Cloning of a gene (zot) encoding a new toxin produced by Vibrio cholerae.

Live oral candidate cholera vaccines have previously been constructed by deletion of Vibrio cholerae sequences encoding the enzymatically active A subunit of the cholera toxin. However, volunteer studies have shown that these non-cholera toxin-producing strains still provoke mild to moderate diarrhea in some individuals. We recently reported the identification of a second toxin produced by V. cholerae which may be responsible for this residual diarrhea (A. Fasano, B. Baudry, D. W. Pumplin, S. S. Wasserman, B. D. Tall, J. M. Ketley, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 88:5242-5246, 1991). This new toxigenic factor increases the permeability of rabbit ileal mucosa by affecting the structure of the intercellular tight junctions (zonula occludens). We now report the identification and cloning of the gene encoding this new toxin. This gene, named zot (for zonula occludens toxin), consists of a 1.3-kb open reading frame which could potentially encode a 44.8-kDa polypeptide. The location of the zot gene encoding the new toxin is immediately upstream of the ctx operon encoding cholera toxin.

Tissue culture-adherent Escherichia coli in infantile diarrhea.

To determine the association of tissue culture-adherent Escherichia coli with diarrhea, serotyped E. coli strains isolated in a yearlong case-control study of infantile diarrhea in Bangkok, Thailand, were examined for adherence to HeLa cells and for hybridization with the enteropathogenic E. coli adherence factor, the F1845, and the enteroaggregative E. coli (EAggEC) DNA probes. E. coli that adhered to HeLa cells in a localized adherence (LA) pattern (LA E. coli) was isolated from 26 of 509 infants with diarrhea (cases) and 9 of 509 age-matched controls (P = .006); E. coli with diffuse or aggregative adherence (DA or AA) to HeLa cells or that hybridized with the F1845 or EAggEC probes was not associated with infantile diarrhea. LA E. coli of classical enteropathogenic E. coli (EPEC) serotypes was isolated from 11 cases and 1 control (P = .003). EPEC O44:H18 that adhered to HeLa cells in a DA pattern and hybridized with the F1845 DNA probe was the predominant E. coli (five of five colonies tested) isolated from a 5-month-old girl with diarrhea in whom no other enteric infections were identified. Although LA E. coli was highly associated with infantile diarrhea, the role of DA and AA E. coli was uncertain in this setting.

Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions.

Attenuated Vibrio cholerae vaccine strains specifically mutated in genes encoding cholera toxin (CT) are still capable of causing mild to moderate diarrhea. Culture supernatants of V. cholerae strains, both CT-positive and CT-negative, were examined in Ussing chambers, and a toxin was found that increases the permeability of the small intestinal mucosa by affecting the structure of the intercellular tight junction, or zonula occludens. The activity of this toxin is reversible, heat-labile, sensitive to protease digestion, and found in culture supernatant fractions containing molecules between 10 and 30 kDa in size. Production of this factor (named ZOT for zonula occludens toxin) correlates with diarrheagenicity of V. cholerae strains in volunteers and may represent another virulence factor of infectious diarrhea that must be eliminated to achieve a safe and effective live oral vaccine against cholera.

Diffuse-adhering Escherichia coli (DAEC) as a putative cause of diarrhea in Mayan children in Mexico.

Diarrhea is a major cause of infantile morbidity and mortality in developing countries. A community-based, case control study was conducted in a southern Mexican Mayan village for 3 weeks during the peak diarrhea period to prospectively identify the infectious agents associated with childhood diarrheal disease. Several enteropathogens were isolated from stools of 34 of 58 cases, although none was significantly associated with diarrhea. For the 24 cases from which no enteropathogens were isolated, diffuse-adhering Escherichia coli (DAEC) strains were significantly associated with diarrheal disease (P less than .02; odds ratio = 6; 95% confidence limit, 1.08-99.0). DAEC were highly heterogeneous with respect to plasmid content and serotype. Three DNA probes designed to differentiate E. coli exhibiting localized, diffuse, or aggregative adherence were compared with results from a standard HeLa cell binding assay to assess the utility of these probes in the field. This study provides evidence for the potential pathogenic capacity of DAEC and underscores the variety of diarrheal agents operating within a community.

A sensitive and specific DNA probe to identify enteroaggregative Escherichia coli, a recently discovered diarrheal pathogen.

The epidemiologic significance of enteroaggregative Escherichia coli (EAggEC) as a diarrheal pathogen has only recently come under study. Although EAggEC has been associated with persistent diarrhea in infants in some developing countries, additional studies are clearly needed. Until now, the only means of identifying EAggEC strains has been the cumbersome HEp-2 cell adhesion assay. The isolation and cloning of a 1-kilobase fragment from the plasmid of EAggEC strain 17-2 is described. This probe is 89% sensitive and 99% specific for EAggEC identification. Thus, this probe should greatly facilitate epidemiologic studies assessing the importance of EAggEC as a diarrheal pathogen.

Nucleotide sequence of the invasion plasmid antigen B and C genes (ipaB and ipaC) of Shigella flexneri.

The nucleotide sequence of a 4.8 kilobase (kb) HindIII fragment from pWR100, the virulence plasmid of Shigella flexneri 5, was determined and analysed. This fragment encodes polypeptides b (62 kilodalton, kD) and c (43 kD) which have already been described as two of the four immunogenic polypeptides of Shigellae. The nucleotide sequence revealed that in addition to the ipaB and ipaC genes encoding polypeptides b and c, a third complete open reading frame was found within the fragment. The gene, named ippI, encoded a 17 kD polypeptide. The deduced amino acids sequence of polypeptides b and c showed no signal peptide but presence of highly hydrophobic domains compatible with a transmembraneous location. The surprising A and T richness of the three genes as compared with the Escherichia coli and Shigella genomes, resulted in a biased codon usage, and raises the question of the origin of the sequences.

Localization of plasmid loci necessary for the entry of Shigella flexneri into HeLa cells, and characterization of one locus encoding four immunogenic polypeptides.

We have previously cloned a 44 kb fragment from the virulence plasmid of Shigella flexneri serotype 5 strain M90T which is capable of restoring invasiveness to an avirulent, plasmidless mutant. This report presents a genetic and physical analysis of Tn5 mutations in recombinant clone pHS4108. Tn5 mutagenesis allowed identification of at least five regions implicated in the entry phenotype. These regions were located on a 20 kb portion of pHS4108. Expression of the insertion mutants was studied by immunoblots using the serum of a convalescent monkey infected by S. flexneri 2a, which recognized four plasmid-associated polypeptides. We propose that the four immunogenic polypeptides, a, b, c, and d, are encoded an operon.

Plasmid-mediated contact haemolytic activity in Shigella species: correlation with penetration into HeLa cells.

The main feature of virulent strains of Shigella is their ability to invade eucaryotic cells. This phenotype is both plasmid-mediated and temperature-regulated. In the present report, we demonstrate a plasmid-mediated and temperature-regulated haemolytic activity in S. flexneri, S. dysenteriae and S. sonnei. Detection of this haemolytic activity requires centrifugation of suspensions containing bacteria and erythrocytes, followed by incubation of the pellets at 37 degrees C. No soluble intra- or extracellular haemolytic activity could be detected. Dose-effect and electron microscopic studies demonstrated that direct contact of several virulent bacteria per erythrocyte was critical for haemolysis to occur. However, no local morphological alteration of the erythrocyte membrane at the site of contact with bacteria could be detected. Intensity of this haemolytic activity was fully correlated with the efficiency of penetration within HeLa cells, suggesting a common mechanism for induction of phagocytosis and lysis of erythrocytes.

Cloning of plasmid DNA sequences involved in invasion of HeLa cells by Shigella flexneri.

A large plasmid is found in virulent isolates of Shigella sp. and encodes functions essential for invasion of mammalian cells. To identify plasmid sequences necessary for invasion, we isolated a series of Tn5 insertions in pWR100, the virulence plasmid of Shigella flexneri serotype 5. These insertions demonstrated that three separate EcoRI fragments of pWR100 were required for invasion of HeLa cells. However, the corresponding native EcoRI fragments, when cloned into pBR325, did not restore virulence to plasmidless strains. Construction of a lambda-sensitive, plasmidless Shigella recipient enabled us to shotgun clone plasmid DNA directly into S. flexneri by using the cosmid vector pJB8 and score for expression of invasive functions. In this fashion, we succeeded in isolating six independent recombinants which restored invasion of HeLa cells in plasmidless Shigella recipients. The cloned inserts all contained a common core of ca. 37 kilobases, thus defining a minimum sequence necessary for invasion of HeLa cells. Virulence-associated peptides produced by wild-type S. flexneri were also produced by the recombinants. Expression of these peptides and expression of invasiveness by the clones were regulated by growth temperature, as is expression of these traits in wild-type S. flexneri. A complete invasive phenotype was not expressed by the recombinants in that they failed to produce a positive Sereny test. Possible explanations for this behavior as it relates to the mechanism of bacterial invasion are discussed.