Author: Mohsen Chitsaz
Chitsaz, Mohsen, 2024 Multidrug resistance afforded by the MtrD efflux transporter protein from Neisseria gonorrhoeae, Flinders University, College of Science and Engineering
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The rise of antimicrobial resistance poses a significant threat to global public health, demanding urgent action. Neisseria gonorrhoeae, a sexually transmitted bacterium, has developed resistance to multiple compounds resulting in multidrug and extensively drug-resistant strains. The MtrD protein in N. gonorrhoeae, a member of the RND transporter family, plays a crucial role in this characteristic. This PhD thesis focuses on characterising the MtrD protein to gain insights into the corresponding mechanism and identify potential drug targets.
The study began by developing a mutagenesis system specific to N. gonorrhoeae for analysing MtrD activity. A panel of N. gonorrhoeae strains was constructed to screen MtrD substrates and compounds that can potentially inhibit the systems. Using this panel, several new MtrD substrates were identified. A recombinant wild-type MtrD clone, and a cysteine-less MtrD variant were also created, enabling site-directed mutagenesis and resistance profiling to be performed.
To understand the substrate pathway and binding sites of MtrD, molecular dynamics simulations and molecular docking studies were conducted. These investigations provide insights into the interactions between MtrD and its substrates, identifying key residues involved in drug binding. Notably, conserved phenylalanine residues were found to play a critical role in substrate binding, similar to their counterparts in other RND efflux pumps. Functional roles of these residues were explored and confirmed through the site-directed mutagenesis developed in this study shedding light on the structure-function relationship of MtrD.
Through additional molecular dynamics simulation studies, we illustrated that protonation within the transmembrane domain induces a transition from a symmetric to an asymmetric configuration of the trimer, thereby activating allosteric couplings that regulate MtrD efflux. Our findings demonstrate that MtrD employs a functional rotation mechanism similar to that described in AcrB.
One of the unique structural characteristics of the MtrD protein, the N917-P927 region, was investigated. We demonstrated that this region is crucial for MtrD activity and may have implications for drug design and resistance mechanisms.
Studying the function of the MtrD protein is challenging due to the fastidious nature of the bacterium. To overcome these limitations, an optimised system for overexpressing MtrD by itself or in combination with the other proteins in the mtr operon, the MtrCDE, was developed for expressing in Escherichia coli using a variety of expression vectors and E. coli strains. However, expressing MtrD in E. coli strains with native efflux systems could yield conflicting results. To address this, the acrAB region encoding a homologous efflux system was deleted from E. coli strains, resulting in hyper-susceptibility to hydrophobic antimicrobials. Furthermore, the mtrD gene was cloned into the pET21a vector, allowing for high-yield production of MtrD protein in E. coli. Batch-style purification of the MtrD protein from this system yielded substantial quantities of high-quality protein, suitable for various downstream investigations.
To streamline the creation of mtrD gene variants, a modified pBluescript II vector was developed, facilitating the construction of site-directed mutants. The expanded multiple cloning site in this vector allowed for efficient cloning of mtrD gene fragments, simplifying the generation of mtrD mutants and enhancing our understanding of MtrD and other RND transporters' efflux mechanisms.
Overall, this PhD thesis provides valuable insights into the structure, function, and role of MtrD in antibiotic resistance. The findings contribute to our understanding of AMR mechanisms in N. gonorrhoeae and offer a foundation for future research in combating drug resistance in this pathogen. Furthermore, understanding the distinct features of MtrD among RND efflux pumps can guide the development of targeted drugs and inhibitors.
Keywords: Neisseria gonorrhoeae RND transporter MtrD protein Antimicrobial resistance Efflux pump
Subject: Microbiology & Infectious Diseases thesis
Thesis type: Doctor of Philosophy
Completed: 2024
School: College of Science and Engineering
Supervisor: Professor Melissa Brown