The transmembrane K+/H+ antiporters of NhaP type of Vibrio cholerae (Vc-NhaP1, 2, and 3) are critical for maintenance of K+ homeostasis in the cytoplasm. The entire functional NhaP group is indispensable for the survival of V. cholerae at low pHs suggesting their possible role in the acid tolerance response (ATR) of V. cholerae. Our findings suggest that the Vc-NhaP123 group, and especially its major component, Vc-NhaP2, might be a promising target for the development of novel antimicrobials by narrowly targeting V. cholerae and other NhaP-expressing pathogens. On the basis of Vc-NhaP2 in silico structure modeling, Molecular Dynamics Simulations, and extensive mutagenesis studies, we suggest that the ion-motive module of Vc-NhaP2 is comprised of two functional regions: (i) a putative cation-binding pocket that is formed by antiparallel unfolded regions of two transmembrane segments (TMSs V/XII) crossing each other in the middle of the membrane, known as the NhaA fold; and (ii) a cluster of amino acids determining the ion selectivity.
The genome of Vibrio cholerae encodes three cation-proton antiporters of NhaP-type, Vc-NhaP1, 2, and 3. To examine physiological roles of Vc-NhaP antiporters, triple ΔnhaP1ΔnhaP2ΔnhaP3 and single ΔnhaP3 deletion mutants of V. cholerae were constructed and characterized. Vc-NhaP3 was, for the first time, cloned and biochemically characterized. Activity measurements on the inside-out membrane vesicle experimental model defined Vc-NhaP3 as a potassium-specific cation-proton antiporter. While elimination of functional Vc-NhaP3 resulted in only minor growth defect in potassium-rich medium at pH 6.0, the triple Vc-NhaP mutant demonstrated severe growth defects at both low and high [K] at pH 6.0 and failed to grow at high [K] in mildly alkaline (pH 8.0 and 8.5) media, as well. Expressed from a plasmid, neither of the Vc-NhaP paralogues was able to complement the severe potassium-sensitive phenotype of the triple deletion mutant completely. Vc-NhaP1 provided much better complementation at acidic pH compared to Vc-NhaP2, despite the fact that Vc-NhaP2 showed much higher antiport activity in sub-bacterial vesicles. In mildly alkaline pH only Vc-NhaP2 complemented the potassium-sensitive phenotype of the triple deletion mutant. Taken together, these data suggest that in vivo all three isoforms operate in concert, contributing to K resistance of V. cholerae. We suggest that the Vc-NhaP paralogue group might play a role in passing gastric acid barrier by ingested V. cholerae cells.
The Vc-NhaP2 antiporter from Vibrio cholerae exchanges H for K or Na but not for the smaller Li. The molecular basis of this unusual selectivity remains unknown. Phyre and Rosetta software were used to generate a structural model of the Vc-NhaP2. The obtained model suggested that a cluster of residues from different transmembrane segments (TMSs) forms a putative cation-binding pocket in the middle of the membrane: D133 and T132 from TMS V together with D162 and E157 of TMS VI. The model also suggested that L257, G258, and N259 from TMS IX together with T276, D273, Q280, and Y251 from TMS X as well as L289 and L342 from TMS XII form a transmembrane pathway for translocated ions with a built-in filter determining cation selectivity. Alanine-scanning mutagenesis of the identified residues verified the model by showing that structural modifications of the pathway resulted in altered cation selectivity and transport activity. In particular, L257A, G258A, Q280A, and Y251A variants gained Li/H antiport capacity that was absent in the nonmutated antiporter. T276A, D273A, and L289A variants exclusively exchanged K for H, while a L342A variant mediated Na/H exchange only, thus maintaining strict alkali cation selectivity.
Coronavirus disease-2019 has become an immense threat to global public health. The causative agent of this disease is a novel zoonotic pathogen called Severe Acute Respiratory Syndrome related Coronavirus-2 (SARS-CoV-2). Since this is a newly evolved pathogen, very limited information is available to develop effective therapeutics against this deadly virus. Although bioinformatics based analysis could be handy to unveil drugs or vaccines against bacteria and fungus, such approaches are hardly seen for acellular viruses. However, in this study we rationally merged several powerful in silico techniques and proposed prospective therapeutics based on available omics data for COVID-19. Through meticulous analysis of conserved regions of 67 SARS-CoV-2 strains, spike and membrane glycoproteins were chosen to develop and propose a chimeric vaccine against this virus. siRNAs were also designed against these glycoprotein genes to silence them. Moreover, six drug compound candidates were suggested to inhibit the conserved RNA-directed RNA polymerase protein. Finally, due to the close relationship of SARS-CoV-2 and SARS-CoV, publicly available gene expression datasets of SARS-CoV were analyzed to identify 13 immunoregulatory genes that might develop interferon based therapy. Our study will quicken the researches among pharmaceuticals, researchers and clinicians to develop rapid therapeutics for controlling this notorious pandemic disease.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.