Abstract
The genome of Vibrio cholerae contains three structural genes for the NhaP-type cation-proton antiporter paralogues, Vc-NhaP1, Vc-NhaP2, and Vc-NhaP3, mediating exchange of K+ and or Na+ for protons across the membrane. Based on phenotypic analysis of chromosomal Vc-NhaP1, Vc-NhaP2, and Vc-NhaP3 triple deletion mutants, we suggest that Vc-NhaP paralogues are primarily K+/H+ antiporters and might play a role in the acid tolerance response of V.cholerae as it passes through the gastric acid barrier of the stomach. Comparison of the biochemical properties of Vc-NhaP isoforms revealed that Vc-NhaP2 was the most active among all three paralogues. Therefore, the Vc-NhaP2 antiporter is a plausible therapeutic target for developing novel inhibitors targeting these ion exchangers. Our structural and mutational analysis of Vc-NhaP2 identified a putative cation-binding pocket formed by antiparallel extended regions of two transmembrane segments (TMSs V and XII) along with TMS VI. Molecular dynamics simulations suggested that the flexibility of TMSs V and XII is crucial for intramolecular conformational events in Vc-NhaP2. In this study, we developed putative Vc-NhaP2 inhibitors from amiloride analogs. Molecular docking of the modified amiloride analogs revealed promising binding properties. The four selected drugs potentially interacted with functionally important amino acid residues located on the cytoplasmic side of TMS VI, the extended chain region of TMSs V and XII, and the loop region between TMSs VIIII and IX. Molecular dynamics simulations revealed that binding of the selected drugs can potentially destabilize Vc-NhaP2 and alter the flexibility of functionally important TMS VI. This work presents the utility of in silico approaches for the rational identification of potential targets and drugs that could target NhaP2 cation proton antiporters to control V. cholerae. The goal was to identify potential drugs that could be validated in future experiments.
Highlights
Vibrio cholerae, the causative agent of the diarrheal disease cholera, is considered an emerging and re-emerging disease with epidemic and pandemic potential (Mandal et al 2011; Faruque et al 2004)
The membrane of V. cholerae contains a trio of cation-proton antiporters of a specific type, NhaP, which are responsible for the transport of both K+ and Na+ (Resch et al 2010, 2011; Mourin et al 2017, 2019a, 2019b; Quinn et al 2012; Schubiger et al 2017)
Preliminary acid tolerance response (ATR) tests conducted with the wild-type parental strain V. cholerae O395N1 and its triple deletion mutant VcDNhaP123, showed that the triple mutant died at a much higher rate than the isogenic wild-type strain upon challenge with HCl (Mourin et al 2017)
Summary
The causative agent of the diarrheal disease cholera, is considered an emerging and re-emerging disease with epidemic and pandemic potential (Mandal et al 2011; Faruque et al 2004). Vibrio cholerae has remarkable genetic features that enable the human pathogen to survive under harsh environmental conditions (Faruque et al 2004). In recent decades, this enteric pathogen has developed multidrug resistance by acquiring numerous mobile genetic elements (Das et al 2020). A simulation based on an artificial neural network demonstrated that climate change might drastically elevate cholera outbreaks in the near future (Asadgol et al 2019). The combination of elevated outbreaks and a higher prevalence of multidrug resistant V. cholerae could lead to a serious situation. The development of new antimicrobial treatments against this deadly pathogen is paramount
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