Na+/H+ antiport is essential for Yersinia pestis virulence.

Na(+)/H(+) antiporters are ubiquitous membrane proteins that play a central role in the ion homeostasis of cells. In this study, we examined the possible role of Na(+)/H(+) antiport in Yersinia pestis virulence and found that Y. pestis strains lacking the major Na(+)/H(+) antiporters, NhaA and NhaB, are completely attenuated in an in vivo model of plague. The Y. pestis derivative strain lacking the nhaA and nhaB genes showed markedly decreased survival in blood and blood serum ex vivo. Complementation of either nhaA or nhaB in trans restored the survival of the Y. pestis nhaA nhaB double deletion mutant in blood. The nhaA nhaB double deletion mutant also showed inhibited growth in an artificial serum medium, Opti-MEM, and a rich LB-based medium with Na(+) levels and pH values similar to those for blood. Taken together, these data strongly suggest that intact Na(+)/H(+) antiport is indispensable for the survival of Y. pestis in the bloodstreams of infected animals and thus might be regarded as a promising noncanonical drug target for infections caused by Y. pestis and possibly for those caused by other blood-borne bacterial pathogens.

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

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.

The putative Na+/H+ antiporter of Vibrio cholerae, Vc-NhaP2, mediates the specific K+/H+ exchange in vivo.

The existence of bacterial K(+)/H(+) antiporters that prevent the overaccumulation of potassium in the cytoplasm was predicted by Peter Mitchell almost 50 years ago. The importance of K(+)/H(+) antiport for bacterial physiology is widely recognized, but its molecular mechanisms remain underinvestigated. Here, we demonstrate that a putative Na(+)/H(+) antiporter, Vc-NhaP2, protects cells of Vibrio cholerae growing at pH 6.0 from high concentrations of external K(+). Resistance of V. cholerae to Na(+) was found to be independent of Vc-NhaP2. When assayed in inside-out membrane vesicles derived from antiporter-deficient Escherichia coli, Vc-NhaP2 catalyzed the electroneutral K(+)(Rb(+))/H(+) exchange with a pH optimum of approximately 7.75 with an apparent K(m) for K(+) of 1.62 mM. In the absence of K(+), it exhibited Na(+)/H(+) antiport, albeit rather weakly. Interestingly, while Vc-NhaP2 cannot exchange Li(+) for protons, elimination of functional Vc-NhaP2 resulted in a significantly higher Li(+) resistance of V. cholerae cells growing at pH 6.0, suggesting the possibility of Vc-NhaP2-mediated Li(+)/K(+) antiport. The peculiar cation specificity of Vc-NhaP2 and the presence of its two additional paralogues in the same genome make this transporter an attractive model for detailed analysis of the structural determinants of the substrate specificity in alkali cation exchangers.

Virulence of metalloproteases produced by Vibrio species on Pacific oyster Crassostrea gigas larvae.

Vibrio tubiashii, a pathogen of shellfish larvae and juveniles, produces several extracellular products. Here, we document that culture supernatants of several marine Vibrio species showed toxicity to oyster larvae. Treatment of these supernatants with EDTA not only severely diminished proteolytic activities, but also dramatically reduced toxicity to the larvae. Culture supernatants of metalloprotease-deficient mutants of V. tubiashii, V. cholerae, and V. splendidus were impaired in their ability to cause larval death compared to the wild type strains. Culture supernatants of Pseudomonas aeruginosa, known to contain several secreted proteases, showed virtually no toxicity to oyster larvae. Purified V. tubiashii protease A (VtpA), but not the prototype metalloprotease, thermolysin from Bacillus thermoproteolyticus, was highly toxic to the larvae. In addition, toxicity of purified VtpA was much greater for 6-d-old oyster larvae than for 16-d-old larvae. Together, these results indicated that culture supernatants of a variety of Vibrio species are highly toxic to oyster larvae and that the production of a metalloprotease is required for this effect. We propose that there are, as yet uncharacterized, specific substrates contained in larval tissue that are degraded by VtpA as well as certain homologous metalloproteases produced by other marine Vibrio species which, in turn, may contribute to vibriosis.

The extracellular metalloprotease of Vibrio tubiashii is a major virulence factor for pacific oyster (Crassostrea gigas) larvae.

Vibrio tubiashii is a recently reemerging pathogen of larval bivalve mollusks, causing both toxigenic and invasive disease. Marine Vibrio spp. produce an array of extracellular products as potential pathogenicity factors. Culture supernatants of V. tubiashii have been shown to be toxic to oyster larvae and were reported to contain a metalloprotease and a cytolysin/hemolysin. However, the structural genes responsible for these proteins have yet to be identified, and it is uncertain which extracellular products play a role in pathogenicity. We investigated the effects of the metalloprotease and hemolysin secreted by V. tubiashii on its ability to kill Pacific oyster (Crassostrea gigas) larvae. While V. tubiashii supernatants treated with metalloprotease inhibitors severely reduced the toxicity to oyster larvae, inhibition of the hemolytic activity did not affect larval toxicity. We identified structural genes of V. tubiashii encoding a metalloprotease (vtpA) and a hemolysin (vthA). Sequence analyses revealed that VtpA shared high homology with metalloproteases from a variety of Vibrio species, while VthA showed high homology only to the cytolysin/hemolysin of Vibrio vulnificus. Compared to the wild-type strain, a VtpA mutant of V. tubiashii not only produced reduced amounts of protease but also showed decreased toxicity to C. gigas larvae. Vibrio cholerae strains carrying the vtpA or vthA gene successfully secreted the heterologous protein. Culture supernatants of V. cholerae carrying vtpA but not vthA were highly toxic to Pacific oyster larvae. Together, these results suggest that the V. tubiashii extracellular metalloprotease is important in its pathogenicity to C. gigas larvae.