Cholera vibrios produce a single polymeric protein that (i) causes hemagglutination; (ii) appears to participate in their attachment to gut epithelium; (iii) may mediate their detachment from gut epithelium; and (iv) is a protease that hydrolyzes fibronectin and mucin, cleaves lactoferrin, and nicks the A subunit of the choleragen-related heat-labile enterotoxin of Escherchia coli.In 1947, F. M. Burnet reported the discovery of a mucinase, elaborated by Vibrio cholerae, which participated in the desquamation of epithelium from pieces of guinea pig intestine in vitro (1, 2). Even though Burnet meticulously avoided overstating his conclusions, the observations inspired a flurry of activity among cholera researchers, which led to recommendations that cholera mucinase be included in cholera vaccines (3-6). The concept that the cholera mucinase played a significant direct role in the pathogenesis of choleraic diarrhea was, however, short-lived. Gangarosa et al. (7) showed that the integrity of the intestinaltepithelium was unaltered during the disease in human beings; Gordon (8) found that the disease was not an "exudative enteropathy"; Phillips (9) observed. that the intestinal secretions were low in protein and isotonic; and Finkelstein et al. (10) found that the enterotoxin (choleragen) which caused the diarrhea of cholera could be separated from the cholera mucinase and that the mucinase was not diarrheagenic. Our current observations, however, indicate that Burnet's mucinase may still be involved in the pathogenesis of cholera even though it is not responsible directly for the diarrhea of cholera.Recent efforts in our laboratory have been directed toward understanding the mechanism(s) by which the cholera vibrios elude the host intestinal clearance mechanisms of mucus secretion and peristalsis (11). We have isolated (12), from broth cultures of V. cholerae, a hemagglutinin (HA), which we previously called cholera lectin (13). This protein, a polymer of 32,000 Mr subunits, which appears to participate in the attachment of cholera vibrios to intestinal epithelium, has inherent protease activity (12). Specific antibody against the purified HA inhibits its protease function and also inhibits attachment of V. cholerae to intestinal epithelium (12).The present report shows that the HA/lectin/protease is produced in both a cell-associated and a soluble form in vivo and that it hydrolyzes fibronectin, mucin, -and lactoferrin-three proteins that may participate in host defense against cholera. It also nicks the A subunit of the choleragen-related heat-labile enterotoxin (LT) of Escherichia coli (14) 18)]. The infant rabbits (nine were inoculated with CA 401, three with 569B, and five with 3083) were sacrificed approximately 18 hr later and intestinal fluids ranging between 6 and 17.5 ml and containing 2.0-42 x 107 live cholera vibrios per ml, were harvested and lightly centrifuged (200 x g for 5 min) to sediment the nonbacterial solids. The supernates were subjected to higher speed (12,000 x g for 20 min) cent...

The structural gene, hap, for the secreted hemagglutinin/protease (HA/protease), a putative virulence factor of Vibrio cholerae, has recently been cloned and sequenced (C. C. Hase and R. A. Finkelstein, J. Bacteriol. 173:3311-3317, 1991). The availability of the null mutant, HAP-1, and HAP-1 complemented with pCH2 (which expresses HA/protease), enabled an examination of the role of HA/protease in the virulence of V. cholerae in an animal model. However, the mutants exhibited reversible colonial variation similar but not identical to that which was previously associated with dramatic changes in virulence of parental strain 3083. Regardless of colonial morphology, the mutants were found to be fully virulent in infant rabbits. Thus, the HA/protease is not a primary virulence factor (for infant rabbits). Observations using cultured human intestinal cells indicated, instead, that the HA/protease is responsible for detachment of the vibrios from the cultured cells by digestion of several putative receptors for V. cholerae adhesins.

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Unnicked cholera enterotoxin was isolated from culture supernatants of Vibrio cholerae 569B by either rapid processing of flask-grown cultures or by growing and processing fermentor cultures in the presence of ethylene glycol-bis(O-aminoethyl ether)-N,N,N',N'-tetra acetic acid, an inhibitor of the previously described V. cholerae hemagglutinin/protease. When unnicked cholera enterotoxin was incubated with purified hemagglutinin/protease, the unnicked A subunit was converted to a molecular weight consistent with that of the Al subunit as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its specific activity for Y1 adrenal cells increased.

A soluble hemagglutinin/protease produced by Vibrio cholerae, which has previously been shown to hydrolyze fibronectin and ovomucin and to cleave lactoferrin and the A subunit of the heat-labile enterotoxin of Escherichia coli, appears to be a zinc metalloendopeptidase. Both its hemagglutinative and protease functions are inhibited by chelating agents, including Zincov, a hydroxamic acid derivative specifically designed to inhibit zinc metalloproteases. Thermolysin, a known zinc-containing protease, also causes hemagglutination of responder chicken erythrocytes. This activity is inhibited by Zincov, which does not affect the hemagglutination activity of trypsin and pronase. The hemagglutinin/protease is active on furylacryloyl-Gly-Leu-NH2, a synthetic substrate for thermolysin and other similar proteases. The hemagglutination activity of V. cholerae-infected or cholera toxin-treated infant rabbit intestinal fluid is not inhibited by Zincov, which suggests that this activity is not due to the hemagglutinin/protease, as formerly proposed.