CTXφ immunity:: Application in the development of cholera vaccines

CTX phi is a filamentous bacteriophage that encodes cholera toxin, the principal virulence factor of Vibrio cholerae. CTX phi is unusual among filamentous phages because it encodes a repressor and forms lysogens. CTX phi can infect the existing live-attenuated V. cholerae vaccine strains derived from either the El Tor or classical V. cholerae biotypes and result in vaccine reversion to toxinogenicity. Intraintestinal CTX phi transduction assays were used to demonstrate that El Tor biotype strains of V. cholerae are immune to infection with the El Tor derived CTX phi, whereas classical strains are not. The El Tor CTX phi repressor, RstR, was sufficient to render classical strains immune to infection with the El Tor CTX phi. The DNA sequences of the classical and EI Tor CTX phi repressors and their presumed cognate operators are highly diverged, whereas the sequences that surround this "immunity" region are nearly identical. Transcriptional fusion studies revealed that the El Tor RstR mediated repression of an El Tor rstA-lacZ fusion but did not repress a classical rstA-lacZ fusion, Likewise, the classical RstR only repressed a classical rstA-lacZ fusion, Thus, similar to the mechanistic basis for heteroimmunity among lambdoid phages, the specificity of CTX phi immunity is based on the divergence of the sequences of repressors and their operators. Expression of the El Tor rstR in either El Tor or classical live-attenuated V. cholerae vaccine strains effectively protected these vaccines from CTX phi infection. Introduction of rstR into V. cholerae vaccine strains should enhance their biosafety.