Expression of virulence factors by classical and El Tor Vibrio cholerae / Gunhild Jonson.

Tese (doutor) - University of Göteborg, 1991

Vibrio cholerae 01 bacteria cause disease through the elaboration of an enterotoxin, cholera toxin (CT). Fimbriae and other cell surface components as well as secreted factors, e.g. enzymes with protease and mucinase activity, have been proposed to be additional virulence factors that may contribute to bacterial colonization of the intestinal mucosa. The present study was undertaken to investigate the expression of some established and putative virulence factors in the two biotypes of V. cholerae 01, classical and El Tor, both under "optimal" growth conditions in vitro and during intestinal infection in vivo. Two cell-associated hemagglutinins (HAs), one sensitive to L-fucose (FSHA) and another to D-mannose (MSHA) were found to be associated with the classical and El Tor biotypes, respectively. A few strains of either biotype expressed both of those HAs as shown by the binding of bacteria to L-fucose- and D-mannose-derivatized agarose beads. HAs could also be detected in agglutination tests using biotype-"specific" polyclonal antisera or a monoclonal antibody (MAb) with specificity for MSHA. MSHA was found to be associated with a pilus expressed on El Tor strains; the pilus was identified by the MSHA-specific MAb which bound along the entire pilus as shown by immunoelectron microscopy (IEM). The expression of a toxin-coregulated pilus (TCP) has been shown to be important for intestinal colonization by classical strains in humans. TCP was not produced by El Tor strains under culture conditions stimulating production of TCP in classical strains. However, when using a special culturing technique, i.e. the AKI-SW method at 30°C, the structural subunit of TCP, TcpA, was produced by both biotypes as shown by immunoblotting. TcpA from the two biotypes cross-reacted immunologically. In addition, TcpA of classical strains had epitopes that were missing, or poorly expressed, in El Tor TcpA. TCP was exposed on the bacterial surface of classical strains as evident from studies with an inhibition ELISA and with IEM. In contrast, although several of the El Tor strains tested contained comparable amounts of TcpA as the classical bacteria, only a few El Tor strains expressed TCP antigen on the bacterial surface. Studies of the protein profiles of envelope preparations derived from bacteria grown in rabbit intestine suggested that there is a down-regulation of several proteins during infection. At the same time novel in vivo-induced proteins were seen; séveral of these proteins having molecular masses of 62-200 kDa or 20 kDa were immunogenic. All strains that produced FSHA or MSHA during growth in vitro were positive for these HAs also after growth in rabbit intestine. However, the formation of a soluble hemagglutinin (SHA)/protease in vivo was considerably reduced. TcpA was induced in vibrios of both biotypcs after entering the intestinal milieu. Whereas classical strains produced comparable amounts of TCP antigen in vivo and in vitro, surface expression of TCP antigen on El Tor strains was poor during growth under either condition. Many of the classical strains produced more CT during infection, while production by El Tor strains tended to be lower in vivo than in vitro. These results suggest that a more precise definition of the role of postulated virulence factors in V. cholerae should include studies of their expression in vivo.