Fellow's research: Flexibility of efflux pumps in the bacterial membrane help superbugs acquire multi-drug resistance
12 Apr 2019
Dr Aravind Penmatsa, Intermediate Fellow
Indian Institute of Science, Bengaluru
Our recently published study addresses the puzzle of how multidrug efflux transporters on the membrane of a cell have the ability to recognize and transport numerous antibacterial compounds and help superbugs like methicillin resistant Staphylococcus aureus (MRSA) survive under antibacterial stress. Efflux or outward flow of antibacterial compounds is a major mechanism of acquiring multi-drug resistance in many pathogens.
We investigated this phenomenon using QacA, a protein embedded in the cell membrane, that has a promiscuous ability to transport nearly thirty different antibacterial compounds using proton gradients across the bacterial membrane. Proton-driven transport is facilitated by charged residues in the core of QacA that can help in the recognition of antibacterial compounds.
In this study, we employed a homology model of QacA and sought to dissect the roles of residues involved in proton driven efflux of structurally different substrates of the transporter.
In the process, we successfully incorporated purified QacA into lipid vesicles to demonstrate its transport activity and used vesicle-based studies, substrate interaction analyses and competition driven proton release assays to reveal the discrete roles of protonatable residues in QacA’s antibacterial efflux properties.
Our experiments demonstrated that two acidic amino acid residues inside the transport vestibule were important for substrate (an antibacterial) recognition. We observed that an acidic residue in the vicinity provides extra protons for aiding transport for some antibacterials. Additionally, our experiments showed that for one of the antibacterial compounds, dequalinium, the general substrate recognition site ceases to act as it should and is replaced by the nearby proton carrier site, suggesting substrate-dependent flexibility in the binding pocket. We also observe that a small subset of antibacterial compounds like pentamidine require a fourth residue in the middle of the passage for efflux.
Different substrates of QacA bind to distinct charged residues within the transporter, prior to efflux
The findings demonstrate the remarkable flexibility of QacA membrane protein and its homologues to accommodate and transport diverse antibacterial compounds, in drug-resistant pathogens. Many of these compounds are popular bactericides used in numerous antibacterial formulations including mouthwashes, lozenges and cleaning agents.
The findings from this study uncovered the mechanistic basis of promiscuous drug efflux in QacA, which would further aid in the design of efflux pump inhibitors for use as adjuvants in improving the efficacy of antibiotics and antibacterial compounds.
Dissection of Protonation Sites for Antibacterial Recognition and Transport in QacA, a Multi-Drug Efflux Transporter. Puja Majumder Shashank Khare, Arunabh Athreya, Nazia Hussain, Ashutosh Gulati, Aravind Penmatsa. Journal of Molecular Biology. March 2019