component of the tripartite MtrCDE system by: ascertaining the full antimicrobial resistance spectrum of MtrD; identifying amino acids and domains of MtrD involved in the binding and transport of antimicrobial drugs and peptides and developing an over-expression and purification regime to obtain significant quantities of MtrD for structural and crystallisation studies.

(3) The QacR multidrug binding repressor protein

The majority of bacterial drug-transporter genes known to be subject to regulation have their expression controlled by transcriptional regulatory proteins. These local transcriptional regulators act by directly binding to a similar wide range of toxic compounds to that exported by the membrane protein whose expression they control. This finding has had important ramifications for the field of protein:drug interactions, since, in contrast to the membrane-bound transport proteins that are notoriously difficult to purify and study in vitro, the soluble cytosolic regulators of drug resistance have provided much more amenable systems for the study of drug recognition and binding.

Regulation of expression of qacA is via the divergently-encoded trans‑acting repressor protein QacR; in a qacR-dependent manner the transcription of qacA increases in response to a structurally-diverse range of monovalent or bivalent compounds that represents almost all of the substrate classes exported by the QacA pump. Crystal structures for QacR complexed to a number of different ligands have led to major advances in defining the molecular basis of multidrug recognition, as it was the first structure of any protein bound to multiple ligands. It has been shown that the QacR multi-faceted ligand binding pocket is comprised of at least 2 distinct but overlapping drug-binding sites. These structures have allowed us to make deductions regarding the residues of the QacR-ligand binding pocket that intimately contact drugs. The specific aims of this project are to: define the multidrug-binding pocket of QacR and characterise the architectural features of the individual ligand-binding sites; characterise the mechanism of induction and ligand-induced conformational transitions in the QacR protein; and examine QacR interactions with operator DNA, including the specific nucleotides and amino acid resides involved.

Current Vacancies

My laboratory is an ideal environment to undertake postgraduate studies, providing state-of-the-art-facilities with support from an active team of postdoctoral researchers, research assistants and PhD and Honours students. Currently there are vacancies for Honours and PhD student projects. If you are interested in undertaking a research project with us, please contact us to come in for a chat, meet the crew and tour the facilities.

Laboratory Alumni

Jessica Boros (School of Medical Sciences; The University of Sydney)

Joanne Gibson (The Picower Institute for Learning and Memory; Massachusetts Institute of Technology)

Kate Hardie (School of Biological Sciences, The University of Sydney)

Karl Hassan (School of Biological Sciences, The University of Sydney)

Libby Kerr

Melanie Galea

Angela Ho (School of Biological Sciences, The University of Sydney)

Penny Ho (Growth Research Group; Kolling Institute of Medical Research)

Samantha Ginn (Gene Therapy Research Unit; Children's Medical Research Institute)

Steve Grkovic (Growth Research Group; Kolling Institute of Medical Research)

Ela Martin (Centre for Immunology and Cancer Research; University of Queensland)

Bernadette Mitchell (Faculty of Pharmacy; The University of Sydney)

Brendon O’Rourke (Genetics Laboratory; Elizabeth Macarthur Agricultural Institute)

Natalie Roberts

George Sharbeen (The Centenary Institute)

Alison Smith (Expert Committees Sections; NHMRC)

Rebecca Smith (School of Biotechnology and Biomolecular Sciences; University of NSW)

Talal Souhani

Torsten Theis (Institute for the Biotechnology of Infectious Diseases; University of Technology)

Yewlan Wanigasekara-Mohoti

Kate Wilson

Jingqin Wu (Retro Viral Genetics Research Group; Westmead Millennium Institute)

Zhiqiang Xu (Entomology; CSIRO)

Wu Yan

Christine Yeates (Advanced Wastewater Management Centre; The University of Queensland)

See publication list

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