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1.  Genomic and Phenotypic Characterization of Vibrio cholerae Non-O1 Isolates from a US Gulf Coast Cholera Outbreak 
PLoS ONE  2014;9(4):e86264.
Between November 2010, and May 2011, eleven cases of cholera, unrelated to a concurrent outbreak on the island of Hispaniola, were recorded, and the causative agent, Vibrio cholerae serogroup O75, was traced to oysters harvested from Apalachicola Bay, Florida. From the 11 diagnosed cases, eight isolates of V. cholerae were isolated and their genomes were sequenced. Genomic analysis demonstrated the presence of a suite of mobile elements previously shown to be involved in the disease process of cholera (ctxAB, VPI-1 and -2, and a VSP-II like variant) and a phylogenomic analysis showed the isolates to be sister taxa to toxigenic V. cholerae V51 serogroup O141, a clinical strain isolated 23 years earlier. Toxigenic V. cholerae O75 has been repeatedly isolated from clinical cases in the southeastern United States and toxigenic V. cholerae O141 isolates have been isolated globally from clinical cases over several decades. Comparative genomics, phenotypic analyses, and a Caenorhabditis elegans model of infection for the isolates were conducted. This analysis coupled with isolation data of V. cholerae O75 and O141 suggests these strains may represent an underappreciated clade of cholera-causing strains responsible for significant disease burden globally.
PMCID: PMC3974666  PMID: 24699521
2.  Temperature regulation of virulence factors in the pathogen Vibrio coralliilyticus 
The ISME Journal  2011;6(4):835-846.
Sea surface temperatures (SST) are rising because of global climate change. As a result, pathogenic Vibrio species that infect humans and marine organisms during warmer summer months are of growing concern. Coral reefs, in particular, are already experiencing unprecedented degradation worldwide due in part to infectious disease outbreaks and bleaching episodes that are exacerbated by increasing SST. For example, Vibrio coralliilyticus, a globally distributed bacterium associated with multiple coral diseases, infects corals at temperatures above 27 °C. The mechanisms underlying this temperature-dependent pathogenicity, however, are unknown. In this study, we identify potential virulence mechanisms using whole genome sequencing of V. coralliilyticus ATCC (American Type Culture Collection) BAA-450. Furthermore, we demonstrate direct temperature regulation of numerous virulence factors using proteomic analysis and bioassays. Virulence factors involved in motility, host degradation, secretion, antimicrobial resistance and transcriptional regulation are upregulated at the higher virulent temperature of 27 °C, concurrent with phenotypic changes in motility, antibiotic resistance, hemolysis, cytotoxicity and bioluminescence. These results provide evidence that temperature regulates multiple virulence mechanisms in V. coralliilyticus, independent of abundance. The ecological and biological significance of this temperature-dependent virulence response is reinforced by climate change models that predict tropical SST to consistently exceed 27 °C during the spring, summer and fall seasons. We propose V. coralliilyticus as a model Gram-negative bacterium to study temperature-dependent pathogenicity in Vibrio-related diseases.
PMCID: PMC3309362  PMID: 22158392
Vibrio pathogens; coral disease; genome and proteome; quorum sensing; global climate change; temperature
3.  Purification and crystallization of Cor a 9, a major hazelnut allergen 
The major hazelnut allergen Cor a 9 was purified from the natural source and crystallized. Diffraction data were collected to 1.9 Å resolution using a synchrotron-radiation source.
Hazelnut (Corylus avellana) is one of the food sources that induce allergic reaction in a subpopulation of people with food allergy. The 11S legumin-like seed-storage protein from hazelnut has been identified as one of the major hazelnut allergens and named Cor a 9. In this study, Cor a 9 was extracted from hazelnut kernels using a high-salt solution and was purified by desalting out and FPLC to a highly purified state. Diffraction-quality single crystals were obtained using the hanging-drop vapour-diffusion method. Diffraction data were collected and a structure solution has been obtained by molecular-replacement calculations. Further refinement of the structure is currently in progress.
PMCID: PMC2628846  PMID: 19153454
hazelnuts; 11S seed-storage proteins; food allergies; tree-nut allergens
4.  Crystallization and initial crystallographic characterization of a vicilin-type seed storage protein from Pinus koraiensis  
In this study, the Korean pine (Pinus koraiensis) vicilin-type 7S seed storage protein was isolated from defatted pine-nut extract and purified by sequential gel-filtration and anion-exchange chromatography. Well diffracting single crystals were obtained by the vapour-diffusion method in hanging drops.
The cupin superfamily of proteins includes the 7S and 11S seed storage proteins. Many members of this family of proteins are known allergens. In this study, the Korean pine (Pinus koraiensis) vicilin-type 7S seed storage protein was isolated from defatted pine-nut extract and purified by sequential gel-filtration and anion-exchange chromatography. Well diffracting single crystals were obtained by the vapor-diffusion method in hanging drops. The crystals belong to the primitive cubic space group P213, with unit-cell parameters a = b = c = 148.174 Å. Two vicilin molecules were present in the asymmetric unit and the Matthews coefficient was determined to be 2.90 Å3 Da−1, with a corresponding solvent content of ∼58%. A molecular-replacement structural solution has been obtained using the program Phaser. Refinement of the structure is currently under way.
PMCID: PMC2344101  PMID: 18084088
allergens; pine nuts; seed storage protein
5.  Purification and Characterization of Enterotoxigenic El Tor-Like Hemolysin Produced by Vibrio fluvialis  
Infection and Immunity  2003;71(6):3213-3220.
The halophilic bacterium Vibrio fluvialis is an enteric pathogen that produces an extracellular hemolysin. This hemolysin was purified to homogeneity by using sequential hydrophobic-interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. It has a molecular weight of 63,000 and an isoelectric point of 4.6, and its hemolytic activity is sensitive to heat, proteases, and preincubation with zinc ions. The hemolysin lyses erythrocytes of the eight different animal species that we tested, is cytotoxic against Chinese hamster ovary cells in tissue culture, and elicits fluid accumulation in suckling mice. Lysis of erythrocytes occurs by a temperature-dependent binding step followed by a temperature- and pH-dependent lytic step. Fourteen of the first 20 N-terminal amino acid residues (Val-Ser-Gly-Gly-Glu-Ala-Asn-Thr-Leu-Pro-His-Val-Ala-Phe-Tyr-Ile-Asn-Val-Asn-Arg) are identical to those of the El Tor hemolysin of Vibrio cholerae and the heat-labile hemolysin of Vibrio mimicus. This homology was further confirmed by PCR analysis using a 5′ primer derived from the amino-terminal sequence of the hemolysin and a 3′ primer derived from the El Tor hemolysin structural gene. The hemolysin also reacts with antibodies to the El Tor-like hemolysin of non-O1 V. cholerae.
PMCID: PMC155747  PMID: 12761101
6.  Purification and Characterization of a Vulnificolysin-Like Cytolysin Produced by Vibrio tubiashii 
An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.
PMCID: PMC93075  PMID: 11472951
7.  Purification and Characterization of a Cytotonic Protein Expressed In Vitro by the Live Cholera Vaccine Candidate CVD 103-HgR 
Infection and Immunity  2000;68(10):6062-6065.
Cholera vaccines developed by the deletion of CTX genes from Vibrio cholerae induce a residual reactogenicity in up to 10% of vaccinees. A novel cytotonic agent named secreted CHO cell elongating protein (S-CEP) was purified from culture supernatants of CVD 103-HgR (Levine et al., Lancet ii:467–470, 1988). Five fractionation steps yielded electrophoretically pure S-CEP with an Mr of 79,000. A partially purified preparation caused fluid accumulation in the sealed infant mouse model. The amino terminus bore a unique sequence with strong homology to a cytotonic toxin of El Tor V. cholerae.
PMCID: PMC101575  PMID: 10992523

Results 1-7 (7)