Purification and Characterization of Enterotoxigenic El Tor-Like Hemolysin Produced by Vibrio fluvialis

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

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

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

Crystal Structure of Korean Pine (Pinus koraiensis) 7S Seed Storage Protein with Copper Ligands

The prevalence of food allergy has increased in recent years, and Korean pine vicilin is a potential food allergen. We have previously reported the crystallization of Korean pine vicilin purified from raw pine nut. Here we report the isolation of vicilin mRNA and the crystal structure of Korean pine vicilin at 2.40 Å resolution. The overall structure of pine nut vicilin is similar to the structures of other 7S seed storage proteins and consists of an N-terminal domain and a C-terminal domain. Each assumes a cupin fold, and they are symmetrically related about a pseudodyad axis. Three vicilin molecules form a doughnut-shaped trimer through head-to-tail association. Structure characterization of Korean pine nut vicilin unexpectedly showed that, in its native trimeric state, the vicilin has three copper ligands. Sequence alignments suggested that the copper-coordinating residues were conserved in winter squash, sesame, tomato, and several tree nuts, while they were not conserved in a number of legumes, including peanut and soybean. Additional studies are needed to assess whether the copper-coordinating property of vicilins has a biological function in the relevant plants. The nutritional value of this copper-coordinating protein in tree nuts and other edible seeds may be worth further investigations

Analysis of the molecular diversity among cronobacter species isolated from filth flies using targeted PCR, pan genomic DNA microarray, and whole Genome sequencing analyses

Cronobacter species are opportunistic pathogens capable of causing life-threatening infections in humans, with serious complications arising in neonates, infants, immuno-compromised individuals, and elderly adults. The genus is comprised of seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti. Despite a multiplicity of genomic data for the genus, little is known about likely transmission vectors. Using DNA microarray analysis, in parallel with whole genome sequencing, and targeted PCR analyses, the total gene content of two C. malonaticus, three C. turicensis, and 14 C. sakazaki isolated from various filth flies was assessed. Phylogenetic relatedness among these and other strains obtained during surveillance and outbreak investigations were comparatively assessed. Specifically, microarray analysis (MA) demonstrated its utility to cluster strains according to species-specific and sequence type (ST) phylogenetic relatedness, and that the fly strains clustered among strains obtained from clinical, food and environmental sources from United States, Europe, and Southeast Asia. This combinatorial approach was useful in data mining for virulence factor genes, and phage genes and gene clusters. In addition, results of plasmidotyping were in agreement with the species identity for each strain as determined by species-specific PCR assays, MA, and whole genome sequencing. Microarray and BLAST analyses of Cronobacter fly sequence datasets were corroborative and showed that the presence and absence of virulence factors followed species and ST evolutionary lines even though such genes were orthologous. Additionally, zebrafish infectivity studies showed that these pathotypes were as virulent to zebrafish embryos as other clinical strains. In summary, these findings support a striking phylogeny amongst fly, clinical, and surveillance strains isolated during 2010-2015, suggesting that flies are capable vectors for transmission of virulent Cronobacter spp.; they continue to circulate among United States and European populations, environments, and that this "pattern of circulation" has continued over decades