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.
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.
Delta-crystallin is the major soluble protein in avian eye lenses with a structural role in light scattering. Dissociation and unfolding of the tetrameric protein in guanidinium chloride (GdmCl) can be sensitively monitored by the intrinsic tryptophan fluorescence. In this study refolding of GdmCl-denatured delta-crystallin was investigated. A marked hysteresis was observed while refolding by dilution of the 5 M GdmCl-denatured delta-crystallin. The secondary structure of the refolded protein was largely restored. However, monitoring intrinsic fluorescence of single tryptophan mutants indicated that the microenvironment of domain 1 (W74) was not restored. The region containing W169, which is close to the dimer interface, remained exposed following refolding. During refolding of the wild-type protein, dimeric, tetrameric, and aggregate forms were identified. The ratio of tetramer to dimer increased with time, as judged by gel-filtration chromatography and nondenaturing gel electrophoresis. However the observed levels of tetramer did not return to the same levels as observed before GdmCl treatment. The proportion of tetramer was significantly decreased in the N-25 deletion mutant and it did not increase with time. These results suggest that there is a kinetic barrier for assembly of dimers into tetramers. The consequence of this is that dimers refold to form aggregates. Aggregation seems to follow a nucleation mechanism with an apparent reaction order of 4.7+/-0.2, suggesting four or five monomers constitute the core structure of nucleus, which propagate to form high molecular weight aggregates. Addition of alpha-crystallin during refolding prevents aggregation. Thioflavin T and Congo red assays indicated a regular structure for the protein aggregates, which appear as hollow tubules packed into helical bundles. Aggregate formation was protein concentration dependent that progressed via two stages with rate constants of 0.0039+/-0.0006 and 0.00043+/-0.00003 s(-1), respectively. We propose that the N-terminal segment of delta-crystallin plays a critical role in proper double dimer assembly and also in the assembly of nucleus to aggregate formation.
Pathogenic vibrios are important etiologic agents in tropical regions and have been frequently recovered from seafoods and aquacultured foods. A number of psychrotrophic vibrios were isolated and selected from frozen seafoods and their survivals in tryptic soy broth (TSB) at 4°C and −30°C were studied. These psychrotrophic strains showed good survival at low temperatures and could probably enhance the risk of vibrios in frozen foods. Vibrio mimicus 70 and 198, Vibrio fluvialis 52 and Vibrio parahaemolyticus 205 survived well at 10°C, 4°C and −30°C, while the non-cold fitter V. fluvialis 28 was completely inactivated in the test periods. These strains were not heat resistant and could be easily inactivated by heat treatment. Effect of phosphates may be different for. various Vibrio species at low temperatures. Survival of V. parahaemolyticus 205 was significantly protected by the heated metaphosphate at 4°C by lowering the lethality of cold injured cells but not by increasing the level of uninjured viable cells. Pyrophosphate was inhibitory to this V. parahaemolyticus strain at −30°C.
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