Dataset Information

Multidrug Resistance in Neisseria gonorrhoeae: Identification of Functionally Important Residues in the MtrD Efflux Protein.

ABSTRACT: A key mechanism that Neisseria gonorrhoeae uses to achieve multidrug resistance is the expulsion of structurally different antimicrobials by the MtrD multidrug efflux protein. MtrD resembles the homologous Escherichia coli AcrB efflux protein with several common structural features, including an open cleft containing putative access and deep binding pockets proposed to interact with substrates. A highly discriminating N. gonorrhoeae strain, with the MtrD and NorM multidrug efflux pumps inactivated, was constructed and used to confirm and extend the substrate profile of MtrD to include 14 new compounds. The structural basis of substrate interactions with MtrD was interrogated by a combination of long-timescale molecular dynamics simulations and docking studies together with site-directed mutagenesis of selected residues. Of the MtrD mutants generated, only one (S611A) retained a wild-type (WT) resistance profile, while others (F136A, F176A, I605A, F610A, F612C, and F623C) showed reduced resistance to different antimicrobial compounds. Docking studies of eight MtrD substrates confirmed that many of the mutated residues play important nonspecific roles in binding to these substrates. Long-timescale molecular dynamics simulations of MtrD with its substrate progesterone showed the spontaneous binding of the substrate to the access pocket of the binding cleft and its subsequent penetration into the deep binding pocket, allowing the permeation pathway for a substrate through this important resistance mechanism to be identified. These findings provide a detailed picture of the interaction of MtrD with substrates that can be used as a basis for rational antibiotic and inhibitor design.IMPORTANCE With over 78 million new infections globally each year, gonorrhea remains a frustratingly common infection. Continuous development and spread of antimicrobial-resistant strains of Neisseria gonorrhoeae, the causative agent of gonorrhea, have posed a serious threat to public health. One of the mechanisms in N. gonorrhoeae involved in resistance to multiple drugs is performed by the MtrD multidrug resistance efflux pump. This study demonstrated that the MtrD pump has a broader substrate specificity than previously proposed and identified a cluster of residues important for drug binding and translocation. Additionally, a permeation pathway for the MtrD substrate progesterone actively moving through the protein was determined, revealing key interactions within the putative MtrD drug binding pockets. Identification of functionally important residues and substrate-protein interactions of the MtrD protein is crucial to develop future strategies for the treatment of multidrug-resistant gonorrhea.

SUBMITTER: Chitsaz M

PROVIDER: S-EPMC6867893 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Multidrug Resistance in Neisseria gonorrhoeae: Identification of Functionally Important Residues in the MtrD Efflux Protein.

Chitsaz Mohsen M Booth Lauren L Blyth Mitchell T MT O'Mara Megan L ML Brown Melissa H MH

mBio 20191119 6

A key mechanism that Neisseria gonorrhoeae uses to achieve multidrug resistance is the expulsion of structurally different antimicrobials by the MtrD multidrug efflux protein. MtrD resembles the homologous Escherichia coli AcrB efflux protein with several common structural features, including an open cleft containing putative access and deep binding pockets proposed to interact with substrates. A highly discriminating N. gonorrhoeae strain, with the MtrD and NorM multidrug e ...[more]

PMID: 31744915

Similar Datasets

Project description:Antimicrobial resistance in Neisseria gonorrhoeae has reached an alarming level, severely impacting the effective treatment of gonorrhea. Belonging to the resistance-nodulation-cell division (RND) superfamily of efflux transporters, the MtrD membrane protein of N. gonorrhoeae provides resistance to a broad range of antimicrobial compounds. A unique feature of MtrD is an 11-residue sequence (from N917 to P927 [N917-P927]) that connects transmembrane helices (TMS) 9 and 10; this sequence is not present in homologous RND proteins. This study explores the structural and functional roles of the N917-P927 region by means of mutant analysis and molecular dynamics simulations. We show that N917-P927 plays a key role in modulating substrate access to the binding cleft and influences the overall orientation of the protein within the inner membrane necessary for optimal functioning. Removal of N917-P927 significantly reduced MtrD-mediated resistance to a range of antimicrobials and mutations of three single amino acids impacted MtrD-mediated multidrug resistance. Furthermore, molecular dynamics simulations showed deletion of N917-P927 in MtrD may dysregulate access of the substrate to the binding cleft and closure of the substrate-binding pocket during the transport cycle. These findings indicate that N917-P927 is a key region for interacting with the inner membrane, conceivably influencing substrate capture from the membrane-periplasm interface and thus is essential for full multidrug resistance capacity of MtrD. IMPORTANCE The historical sexually transmitted infection gonorrhea continues to be a major public health concern with an estimated global annual incidence of 86.9 million cases. N. gonorrhoeae has been identified by the World Health Organization as one of the 12 antimicrobial-resistant bacterial species that poses the greatest risk to human health. As the major efflux pump in gonococci, the MtrD transporter contributes to the cell envelope barrier in this organism and pumps antimicrobials from the periplasm and inner membrane, resulting in resistance. This study demonstrates that a unique region of the MtrD protein that connects TMS 9 and TMS 10 forms a structure that may interact with the inner membrane positioning TMS 9 and stabilizing the protein facilitating substrate capture from the inner membrane-periplasm interface. Analysis of mutants of this region identified that it was essential for MtrD-mediated multidrug resistance. Characterization of the structure and function of this unique local region of MtrD has implications for drug efflux mechanisms used by related proteins and is important knowledge for development of antibiotics that bypass efflux.

Dataset Information

Multidrug Resistance in Neisseria gonorrhoeae: Identification of Functionally Important Residues in the MtrD Efflux Protein.

Publications

Multidrug Resistance in Neisseria gonorrhoeae: Identification of Functionally Important Residues in the MtrD Efflux Protein.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets