Purification and characterization of a Chinese hamster ovary cell elongation factor of Vibrio hollisae

Abstract

The halophilic bacterium Vibrio hollisae, isolated from patients with diarrhea, produces an extracellular toxin which elongates Chinese hamster ovary (CHO) cells. We purified this toxin to homogeneity by sequential ammonium sulfate precipitation, gel filtration with Sephacryl S-200, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, ion-exchange chromatography with DEAE-Sephadex A-50, and affinity chromatography. The toxin is heat labile and sensitive to proteases, with an isoelectric point of about 6.5 and molecular weights of about 83,000 and 80,000, as estimated by gel filtration and sodium dodecyl sulfatepolyacrylamide gel electrophoresis, respectively. The toxin did not react with immunoaffinity-purified antibodies to cholera toxin in a plate enzyme-linked immunosorbent assay and in a Western blot, and its activity could not be neutralized by anti-cholera toxin serum. Mixed gangliosides and gangliosides

Enteric bacterial toxins: mechanisms of action and linkage to intestinal secretion.

“…The toxin is heat labile (30 min at 56ЊC) and causes fluid accumulation in suckling mice. HT does not react with antisera against CT, and no DNA sequences homologous to ctx have been found (293). Although HT can cause elongation of CHO cells, there is no accompanying increase in intracellular cyclic AMP levels in this cell line and the mechanisms by which HT leads to CHO elongation and intestinal secretion are unknown.…”

“…Recently, however, the toxin of Vibrio hollisae (HT) and a 70-kDa heat-stable secretory toxin produced by Aeromonas spp. have been shown to cause elongation of CHO cells in the absence of increased cyclic AMP levels (293,347), thus demonstrating that morphological changes in nonintestinal cell lines cannot be assumed to identify the biochemical mediator by which the enteric toxin acts. Another example of the use of nonintestinal cell lines to study enteric toxins is the use of HeLa or Vero cells for demonstrating cytotoxicity due to inhibition of protein synthesis by Shiga and Shiga-like toxins (401).…”

“…hollisae causes diarrhea and septicemia in humans, with most cases linked to the consumption of raw seafood. In addition to producing a toxin similar to the thermostable direct hemolysin of V. parahaemolyticus (see below), V. hollisae produces a toxin which causes elongation of CHO cells (293). The purified V. hollisae toxin (HT) has a molecular weight of 80,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and no apparent subunit structure.…”

Characterization of the Secretomes of Two Vibrios Pathogenic to Mollusks

“…The secretomes of both vibrio species contained an extracellular triacylglycerol lipase ( Table 1 ). This protein belongs to the same family as the phospholipase Pla1, a secreted virulence factor of Aeromonas hydrophila [29] and Cef, a toxin with cell elongation activity produced by Vibrio hollisae , which causes diarrhea in humans [30] . Phospholipases can act as potent membrane destructors and can manipulate host signalling pathways [31] .…”

Immunization with recombinant fusion of LTB and linear epitope (40–62) of epsilon toxin elicits protective immune response against the epsilon toxin of Clostridium perfringens type D

“…Wild type holotoxin, made up of wild type LTB and LTA has been reported to alter CHO-K1 cells morphology (elongation of cells) (Guerrant et al 1974; Kothary et al 1995). We have earlier shown that the rLTB.Etx 40–62 could bind to the GM1 receptor which is crucial for the biological activity of LTB (Kaushik et al 2013).…”