Use of Monoclonal Antibodies To Identify Phospholipase C as the Enterotoxic Factor of the Bifunctional Hemolysin-Phospholipase C Molecule of Vibrio cholerae O139

Abstract

Two hybrid clones producing monoclonal antibodies (MAbs) raised against the purified enterotoxic hemolysin-phospholipase C (HlyPC) bifunctional molecule of a Vibrio cholerae O139 strain were used to study its enterotoxicity in relation to its hemolytic and enzymatic activities. Fab fragments of MAbs from ascites produced by the two hybrids neutralized the hemolytic activity of HlyPC, leaving the enzymatic activity unaffected. In ligated rabbit ileal loop and infant mouse intestine, the Fab fragments of the MAbs were not able to neutralize the enterotoxicity of HlyPC, suggesting that PC rather than Hly is the enterotoxic moiety of the molecule. The enterotoxicity of the purified PC molecule isolated from an Hly− spontaneous mutant of the HlyPC-producing parent strain further confirms this contention. The Hly molecule isolated from a PC− mutant was not diarrheagenic.

Comparative analysis of cytotoxin, hemolysin, hemagglutinin and exocellular enzymes among clinical and environmental isolates of Vibrio cholerae O139 and non-O1, non-O139

“…Hemolysin of V. cholerae is suggested to be a virulence factor contributing towards cholera pathogenesis [20]. Recently, we pu-ri¢ed a bifunctional hemolysin-phospholipase C molecule from V. cholerae O139 showing enterotoxic activity, as shown by £uid accumulation in the ligated rabbit ileal loop and in the intestines of suckling mice [21]. All the O139 and non-O1, non-O139 strains produced considerable amounts of extracellular enzymes such as elastase, protease, neuraminidase, phospholipase A and phospholipase C. However, expression of these enzymes did not vary signi¢cantly [ Table 3] between the strains isolated from clinical and environmental sources.…”

“…Bacterial phospholipase C have been implicated in the pathogenicity of a number of bacteria [22]. Phospholipase C from V. cholerae O139 is known to be enterotoxic and may have some involvement in cholera pathogenesis [21]. Further investigation is needed to con-¢rm and clarify the role of extracellular enzymes, if any, in the disease.…”

Exogenous Polyunsaturated Fatty Acids Impact Membrane Remodeling and Affect Virulence Phenotypes among Pathogenic Vibrio Species

“…This study also discussed the additional molecular machinery possessed by Vibrio cholerae, including multiple homologs to FadL, FadD, and acyltransferases (10). In addition to scavenging free fatty acids, Vibrio species express a variety of lipases that can generate fatty acids through catalysis of larger lipid molecules (3,(37)(38)(39). In particular, a recent discovery described the surface-exposed lysophospholipase VolA, a lipoprotein conserved among Vibrio species that is transcriptionally and perhaps physically coupled to a fatty acid transporter (40).…”

Comparative analysis of cytotoxin, hemolysin, hemagglutinin and exocellular enzymes among clinical and environmental isolates ofVibrio choleraeO139 and non-O1, non-O139

“…Hemolysin of V. cholerae is suggested to be a virulence factor contributing towards cholera pathogenesis [20]. Recently, we purified a bifunctional hemolysin‐phospholipase C molecule from V. cholerae O139 showing enterotoxic activity, as shown by fluid accumulation in the ligated rabbit ileal loop and in the intestines of suckling mice [21]. All the O139 and non‐O1, non‐O139 strains produced considerable amounts of extracellular enzymes such as elastase, protease, neuraminidase, phospholipase A and phospholipase C. However, expression of these enzymes did not vary significantly [Table 3] between the strains isolated from clinical and environmental sources.…”

“…Bacterial phospholipase C have been implicated in the pathogenicity of a number of bacteria [22]. Phospholipase C from V. cholerae O139 is known to be enterotoxic and may have some involvement in cholera pathogenesis [21]. Further investigation is needed to confirm and clarify the role of extracellular enzymes, if any, in the disease.…”