Biomedical subjects

J B Kaper

Publications and source records attributed to J B Kaper.

At least 109 records · Page 6Linked to original sources

PubMed1994

Cloning and sequencing of the genes encoding Escherichia coli cytolethal distending toxin.

Escherichia coli strains expressing cytolethal distending toxin (CDT) cause elongation of CHO cells at 24 h, followed by progressive cellular distention and death for up to 120 h. Similar distention and cytotoxicity are seen in HeLa, HEp-2, and, to a lesser extent, Vero cells. The initial elongation in CHO cells is indistinguishable from that caused by E. coli heat-labile toxin (LT). In contrast to those from LT strains, supernatants from these strains have no effect on Y-1 adrenal cells. TnphoA was introduced into CDT-positive E. coli E6468/62 (O86:H34), isolated from a child with diarrhea, and 13 CDT-negative transconjugants were identified. DNA probes constructed from DNA flanking the TnphoA insertion sites of CDT-negative mutants were used to identify a CDT-positive clone from an E6468/62 genomic library with a 5.5-kb insert. Exonuclease deletions were created and assayed in CHO cells. In this manner, a 2.3-kb CDT-active region was defined, and the nucleotide sequence was determined. Sequence analysis identified three open reading frames (ORFs), designated cdtA, cdtB, and cdtC. These contain 711, 819, and 570 bp, respectively, and encode polypeptides with predicted molecular masses of 25.5, 29.8, and 20.3 kDa, respectively. Each ORF has a putative signal sequence, and there are 4-bp overlaps between cdtA and cdtB and between cdtB and cdtC. The nucleotide and predicted amino acid sequences have no significant homology with those of any previously reported genes or proteins. By in vitro transcription-translation and an anti-alkaline phosphatase immunoblot, native proteins and/or fusion proteins corresponding to each ORF were identified.

Amino Acid Sequence↗

PubMed1994

Potential for reacquisition of cholera enterotoxin genes by attenuated Vibrio cholerae vaccine strain CVD 103-HgR.

The potential for reacquisition of ctxA genes by attenuated Vibrio cholerae O1 vaccine strain CVD 103-HgR was examined by performing a series of mating experiments under a variety of in vivo and in vitro conditions. We found no evidence that CVD 103-HgR could reacquire ctxA genes from wild-type V. cholerae O1 strains. However, if the donor V. cholerae O1 strains were genetically manipulated to add genes that allow chromosomal gene transfer, then ctxA sequences could be acquired by CVD 103-HgR. The minimal excretion of CVD 103-HgR by vaccinees and the refractoriness to reacquisition of ctxA sequences suggest that this well-tolerated, highly immunogenic live oral cholera vaccine will have a minimal environmental impact.

Cholera Vaccines↗

PubMed1994

Distribution of Zonula occludens toxin (zot) gene among clinical isolates of Vibrio cholerae O1 from Bangladesh and Africa.

Seventy-two clinical isolates of Vibrio cholerae O1 from Bangladesh, and 12 and 9 isolates respectively from Tanzania and Nigeria were screened for sequences homologous to zonula occludens toxin (zot) and cholera toxin (ctx) genes. As observed previously, all isolates in the present study also possessed sequences for both toxins which suggested that zot does not occur independent of ctx. It appears that along with the virulence genes located in the "virulence cassette" region of the bacterial chromosome, zot may play a role in the pathogenesis of cholera.

Africa↗

PubMed1993

Construction of genetically marked Vibrio cholerae O1 vaccine strains.

Attenuated Vibrio cholerae O1 vaccine strains lacking the gene encoding the A subunit of cholera toxin have proven efficacious in preventing experimental cholera. As these strains move from closed, contained testing environments to large-scale field trials, a readily assayable phenotypic trait to distinguish a vaccine strain from wild-type V. cholerae O1 is desirable. We have constructed three derivatives of the attenuated V. cholerae strain CVD 103 which carry a mercury resistance or urease marker in the hlyA gene. CVD 103-HgR was constructed using a protracted marker-exchange procedure; this strain was found to have somewhat lowered colonisation efficiency in infant mice in comparison to its parent strain, CVD 103. The insertion of the resistance marker was repeated using a suicide vector system; CVD 103-HgR2 was found to colonise infant mice as efficiently as CVD 103. Strain CVD 103-UR, in which sequences encoding urease were inserted using a suicide vector, also colonised infant mice as well as CVD 103. The genetically marked strains CVD 103-HgR, CVD 103-HgR2 and CVD 103-UR form the basis for a generation of defined oral vaccines that may give single-dose, long-lasting protection to populations at risk from cholera.

Animals↗

PubMed1993

Accessory cholera enterotoxin (Ace), the third toxin of a Vibrio cholerae virulence cassette.

Vibrio cholerae causes the potentially lethal disease cholera through the elaboration of the intestinal secretogen cholera toxin. A second toxin of V. cholerae, Zot, decreases intestinal tissue resistance by modifying intercellular tight junctions. In this report, a third toxin of V. cholerae, Ace (accessory cholera enterotoxin), is described. Ace increases short-circuit current in Ussing chambers and causes fluid secretion in ligated rabbit ileal loops. The predicted protein sequence of Ace shows striking similarity to eukaryotic ion-transporting ATPases, including the product of the cystic fibrosis gene. The gene encoding Ace is located immediately upstream of the genes encoding Zot and cholera toxin. The ctx, zot, and ace genes, which are located on a dynamic sector of the chromosome, comprise a V. cholerae "virulence cassette."

Amino Acid Sequence↗

PubMed1993

Live oral vaccines against cholera: an update.

One hundred years elapsed between the first (live, parenteral) cholera vaccine that entered clinical trials in 1885 and the field trials of two oral inactivated cholera vaccines undertaken in Bangladesh in the mid-1980s. The oral inactivated vaccines advanced the art by establishing, convincingly, that oral vaccines could protect (although multiple doses were required) and that (at least in adults) protection could last 3 years. Attenuated Vibrio cholerae O1 strain CVD 103-HgR (deleted of the cholera toxin A subunit gene and harbouring a gene encoding resistance to Hg++) constitutes another significant advance. This live oral vaccine is well tolerated and highly immunogenic in adults and children and highly protective (in adult volunteer challenge studies) following ingestion of of a single dose.

Administration, Oral↗

PubMed1993

Identification of the flagellar antigens of Vibrio cholerae El Tor and their role in protection.

Antiserum to the surface antigens of the wild-type flagellate strain KB207 of Vibrio cholerae El Tor was absorbed with isogenic aflagellate mutant CD12. Antibodies remaining in the absorbed serum exhibited specificity to KB207 but not to CD12 and inhibited motility of KB207. Proteins from cell-free lysates of KB207 and CD12 were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CD12 differed from KB207 in the absence of two proteins of 40 and 38 kDa. These proteins were detected in KB207 when electrophoretically separated proteins were immunoblotted with unabsorbed and absorbed sera. These two proteins were isolated by immunoaffinity chromatography using purified antibodies from absorbed serum. Although antigenic, the 40 and 38 kDa proteins did not induce protection against cholera in the rabbit ileal loop model.

Animals↗

PubMed1993

Identification of a 33 kDa antigen associated with an adhesive and colonizing strain of Vibrio cholerae El Tor and its role in protection.

Proteins from the cell-free lysates of the wild-type strain KB207 of Vibrio cholerae El Tor and the isogenic non-adhesive mutant CD11 were analysed by native and denaturing polyacrylamide gel electrophoresis. A protein of 33 kDa present in KB207 was absent from CD11. Antiserum to the surface antigens of KB207 was absorbed with CD11. Antibodies remaining in the serum after absorption reacted to KB207 but not to CD11 as judged by slide agglutination, double gel diffusion and dot blot ELISA. Antibodies in the absorbed serum inhibited adherence of KB207 to rabbit intestinal mucosa and colonization in an infant mice model. The 33 kDa protein was isolated from KB207 by immunoaffinity chromatography. Antibodies present in the absorbed serum were used as ligand. The 33 kDa antigen was immunogenic and conferred protection in the rabbit ileal loop model. Combined administration of 33 kDa protein and B-subunit of cholera toxin offered full protection.

Animals↗

PubMed1993

Distribution of the bundle-forming pilus structural gene (bfpA) among enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) express an inducible bundle-forming pilus (BFP) associated with the presence of the EPEC adherence factor (EAF) plasmid and localized adherence (LA) on HEp-2 cells. The cloned structural gene (bfpA) encoding BFP was found to be specific for EPEC, as homologous sequences were found only in EPEC and not in other enteropathogens. The bfpA probe was slightly more sensitive than the EAF probe; among EPEC strains with LA, the bfpA and EAF probes hybridized with 99% and 96% of the strains, respectively. Immunoblotting of whole cell lysates of BFP-positive, EAF-positive or -negative, and LA-positive or -negative EPEC strains revealed variations in the size (18,500-21,000) of the expressed structural subunit of BFP, suggesting differences in processing that may account for discrepancies between the bfpA, EAF, and LA. Because the bfpA probe consists primarily of sequences encoding an important EPEC virulence factor, in contrast to the unknown function of sequences contained in the EAF probe, the bfpA probe may be useful in epidemiologic studies to detect EPEC.

Carcinoma, Squamous Cell↗

PubMed1993

Safety and immunogenicity of live oral cholera vaccine candidate CVD 110, a delta ctxA delta zot delta ace derivative of El Tor Ogawa Vibrio cholerae.

The current pandemic of cholera is caused primarily by Vibrio cholerae O1 of the El Tor biotype. Live attenuated classical biotype V. cholerae vaccine strains prevent severe and moderate cholera due to either biotype in challenged volunteers but may provide less protection against mild cholera due to El Tor organisms. CVD 110, a new ctxA-deleted vaccine strain derived from an El Tor Ogawa parent, lacks zona occludens toxin (Zot), accessory cholera enterotoxin (Ace), and hemolysin/enterotoxin. Ten healthy adult volunteers were given 10(8) cfu of CVD 110 with buffer; 7 developed diarrhea (mean stool volume, 861 mL). Vaccine organisms were shed in stool by all vaccines and were recovered from duodenal fluid in three-quarters of vaccinees. After vaccination, the geometric mean peak reciprocal vibriocidal titer among vaccinees was 17,829. CVD 110 is a powerful immunogen but insufficiently attenuated despite the absence of known potential enterotoxins of V. cholerae. Another unrecognized toxin or colonization alone may be responsible for diarrhea after ingestion of this strain.

Administration, Oral↗

PubMed1993

CVD110, an attenuated Vibrio cholerae O1 El Tor live oral vaccine strain.

The recent expansion of the seventh cholera pandemic into South America emphasizes the need for a safe, long-lasting, protective, and nonreactogenic vaccine for this disease. Since the predominant Vibrio cholerae O1 strains in the world today are of the El Tor biotype, a bivalent vaccine containing both classical and El Tor biotypes may be desirable. We have constructed a new oral vaccine candidate, V. cholerae CVD110 El Tor, Ogawa, from which all toxin genes so far identified in V. cholerae have been deleted. Three of these genes, those encoding cholera toxin (ctx), zonula occludens toxin (zot), and accessory cholera enterotoxin (ace), are located on a 4.5-kb virulence cassette flanked by repetitive sequences (RS1 elements). Homologous recombination between these RS1 elements resulted in the deletion of this virulence cassette to yield V. cholerae CVD109. Insertion of genes encoding mercury resistance (mer) and the cholera toxin B subunit (ctxB) into the hemolysin locus (hlyA) produced CVD110. This insertion serves three purpose. (i) It genetically tags the vaccine strain so as to distinguish it from wild-type V. cholerae O1. (ii) It produces cholera toxin B subunit in order to elicit antitoxic immunity. (iii) It inactivates the hemolysin gene, rendering the strain nonhemolytic on sheep erythrocyte plates. Supernatants from V. cholerae CVD110 cultures are nonreactogenic when assayed in Ussing chambers.

Bacterial Toxins↗

PubMed1993

Secondary Vibrio cholerae-specific cellular antibody responses following wild-type homologous challenge in people vaccinated with CVD 103-HgR live oral cholera vaccine: changes with time and lack of correlation with protection.

Peripheral blood immunoglobulin A antibody-secreting-cell (ASC) responses are thought to reflect the mucosal immune response to locally presented antigens. We evaluated the ASC response to cholera toxin (CT) and Inaba lipopolysaccharide (LPS) in 26 North American volunteers following immunization with a single oral dose of live attenuated Vibrio cholerae O1 vaccine strain CVD 103-HgR and again upon homologous wild-type challenge with V. cholerae classical Inaba 569B. Challenge occurred at either 7, 30, or 180 days after vaccination. The CT and LPS ASC responses of volunteers following vaccination (83 and 55%, respectively) were similar in magnitude and frequency to those of unvaccinated controls following wild-type challenge (80 and 60%, respectively [0.1 < or = P < or = 0.9]). The responses were primarily immunoglobulin A. Vaccinated volunteers challenged within 30 days of vaccination had reduced or nondetectable CT and LPS ASC responses. Challenge at 6 months resulted in a heightened ASC response to LPS, confirming the existence of mucosal memory. ASC responses to CT upon challenge at 6 months were detectable but not different from that seen following primary immunization, suggesting that secondary ASC responses to different antigens from a single vaccine operate independently. In spite of these variable ASC responses, the vaccine efficacy was 100% following challenge for all vaccinees. V. cholerae-specific ASC responses following antigenic reexposure gave information on the presence of mucosal B memory cells but did not correlate with protective immunity. As such, these ASC assays will have limited usefulness for evaluating vaccine responders in vaccine field trials in cholera-endemic areas where prior V. cholerae O1 exposure is unknown.

Administration, Oral↗

PubMed1993

Enteropathogenic Escherichia coli decreases the transepithelial electrical resistance of polarized epithelial monolayers.

The mechanisms whereby enteropathogenic Escherichia coli (EPEC) causes diarrhea remain undefined. We found that EPEC caused a decrease in transepithelial electrical resistance across polarized monolayers of Caco-2 and MDCK epithelial cells. This occurred approximately 6 to 10 h after bacterial addition and was reversible if the monolayers were treated with tetracycline or gentamicin. Although significant alterations in host actin occurred beneath adherent EPEC, actin filaments supporting tight junctions were not noticeably affected in the epithelial cells, nor was the distribution of ZO-1, a tight junction protein. Despite the decrease in transepithelial electrical resistance, EPEC did not cause an increase in [3H]inulin penetration across MDCK monolayers. Unlike in the parental strain, mutations in any loci involved in adherence or formation of attaching and effacing lesions were unable to cause a decrease in transepithelial resistance. These data indicate that EPEC causes a decrease in transepithelial electrical resistance by disrupting a transcellular (intracellular) pathway rather than by disrupting intercellular tight junctions (paracellular) and that these disruptions occur only when attaching and effacing lesions are formed.

Actins↗

PubMed1993

Safety, immunogenicity, and excretion pattern of single-dose live oral cholera vaccine CVD 103-HgR in Peruvian adults of high and low socioeconomic levels.

Groups of 122 Peruvian adults of low socioeconomic level (SEL) and 125 of high SEL received a randomly allocated 5 x 10(9)- or 5 x 10(8)-CFU dose of CVD 103-HgR live oral cholera vaccine or a placebo. The vaccine was well tolerated. Vibriocidal seroconversions occurred in 78% of high-SEL and 72% of low-SEL subjects who ingested the high dose and in 78 and 49%, respectively, of those who received the low dose.

Administration, Oral↗

PubMed1993

A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells.

Enteropathogenic Escherichia coli (EPEC) is capable of attaching intimately to epithelial cells and effacing their microvilli. A chromosomal locus, eaeA (originally eae), is required for the intimate attachment aspect of this effect. We report the mapping of a region of the EPEC chromosome that is located immediately downstream of the eaeA gene and that is also necessary for intimate attachment. An isogenic in-frame deletion mutation in one of the open reading frames identified in this region was engineered. Because the resulting mutant, like an eaeA deletion mutant, is deficient in the ability to attach intimately to epithelial cells, the mutated gene is designated eaeB. Full activity is restored to the eaeB mutant when the cloned gene is reintroduced on a plasmid. The eaeB mutant remains capable of producing intimin, the product of the eaeA gene. No differences in the fractionation properties or electrophoretic mobility of intimin are apparent in the eaeB mutant. The product of the eaeB locus was identified by in vitro transcription-translation. The nucleotide sequence of the eaeB gene predicts a protein that contains a sequence motif common to several aminotransferase enzymes. These results indicate that the attaching and effacing effect is a complex phenotype dependent on a gene cluster present on the EPEC chromosome.

Amino Acid Sequence↗

PubMed1993

Gene encoding zonula occludens toxin (zot) does not occur independently from cholera enterotoxin genes (ctx) in Vibrio cholerae.

Of 167 Vibrio cholerae isolates screened for sequences homologous with zonula occludens toxin (zot) or cholera toxin (ctx) genes, 3.0% of non-O1, 100.0% of clinical O1, and 0.0% of environmental O1 strains contained both zot and ctx. zot was present only in strains that were ctx positive; all ctx-positive strains carried zot. The absence of zot-positive, ctx-negative strains suggests ZOT is not an independent virulence factor for V. cholerae, although ZOT may play a role in the pathogenesis of toxigenic strains.

Cholera Toxin↗

PubMed1993

Role of the eaeA gene in experimental enteropathogenic Escherichia coli infection.

Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infant diarrhea in developing countries. Recently eaeA, a gene necessary for the characteristic intimate attachment of EPEC to epithelial cells in tissue culture, was described. We conducted a randomized, double-blind study to determine the role of the eaeA gene in human EPEC infection. 11 adult volunteers ingested 2 x 10(10) colony-forming units of O127:H6 EPEC strain E2348/69, and an equal number received the same dose of an isogenic eaeA deletion mutant constructed from E2348/69. Volunteers were monitored for the development of diarrhea, fever, and systemic and gastrointestinal complaints. Diarrhea developed in all 11 volunteers who received E2348/69 and in 4 of 11 who received the mutant (P = 0.002). Fever was more common in recipients of the wild-type strain (P = 0.024). Stool volumes were lower in recipients of the mutant. All volunteers seroconverted to E2348/69 LPS, but the geometric mean peak titers of serum IgG and IgA in recipients of the mutant were lower than those of recipients of the wild-type strain. IgA against LPS was detected in the jejunal fluid of six of six recipients of E2348/69 and 5/6 recipients of the mutant. This study unambiguously assigns a role for eaeA as an EPEC virulence gene, but the residual diarrhea seen in recipients of the mutant indicates that other factors are involved.

Adhesins, Bacterial↗

PubMed1993

The role of the eae gene of enterohemorrhagic Escherichia coli in intimate attachment in vitro and in a porcine model.

The eaeA gene of enteropathogenic Escherichia coli (EPEC) is necessary for intimate attachment to epithelial cells in vitro. Enterohemorrhagic E. coli (EHEC) strains also possess an eae gene and are capable of intimate attachment and microvillus effacement in vitro and in animal models. To assess the role of the EHEC eae gene in intimate attachment, we constructed an eae deletion/insertion mutation in wild-type EHEC O157:H7 strain 86-24 by using linear electroporation of a recombinant allele. The mutant obtained was deficient in inducing f-actin accumulation in HEp-2 cells and was incapable of attaching intimately to colonic epithelial cells in a newborn piglet model of infection. Intimate attachment in vivo was restored when the EHEC eae gene or the eaeA gene of EPEC was introduced into the mutant on a plasmid. These results indicate that the eae gene is necessary for intimate attachment of EHEC in vivo. In addition, the complementation achieved by the EPEC locus indicates that the eae gene of EHEC and the eaeA gene of EPEC are functionally homologous.

Adhesins, Bacterial↗