Abstract:Vibrio parahaemolyticus is an important enteropathogen in Japan, Taiwan and other coastal regions.The influence of the regulation of iron on the pathogenesis of this pathogen has not been well characterized. in Japan and Taiwan (1, 24). Thermostable direct hemolysin (TDH) and other TDH-related hemolysins are the major virulence factors in this pathogen (7-9, 19, 30). Karunasagar et al (11) demonstrated that the addition of lysed erythrocyte extract or ferric ammonium citrate to bacterial cultures significantly enhances the virulence of V parahaemolyticus. Wong and Lee (26) also showed that the in vivo growth of this pathogen is enhanced by the presence of supplementary iron in a peritoneally infected mouse model. Factors involved in the acquisition of iron have been described for V parahaemolyticus. In our previous in vitro study, production of siderophore, specific outer membrane proteins (OMPs) and TDH were all enhanced in V parahaemolyticus by limiting iron in the culture (3). One of the specific OMPs (77 kDa), characterized by a putative TonB box in its N-terminal amino acid sequence, may be involved in the acquisition of iron-containing compounds (15). TonB-dependent proteins usually act as receptors in the iron acquisition systems of Escherichia coli and V cholerae (2, 6). In another study, an 83 kDa OMP was recognized as being associated with the utilization of hemin and hemoglobin in V parahaemolyticus (27).Besides their role in the acquisition of iron, iron-regulated proteins could also be associated with the virulence of pathogens. Iron-regulated IrgA has been demonstrated to be a virulence factor for infection in an animal model with classical V cholerae, and homologous sequences were detected in non-Ol V cholerae, V. parahaemolyticus, V fluvialis and V alginolyticus (5).In this study, we obtained several spontaneous iron-utilizing mutant strains (mutants) of V parahaemolyticus which showed limited growth in iron-limited medium and did not produce iron-regulated OMPs in normal growth medium. The goal of this study was to elucidate the roles of iron acquisition in the pathogenesis of V.parahaemolyticus by examining the changes in the virulence of these iron-utilizing mutants by in vitro and in vivo assays. We also reported on the utilization of different iron sources by different strains of V parahaemolyticus. Materials and MethodsBacterial strains. Pathogenic and non-pathogenic

S-Crystallin is a major protein present in the lenses of cephalopods (octopus and squid). To facilitate the cloning of this crystallin gene, cDNA was constructed from the poly(A)+ mRNA of octopus lenses, and amplified by PCR for nucleotide sequencing. Sequencing of 10 of 15 positive clones coding for this crystallin revealed three distinct S-crystallin isoforms with 61-64% identity in nucleotide sequences and 42-58% similarity in amino acid sequences when compared with homologous crystallins in squid lenses. These charge-isomeric crystallins also show between 26 and 33% amino acid sequence identity to four major classes of glutathione S-transferase (GST), a major detoxification enzyme present in most mammalian tissues. For further analysis, expression of one of the S-crystallin cDNAs was carried out in the bacterial expression system pQE-30, and the S-crystallin protein produced in Escherichia coli was purified to homogeneity to determine the enzymic properties. We found that the expressed octopus S-crystallin possessed much lower GST activity than the authentic GSTs from other tissues. Sequence comparison and construction of phylogenetic trees for S-crystallins from squid and octopus lenses and various classes of GSTs revealed that S-crystallins represent a multigene family which is structurally related to Alpha-class GSTs and probably derived from the ancestral GST by gene duplication and subsequent multiple mutational substitutions.

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Pathogenic vibrios are important etiologic agents in tropical regions and have been frequently recovered from seafoods and aquacultured foods. In this study, commercially frozen seafoods including peeled shrimps and fish and shrimp dumplings were examined. Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio cholerae and Vibria fluvialis were recovered at 36.0%, 15.8%, 14.9% and 13.2%, respectively. A number of psychrotrophic vibrios were selected and their survival in tryptic soy broth (TSB) supplemented with 1% sodium chloride (NaCl) (TSBS medium) and shrimp homogenate at 4°C and −30°C were studied. Two psychrotrophic non-O1 V. cholerae (laboratory stocks no 128 and 129) survived well at these low temperatures. Counts decreased by about 1 log/ml in TSBS medium at 4°C for 6 days and 3 log/ml at −30°C for 3 days. Shrimp homogenate provided better protection than TSBS medium for psychrotrophic V. cholerae at −30°C. Survival of V. cholerae at low temperatures was further increased by the addition of 0.5% of heated pyrophosphate and metaphosphate, probably by decreasing the lethality of the cold injury to the cells. Measures should be taken to minimize the risk from pathogenic vibrios in frozen seafoods, especially if phosphates are used and psychrotrophic strains are present.