In vitro production of toxin-coregulated pili by Vibrio cholerae El Tor

Abstract

Toxin-coregulated pili (TCP) have been shown to be a virulence determinant and protective antigen for Vibrio cholerae strains of classical biotype, but their role in infection by strains of the alternative El Tor biotype remains uncertain. In an attempt to demonstrate TCP production by El Tor vibrios we have over-expressed the El Tor tcpA gene in Escherichia coli, in order to prepare a biotype-specific anti-TcpA serum. This reagent proved to be a very sensitive indicator of TcpA production in immunoblotting studies, but failed to detect polymerized pili on the bacterial surface by immuno-electron microscopy (IEM). However, results with an analogous reagent which detects classical TcpA suggested that antisera to unprocessed TcpA do not efficiently recognize epitopes on native proteins. Accordingly we prepared a serum against a cell envelope fraction rich in processed El Tor TcpA. After extensive absorption this reagent reacted almost exclusively with TcpA by immunoblotting; when used in IEM, it allowed visualization of typical TCP bundles on the surfaces of each of five El Tor strains known to produce TcpA in vitro .We have previously reported that the El Tor strain O17 does not synthesize TcpA during growth in vitro, but that an O17 clone carrying a cosmid of classical origin expresses surface TCP. Using the biotype-specific anti-TcpA reagents in immunoblotting studies it has been possible to detect the product of the host chromosomal tcpA gene in such constructs; transcription of this gene was confirmed using biotype-specific tcpA probes. IEM revealed that El Tor TcpA was present in the TCP bundles associated with the O17 cosmid clones. Further studies suggest that regulation of the genes encoding TcpA and cholera toxin varies between different strains of El Tor biotype.