Pathogenic Vibrio cholerae 01 cause disease by colonizing the human small intestine, where they produce a potent enterotoxin (1). Many elements of the vibrio cell surface have been postulated to be involved in colonization, but an actual molecular component or morphological structure responsible for adherence to the human intestinal mucosa has not previously been conclusively demonstrated .In the classical Ogawa V . cholerae 01 strain 395, a regulatory protein, ToxR, controls expression of cholera toxin (2). ToxR also regulates the expression of a rigid pilus, TcpA, in a coordinate manner with cholera toxin (3). Recently, tc,6A and toxR insertion mutants of V . cholerae Ol were shown to be defective in colonization in suckling mice (3). To determine the role of TcpA in colonization of the human intestine, the bacteriology, and clinical and immunologic responses of healthy adult volunteers who ingested three Ogawa 395 mutants were assessed .
Enteroaggregative Escherichia coli (EAEC) has been implicated as an agent of pediatric diarrhea in the developing world. We have shown previously that EAEC adheres to HEp-2 cells by virtue of a plasmid-encoded fimbrial adhesin designated aggregative adherence fimbria I (AAF/I), the genes for which have been cloned and sequenced. However, not all EAEC strains express AAF/I. Using TnphoA mutagenesis, we have characterized a novel fimbria (designated AAF/II) which mediates HEp-2 adherence of the human-pathogenic strain 042. AAF/II is 5 nm in diameter and does not bind AAF/I antiserum, as determined by immunogold transmission electron microscopy. TnphoA identified a gene (designated aafA) which bears significant homology to aggA, the fimbrial subunit of AAF/I (25% identity and 47% similarity at the amino acid level). When hyperexpressed and purified by polyhistidine tagging, the AafA protein assembled into 5-nm-diameter filaments which bound anti-AAF/II antiserum. The cloned aafA gene complemented a mutation in the aggA gene to confer fimbrial expression from the AAF/I gene cluster, manifesting phenotypes characteristic of AAF/II but not AAF/I. The aafA mutant did not adhere to human intestinal tissue in culture, suggesting a role for AAF/II in intestinal colonization. By using DNA probes for AAF/I and AAF/II derived from fimbrial biosynthesis genes, we show that AAF/I and AAF/II are each found in only a minority of EAEC strains, suggesting that still more EAEC adhesins exist. Our data suggest that AAF adhesins represent a new family of fimbrial adhesins which mediate aggregative adherence in EAEC.
Vibrio cholerae 01 A-Bvaccine strain JBK 70 and A-B' CVD 101 prepared by recombinant DNA techniques from pathogenic El Tor Inaba N16961 and classical Ogawa 395, respectively, were fed to 38 volunteers in single doses of 104 to 1010. Although severe diarrhea did not occur in any vaccinee, more than one-hplf developed mild diarrhea. These attenuated strains colonized well and elicited prominent vibriocidal and antitoxic (CVD 101) antibody responses. Recipients of a single dose of JBK 70 were significantly protected when challenged with 106 wild-type N16961. Diarrhea occurred in 7 of 8 controls but in only 1 of 10 vaccinees (P < 0.003, 89% vaccine efficacy), demonstrating the potency of immune mechanisms that do not involve cholera antitoxin. Further derivatives were prepared to explore the pathogenesis of the residual diarrhea, considering that either intestinal colonization by the vaccine itself or accessory toxins might be responsible. CVD 102, an auxotrophic mutant of CVD 101, did not cause diarrhea but colonized poorly and elicited feeble immune responses. Derivatives of JBK 70 and CVD 101 (CVD 104 and 105) deleted of genes encoding the El Tor hemolysin still caused mild diarrhea. Genetically engineered strains can be colonizing, highly immunogenic, and protective single-dose oral vaccines, but they must be further attenuated before they can be considered for use as public health tools.
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