NhaP1 is a K+(Na+)/H+ antiporter required for growth and internal pH homeostasis of Vibrio cholerae at low extracellular pH

Abstract

Vibrio cholerae has adapted to a wide range of salinity, pH and osmotic conditions, enabling it to survive passage through the host and persist in the environment. Among the many proteins responsible for bacterial survival under these diverse conditions, we have identified Vc-NhaP1 as a K + (Na + )/H + antiporter essential for V. cholerae growth at low environmental pH. Deletion of the V. cholerae nhaP1 gene caused growth inhibition when external potassium was either limited (100 mM and below) or in excess (400 mM and above). This growth defect was most apparent at mid-exponential phase, after 4-6 h of culture. Using a pH-sensitive GFP, cytosolic pH was shown to be dependent on K + in acidic external conditions in a Vc-NhaP1-dependent manner. When functionally expressed in an antiporterless Escherichia coli strain and assayed in everted membrane vesicles, Vc-NhaP1 operated as an electroneutral alkali cation/proton antiporter, exchanging K + or Na + ions for H + within a broad pH range (7.25-9.0). These data establish the putative V. cholerae NhaP1 protein as a functional K + (Na + )/H + antiporter of the CPA1 family that is required for bacterial pH homeostasis and growth in an acidic environment. INTRODUCTIONVibrio cholerae is a Gram-negative pathogen which causes cholera, a dangerous disease that remains a public health concern (Enserink, 2010). As it transitions between the infectious state and its environmental reservoir, the bacterium encounters a dynamic range of osmotic and pH conditions. During human infection, V. cholerae produces a potent enterotoxin, cholera toxin, which promotes accumulation of Na + and Cl 2 ions in the host intestinal lumen and, in turn, causes rapid osmotic dehydration of host tissue and profuse diarrhoea. In the environment, V. cholerae is found in many coastal and estuarine waters, where it is exposed to severe periodic changes in salinity, pH and osmolarity as variable ratios of brackish and fresh water mix at different rates (Miller et al., 1984; Singleton et al., 1982a, b). Thus, both the pathogenic and environmental lifestyles of this organism require that V. cholerae can adapt to rapidly shifting osmolarities, ionic strengths and pH values. These lifestyles require that V. cholerae possess adequate molecular mechanisms to adapt to such environmental challenges.A number of V. cholerae proteins have been described that generate, maintain or use a transmembrane gradient of cations such as Na + (Häse et al., 2001). These proteins are predicted to help the bacterium survive hypo-and hyperosmolar states in addition to exploiting the Na + gradient for solute transport, pH regulation and motility. For example, the NQR complex couples Na + export to electron transport, resulting in the generation of a sodium-motive force that can then be used for various types of membrane work (Tokuda & Unemoto, 1981Zhou et al., 1999). The V. cholerae NhaA antiporter mediates Na + /H + exchange and thus regulates Na + homeostasis at pH 8.5, conditions which are not unusual for seawater in areas where...

Catabolism of N -Acetylneuraminic Acid, a Fitness Function of the Food-Borne Lactic Acid Bacterium Lactobacillus sakei, Involves Two Newly Characterized Proteins

“…The LSA1645 protein might have just such a regulatory role in maintaining internal pH, and we propose to name it NanP. It has recently been reported that in Vibrio cholerae, the NhaP1 described to be an K ϩ (Na ϩ )/H ϩ antiporter is required for growth and internal pH homeostasis (50). Although LSA1645 presents no homology with NhaP1, they could play similar roles.…”

A UPF0118 family protein with uncharacterized function from the moderate halophile Halobacillus andaensis represents a novel class of Na+(Li+)/H+ antiporter

“…In prokaryotes, Na + /H + antiporters are ubiquitous secondary transporters catalyze the efflux of intracellular alkali cations in exchange for external protons, which play a vital role in reducing the cytoplasmic concentration of toxic alkali cations and supporting Na + -dependent intracellular pH homeostasis under alkaline conditions1234. They were also designated Na + (Li + )/H + antiporters, due to Na + /H + antiport activity, together with Li + /H + antiport activity.…”

Na + /H + Antiport Is Essential for Yersinia pestis Virulence

“…1) (4). However, recent detailed biochemical characterization of NhaP-type antiporters NhaP-1 and NhaP-2 from Vibrio cholerae revealed that these antiporters mediate K ϩ /H ϩ exchange rather than Na ϩ /H ϩ exchange under physiological conditions (6,7). By analogy, we predict that the Y. pestis NhaP protein has a similar cation preference ( Fig.…”