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.Vibriosis caused by marine Vibrio species is considered one of the most serious diseases of hatchery-reared oyster larvae (10,11,17,47,52). The disease is characterized by a rapid and dramatic reduction in larval motility, detached vela, and necrotic soft tissue, which lead to high mortality rates, exceeding 90% within 1 day of infection (45). Pathogenic agents that cause larval bivalve vibriosis have intermittently and severely curtailed shellfish hatchery production on the Atlantic and Pacific coasts of the United States, causing substantial losses in the industry (3, 10, 13). Vibrio tubiashii, a bacterial species first reported by Tubiash et al. (51), was identified as a causative agent of vibriosis (originally referred to as bacillary necrosis) in larval and juvenile bivalves of the hard clam (Mercenaria mercenaria) and Eastern oyster (Crassostrea virginica). Estes et al. (14) characterized a number of pathogenic and nonpathogenic bacterial strains from diseased Pacific oysters (Crassostrea gigas) at shellfish hatcheries on the Pacific coast of North America and described some of the highly pathogenic bacterial isolates as V. tubiashii.The genus Vibrio is the largest member of the family Vibrionaceae, which includes gram-negative and curved rod-shaped facultative anaerobes. The genus consists of at least 30 known species, which are w...

Introduction: Cancer survivors are increasingly diagnosed with a syndrome of neurocognitive dysfunction termed cancer-related cognitive impairment (CRCI). Chemotherapy and radiation therapy have been implicated in CRCI; however, its underlying pathogenesis remains unclear, hindering effective prevention or treatment.Methods: We used the hairless strain SKH1 (11–12-week-old) and treated the mice with radiation to the right hindlimb, doxorubicin (a chemotherapy agent), concurrent radiation, and doxorubicin, or no treatment (control). Neurocognition was evaluated via standardized behavioral testing following treatment. Mice were subsequently humanely euthanized, and plasma and brains were collected to identify inflammatory changes.Results: Mice treated with radiation, doxorubicin, or both radiation and doxorubicin demonstrated equivalent hippocampal dependent memory deficits and significant increases in activated microglia and astrocytes compared to control mice. Doxorubicin-treated mice had significantly increased plasma IL-6 and failed to gain weight compared to control mice over the study period.Discussion: This study demonstrates that non-brain directed radiation induces both gliosis and neurocognitive deficits. Moreover, this work presents the first characterization of SKH1 mice as a relevant and facile animal model of CRCI. This study provides a platform from which to build further studies to identify potential key targets that contribute to CRCI such that strategies can be developed to mitigate unintended neuropathologic consequences associated with anticancer treatment.

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...

The existence of bacterial K+/H+ antiporters preventing the over-accumulation of potassium in the cytoplasm was predicted by Peter Mitchell almost fifty 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 pH optimum at ~7.75 with an apparent Km 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 structural determinants of the substrate specificity in alkali cation exchangers.

e 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.