Physiological, structural and functional analysis of the paralogous cation-proton antiporters of NhaP type from Vibrio cholerae

The genome of Vibrio cholerae contains three structural genes for the NhaP-type antiporters paralogues Vc-NhaP1, 2 and 3 supposedly mediating exchange of K+ and or Na+ for protons across the membrane. Individual biochemical and physiological properties of these ion exchangers were analyzed in the presented work. Phenotype analysis of engineered chromosomal Vc-nhaP1, Vc-nhaP2 and Vc-nhaP3 deletion mutants and complementation of each isoform in trans reported in this thesis, has proven that the three NhaP paralogues are essential for maintaining K+ homeostasis in the cytoplasm of V. cholerae in the cell is in vivo. Expressed in trans, neither of the Vc-NhaP paralogues was able to complement the severe potassium-sensitive phenotype of the triple deletion mutant completely. The wild type V. cholerae had much higher survival rates compared to the triple deletion mutant, Vc∆NhaP123, when challenged by HCl (pH 3.5). We therefore suggested that Vc-NhaP paralogues might play a role in the Acid Tolerance Response (ATR) 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 is the most active among all three paralogues. In the course of extensive mutagenesis experiments, we have identified a number of functionally critical residues. In particular, we found that mutation of Gly159 to Ala renders Vc-NhaP2 able to exchange Li+ for protons, introducing a completely new activity. A structural analysis of Vc-NhaP2 based on the mutagenesis data combined with the in silico structure modelling and Molecular Dynamics Simulations yielded information regarding two important elements in the organization of Vc-NhaP2: (1) a putative cation binding pocket formed by antiparallel extended regions of two transmembrane segments (TMSs V/XII) crossing each other in the middle of the membrane, and (2) a cluster of amino acid residues near the putative cation binding pocket possibly determining t