Vibrio cholerae produces a cytolytic toxin named El Tor cytolysin/hemolysin which is encoded by the hlyA gene. This cytolysin is produced as a 79-kDa precursor form (pro-HlyA) into the culture supernatant after cleavage of the signal peptide of the hlyA product (prepro-HlyA). The pro-HlyA is then processed to a 65-kDa mature cytolysin (mature HlyA) after cleavage of the 15-kDa N-terminal peptide (pro region) of the 79-kDa precursor, usually at the bond between Ala-157 and Asn-158. We investigated whether proteases could process the recombinant 79-kDa pro-HlyA to the 65-kDa mature HlyA. We observed that the soluble hemagglutinin/ protease (HA/protease; a major protease of V. cholerae), trypsin, ␣-chymotrypsin, subtilisin BPN, papain, and thermolysin all processed the pro-HlyA to the 65-kDa mature form of the protein. Along with this, the protease-processed HlyA showed drastically increased hemolytic activity. The N-terminal amino acid of the mature form of cytolysin generated by HA/protease was Phe-151, and that due to trypsin was Ser-149. Other proteases also cleaved the pro-HlyA at a nearby site, between Leu-146 and Ser-153, and all the processed cytolysins showed increased hemolytic activity. These data suggest that the active El Tor cytolysin of V. cholerae could be derived from the C-terminal region of a pro-HlyA following proteolytic cleavage of the bonds in the vicinity of Leu-146 to Asn-158 by any of a wide variety of proteases.
Two forms (34 kDa and 32 kDa) of hemagglutinin/protease produced by Vibrio cholerae non-O1 were characterized. The hemagglutinin/protease purified by immunoaffinity column chromatography using a monoclonal antibody was essentially a 34-kDa form. By incubation of the purified 34-kDa form at 37 degrees C, it was processed (autodigested) to the 32-kDa form. The N-terminal 20 amino acid sequences of both the 34- and 32-kDa forms were identical, suggesting that proteolytic processing at the C-terminal region of the 34-kDa hemagglutinin/protease resulted in the 32-kDa form. With this shift, protease activity increased, but hemagglutinating activity decreased, suggesting that the C-terminal region of the hemagglutinin/protease is related to hemagglutinating activity.
Haemaglutinin/protease (HA/P) is one of the virulence factors of Vibrio cholerae O1 and pathogenic strains of V. cholerae non-O1. In this study, we examined protease activity of a new serogroup of Vibrio cholerae recently designated as O139 synonym Bengal. The protease activity was produced by all eight isolates of V. cholerae O139 from Bangladeshi patients. Purification and partial characterization of the protease from V. cholerae O139 demonstrated the purified protease (O139-P) was indistinguishable from that previously reported for HA/P of V. cholerae non-O1 (NAG-HA/P) and V. cholerae O1 (Vc-HA/P). These results prove that V. cholerae O139 produces a protease belonging to HA/P, and suggest that the protease is another virulence factor found in newly emerged V. cholerae O139, as in V. cholerae O1.
A new simple purification method (I) for Vibrio cholerae non-O1 hemagglutinin/protease (NAG-HA/P) was developed. The method (I) requires only an immunoaffinity column chromatography using a monoclonal antibody against NAG-HA/P. The method (I) is much simpler than previously reported purification method (II) (Honda, T. et al, Infection and Immunity 57: 2799-2803) which required four or more complicated chromatographic procedures. Method (I) also gave an improved recovery rate (about 27%) compared with (II). The molecular weight of NAG-HA/P purified by method (I) was mainly 34 kilodaltons (kDa) with a little of 32 kDa, whereas that of NAG-HA/P purified by (II) was usually 32 kDa. Immunological analysis by the Olichterlony double gel diffusion test and Western blotting test using polyclonal antibody against 32 kDa protein revealed that the 34 and 32 kDa proteins are immunologically indistinguishable and thus it is supposed that 34 K protein is an isoform or a preform of the 32 K protein.Vibrio cholerae non-O1 (NAG vibrio), which is similar to V. cholerae O1, but is not agglutinated by V. cholerae O1 antiserum, is an important cause of human diarrheal diseases. Gastroenteritis associated with V. cholerae non-O1 shows various clinical symptom (2, 12), such as abdominal pain, mucous and bloody diarrhea as well as watery diarrhea similar to cholera due to V. cholerae O1. These various symptoms may be explained by the production of a variety of toxins by V.
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