Vibrio cholerae hemagglutinin/lectin/protease hydrolyzes fibronectin and ovomucin: F. M. Burnet revisited

Abstract

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...

Proteomic Analysis of the Vibrio cholerae Type II Secretome Reveals New Proteins, Including Three Related Serine Proteases

“…Moreover, gbpA mutants display defects in adherence to human epithelial cells and in colonization of infant mice, suggesting that GbpA represents a dual colonization factor functioning both in the aquatic environment and the human host (27). Similarly, HAP has been suggested to play a role in infection by facilitating the penetration of V. cholerae through the mucus barrier of the gastrointestinal tract and by causing its detachment from the epithelium following the induction of the secretory diarrhea (37,38).…”

Integration of Global Regulatory Mechanisms Controlling Vibrio Cholerae Behavior

“…Consistently, inactivation of hapA encoding HA/protease enhances adherence to mucin-coated polystyrene plates , adherence to mucin-secreting differentiated HT29-18N2 cultured cells (Benitez et al, 1997), and colonization of the suckling mouse intestine (Robert et al, 1996;Silva et al, 2006). The mucinase activity of HA/protease (Finkelstein et al, 1983), together with its capacity to cleave the mucin-binding adhesin GbpA (Jude et al, 2009) at high cell density, has provided a mechanism supporting the "detachase" function attributed to this protein.…”

“…Numerous V. cholerae strains of the El Tor biotype express a Zn-dependent metalloprotease (mucinase) known as hemagglutinin (HA)/protease (Finkelstein et al, 1983;Hase & Finkelstein, 1991). HA/protease enhances enterotoxicity in the rabbit ileal loop model of cholera (Ichinose et al, 1994;Silva et al, 2006) and contributes to live vaccine candidates' reactogenicity in humans (Benitez et al, 1999;Garcia et al, 2005).…”

Production of Antigenically Related Exocellular Elastolytic Proteases Mediating Hemagglutination by Vibrios

“…Likewise, in the present experiment the caseinolytic as well as HA activities of TEP-inactivated proteases could be reversed by the addition of trace amounts of metal ions. The effect of proteolysis on the HA activities were shown with V cholerae protease (VCP) and VMP (3,5). Based on this data which demonstrated the simultaneous inactivation and reactivation of both caseinolytic and HA activities, it can be concluded that the proteolytic enzymes produced by the above vibrios also possess HA ability.…”

“…Of these, the protease from Vibrio cholerae (5) is well known for its roles in the pathogenesis of deadly cholera (2,4). This protease was first discovered as an adhesion factor due to its hemagglutinating (HA) abilities (6), and was then shown to be a protease (5) possessing the ability to nick and activate the cholera enterotoxin (2). Later, it was demonstrated to be a metalloprotease containing zinc as an essential ligand (1).…”