Biomedical subjects

J B Kaper

Publications and source records attributed to J B Kaper.

At least 145 records · Page 8Linked to original sources

PubMed1991

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.

Bacterial Adhesion↗

PubMed1991

Characterization of interactions of enteropathogenic Escherichia coli O127:H6 with mammalian cells in vitro.

Previous studies have identified two bacterial factors involved in enteropathogenic Escherichia coli (EPEC) infection. A plasmid-mediated EPEC adherence factor (EAF) is responsible for initial and localized adherence. A chromosomally encoded E. coli attachment and effacement factor (eae) is involved in effacement of the eukaryotic cell surface and characteristic "pedestal" formation. By using isogenic strains deficient in either EAF, eae, or both, the process of EPEC adherence and entry in vitro was examined. While EAF proved necessary and sufficient for efficient bacterial association with HEp-2 cells, both EAF and eae were required for efficient effacement of and entry into these cells and other cultured cell lines. Invasion mediated by eae was markedly inhibited by cytochalasin D and colchicine. Afimbrial adhesion or type I pili from uropathogenic strains of E. coli substituted for EAF in EAF-Eae+ strains to provide initial adherence to HEp-2 cells and to facilitate actin condensation.

Adhesins, Escherichia coli↗

PubMed1991

Plasmid and chromosomal elements involved in the pathogenesis of attaching and effacing Escherichia coli.

Attaching and effacing (A/E) intestinal lesions are produced by enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and RDEC-1, a pathogen of weanling rabbits. We recently identified a chromosomal locus (eae[E. coli A/E]) which is required for A/E activity in a wild-type EPEC strain. Sequences homologous to those of an eae gene probe were detected in EPEC, RDEC-1, and EHEC isolates. We report here that the eae gene is chromosomally encoded in all EPEC and EHEC strains tested and in RDEC-1. In addition, the eae probe was found to be 100% sensitive and 98% specific in detecting E. coli of EPEC serogroups that demonstrate A/E activity. Ten percent of E. coli of EPEC serogroups that hybridized with the eae probe and produced A/E activity did not hybridize with the EAF (EPEC adherence factor) probe, a plasmid-associated diagnostic probe which is currently used to identify EPEC. In addition to A/E factors, plasmid-associated adhesins also contribute to the pathogenesis of EPEC and RDEC-1. To further investigate the role of plasmid-associated adherence, a hybrid RDEC-1-EPEC strain containing the adherence plasmid of an EPEC strain in the A/E background of RDEC-1 was constructed. This hybrid strain, unlike the parent RDEC-1 strain, produced A/E lesions on human tissue culture cells, which suggests that the EPEC adherence plasmid provides tissue specificity to the hybrid strain and that the A/E factors of RDEC-1 are not host restricted.

Actin Cytoskeleton↗

PubMed1991

The eae gene of enteropathogenic Escherichia coli encodes a 94-kilodalton membrane protein, the expression of which is influenced by the EAF plasmid.

The production of a characteristic intestinal histopathology called attaching and effacing (A/E) lesions by enteropathogenic Escherichia coli (EPEC) is a major characteristic of EPEC pathogenesis. We previously identified a chromosomal gene (eae) of EPEC necessary for the production of A/E lesions on human tissue culture cells. Using antiserum raised to an Eae-PhoA fusion protein, we found that the eae gene encodes a 94-kDa membrane protein. This antiserum recognized a 94-kDa membrane protein in parent strain E2348/69 and a protein of similar size in E. coli HB101 carrying eae on a plasmid but did not recognize any proteins in E. coli HB101 carrying a plasmid with an internal deletion in the eae gene. Antigenically related proteins of ca. 94 kDa were detected in a collection of EPEC strains representing seven EPEC serogroups and in two EHEC strains of serotype O26:H11. Volunteer sera drawn 28 days after but not before ingestion of strain E2348/69 recognized the 94-kDa Eae protein as well as a 128-kDa Eae-PhoA fusion protein, suggesting that the Eae protein is likely to be a previously reported 94-kDa protein shown to be immunogenic in volunteers. The amount of detectable Eae protein was increased in the presence of a high-molecular-weight plasmid which is associated with the ability to produce localized adherence to tissue culture cells. These data suggest that the virulence plasmid of EPEC strain E2348/69 may have a regulatory role in the production of A/E activity.

Adhesins, Escherichia coli↗

PubMed1991

Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector.

The ability to attach to epithelial cells, efface the microvillus surface, and disrupt the underlying cytoskeleton is characteristic of enteropathogenic Escherichia coli (EPEC). Recently, eae, a gene necessary for this phenomenon, was described (A. E. Jerse, J. Yu, B. D. Tall, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 87:7839-7843, 1990). We report the use of a novel suicide vector containing the pir-dependent R6K replicon and the sacB gene of Bacillus subtilis to construct an eae deletion mutant of EPEC. This system enables positive selection for the loss of vector sequences. The resulting mutant, CVD206, is indistinguishable from the wild-type strain except for the loss of a 94-kDa outer membrane protein and attaching and effacing ability. Both the 94-kDa outer membrane protein and attaching and effacing ability are restored upon reintroduction of the eae gene on a plasmid. These results confirm the role of the eae gene in the attaching and effacing activity of EPEC and establish the utility of a new system for the construction of deletion mutations.

Bacterial Adhesion↗

PubMed1990

Effects of DNase production, plasmid size, and restriction barriers on transformation of Vibrio cholerae by electroporation and osmotic shock.

Attempts to transform wild type strains of V. cholerae with plasmid DNA by traditional osmotic shock methods were not successful. A mutant of V. cholerae that was deficient in extracellular DNase was transformed with plasmid DNA by osmotic shock, demonstrating directly that extracellular DNase is a major barrier to transformation of V. cholerae. Transformation of wild type and DNase-negative strains of V. cholerae was accomplished by electroporation. Efficiency of transformation by electroporation increased with field strength, decreased with plasmid size, and was relatively insensitive to changes in the electrolyte composition of the buffer as long as isotonic sucrose was present. Host-controlled modification/restriction systems also affected transformation efficiency in V. cholerae.

Cell Membrane Permeability↗

PubMed1990

Randomized double-blind placebo controlled trial to evaluate the safety and immunogenicity of the live oral cholera vaccine strain CVD 103-HgR in Swiss adults.

A randomized, double-blind, placebo controlled trial was conducted in 50 healthy Swiss adults to assess the safety and immunogenicity of the live oral attenuated cholera vaccine candidate strain Vibrio cholerae CVD 103-HgR (classical, Inaba). A single dose of 5 x 10(8) viable CVD 103-HgR organisms, administered in a buffered liquid formulation, was well tolerated as compared with individuals who received an equivalent amount of heat-killed Escherichia coli K-12 placebo. Eighty-eight percent of subjects receiving CVD 103-HgR mounted a significant (greater than fourfold) rise in Inaba vibriocidal titre while 68% did so for the heterologous Ogawa serotype. The magnitude of the vibriocidal antibody response (as measured by peak geometric mean titre and by fold-rise in titre over baseline) was greater for the homologous Inaba serotype. Nineteen out of 25 volunteers (76%) responded with a significant (p less than 0.05) rise in serum antitoxin levels. No vaccinee who received the E. coli K-12 placebo mounted a significant rise in either vibriocidal or antitoxin antibody levels. These results corrobrate the safety and immunogenicity of CVD 103-HgR in healthy adult volunteers.

Administration, Oral↗

PubMed1990

Comparative analysis of the hemolysin genes of Vibrio cholerae non-01, V. mimicus, and V. hollisae that are similar to the tdh gene of V. parahaemolyticus.

The genes encoding the hemolysins similar to the thermostable direct hemolysin (tdh gene) of Vibrio parahaemolyticus were cloned from chromosomes of V. mimicus and V. hollisae. These cloned hemolysin genes and previously cloned tdh genes of V. parahaemolyticus and V. cholerae non-01 were compared by physical mapping and by hybridization with oligodeoxyribonucleotide probes. The nucleotide sequences in the coding regions of all the cloned hemolysin genes were very homologous and had only minor variations but the sequences flanking the homolysin genes were dissimilar, indicating that the hemolysin genes have a common ancestor and suggesting that they may have been transferred between Vibrio species as a descrete genetic unit.

Biological Evolution↗

PubMed1990

A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells.

The ability of enteropathogenic Escherichia coli (EPEC) to form attaching and effacing intestinal lesions is a major characteristic of EPEC pathogenesis. Using TnphoA mutagenesis we have identified a chromosomal gene (eae, for E. coli attaching and effacing) that is necessary for this activity. A DNA probe derived from this gene hybridizes to 100% of E. coli of EPEC serogroups that demonstrate attaching and effacing activity on tissue culture cells as well as other pathogenic E. coli that produce attaching and effacing intestinal lesions, such as RDEC-1 (an EPEC of weanling rabbits) and enterohemorrhagic E. coli. The predicted amino acid sequence derived from the nucleotide sequence of eae shows significant homology to that of the invasin of Yersinia pseudotuberculosis.

Animals↗

PubMed1990

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.

Bacterial Adhesion↗

PubMed1990

Transposon-induced non-motile mutants of Vibrio cholerae.

Non-motile mutants of Vibrio cholerae were isolated after transposon insertion mutagenesis with either Tn5 on a plasmid or Tn10ptac mini-kan in bacteriophage lambda. The physical location and number of transposon insertions was determined. Eighteen Tn5 insertion mutants and 11 Tn10ptac mini-kan insertion mutants had single unique insertion sites. The 18 Tn5 insertions were contained within six different EcoRI fragments and the 11 Tn10ptac mini-kan insertions were contained within eight different fragments of V. cholerae chromosomal DNA. These data suggest that multiple genes are involved in motility. Immunoblot analysis of non-motile mutants with antibody to wild-type flagellar core protein indicated that two of the non-motile mutants made flagellar core protein. Three additional mutants reacted weakly with the antibodies. However, these mutants with immunopositive reactions did not produce any structures which resembled flagella by transmission electron microscopy. In addition, none of the other non-motile mutants produced wild-type flagella. However, five mutants which did not react in the immunoblot produced a structure which resembled a flagellar sheath without the internal flagellar core. In addition to having no filamentous core, the sheaths often extended from the sides of the bacteria, rather than from the poles where the flagellum is normally located. The data suggest that sheath formation is independent of flagellar filament formation, but that proper positioning of the sheath may require the flagellar filament.

DNA Transposable Elements↗

PubMed1990

Duplication and variation of the thermostable direct haemolysin (tdh) gene in Vibrio parahaemolyticus.

The relationship between phenotypic variation and nucleotide sequence variation of the gene encoding Vibrio parahaemolyticus thermostable direct haemolysin (tdh gene) was examined. Strains showing a typical haemolysin-positive phenotype carried two chromosomal gene copies (designated tdh1 and tdh2) while tdh-gene-positive strains showing a weakly positive or negative haemolysin phenotype possessed only a single chromosomal gene copy. Both gene copies from a typical haemolysin-positive strain were cloned and sequenced and possessed 97.2% homology. Comparison of the amino acid sequence predicted from the nucleotide sequence with the protein sequence determined by Edman degradation as well as construction of a tdh1-deficient yet haemolytic strain of V. parahaemolyticus suggest that the tdh2 locus is primarily responsible for the haemolytic phenotype. Two other tdh gene copies were cloned from a phenotypically negative strain which was unusual in that it contained one gene copy on a plasmid (designated tdh4) in addition to a single copy on the chromosome (tdh3). Both tdh3 and tdh4 were expressed in Escherichia coli and TDHs with haemolytic activity were produced. These gene copies were sequenced and shared 96.7% homology with the tdh1 gene. The V. parahaemolyticus strain carrying tdh3 and tdh4 gene copies did not produce detectable amount of tdh-specific RNA transcript. It seems, therefore, that differences in the transcriptional control are primarily responsible for the differences seen in haemolytic phenotype.

Amino Acid Sequence↗

PubMed1990

Association of rns homologs with colonization factor antigens in clinical Escherichia coli isolates.

rns is a trans-acting positive regulatory factor required for expression of the colonization factor antigen II (CFA/II) antigens CS1 and CS2 (J. Caron, L. M. Coffield, and J. R. Scott, Proc. Natl. Acad. Sci. USA 86:963-967, 1989). All 35 CFA/II-positive strains hybridized with a rns gene probe, as did all 10 CFA/I strains and all 4 CS4 strains. Hybridization with rns was detected in 25% of non-enterotoxigenic Escherichia coli strains and was not detected in enteric pathogens with low G + C content.

Antigens, Bacterial↗

PubMed1990

Diminished immunogenicity of a recombination-deficient derivative of Vibrio cholerae vaccine strain CVD103.

To address potential concerns over the release of genetically engineered live bacterial vaccines, we constructed a recombination-deficient derivative of the Vibrio cholerae O1 vaccine strain CVD103 (CVD103RM). Oral immunization of adult volunteers with CVD103RM showed that the recA mutation significantly diminished colonization ability and immunogenicity of the vaccine strain.

Antibodies, Bacterial↗

PubMed1990

Construction and analysis of TnphoA mutants of enteropathogenic Escherichia coli unable to invade HEp-2 cells.

Enteropathogenic Escherichia coli (EPEC) strains have recently been shown to invade tissue culture cells. We describe a set of 22 Tn5 IS50L::phoA (TnphoA) insertion mutants of EPEC strain E2348-69 that are unable to invade HEp-2 cells. Each mutant was tested for the ability to adhere to and to induce the polymerization of actin in HEp-2 cells. Southern hybridization of plasmid and total DNA of each strain was performed to determine the location of each TnphoA insert, and each TnphoA insert along with flanking EPEC sequences was cloned. These studies resulted in the grouping of the mutants into five main categories. These include strains with plasmid and chromosomal insertions that alter adherence, chromosomal insertions that alter the ability to induce actin polymerization, and chromosomal insertions that do not affect adherence or actin polymerization. These studies indicate that genes affecting EPEC adherence may be located on both the plasmid and chromosome, that several genes are involved in the induction of actin polymerization in epithelial cells, and that EPEC invasion is a complex process involving multiple genetic loci.

Actin Cytoskeleton↗

PubMed1990

The cytolysin gene of Vibrio vulnificus: sequence and relationship to the Vibrio cholerae E1 Tor hemolysin gene.

A cytolysin of ca. 56 kilodaltons has been suggested as a possible virulence factor in Vibrio vulnificus infections. We sequenced the DNA encoding cytolytic activity and found that the sequence contained two open reading frames, vvhA and vvhB. vvhA encoded the structural gene for the cytolysin and contained the N-terminal amino acid sequence previously reported for the protein. Regions of the vvhA gene showed homology to the structural gene for the Vibrio cholerae E1 Tor hemolysin.

Amino Acid Sequence↗

PubMed1990

Practical and economical method for using biotinylated DNA probes with bacterial colony blots to identify diarrhea-causing Escherichia coli.

A simple and economical method was developed for using biotinylated DNA probes to hybridize with bacterial colonies belonging to the various categories of diarrhea-causing Escherichia coli. Simplification and cost containment were achieved by using Whatman no. 541 filter papers instead of nitrocellulose, by minimizing the concentration of proteinase K (an expensive but necessary reagent used to pretreat the colony blots prior to hybridization with biotin-labeled DNA probes) and by reusing hybridization solution containing labeled probe DNA. After exposing the colony blots to lysing solution and steam, followed by lysozyme (1.5 mg/ml), sucrose (25%), and proteinase K (10 micrograms/ml) treatments, biotinylated probes were used to detect enterotoxigenic, enteropathogenic, enterohemorrhagic, diffuse adherence, and enteroinvasive categories of diarrhea-causing E. coli with a high level of sensitivity and specificity. Three independent observers who were experienced in reading DNA blots recorded remarkably similar results, while less satisfactory results were obtained when the blots were read by an inexperienced observer. This technique will be useful in laboratories in which radioactive isotopes are unavailable or impractical and in which budgets are restricted.

Biotin↗

PubMed1990

Oligonucleotide probe for detection of the enteropathogenic Escherichia coli (EPEC) adherence factor of localized adherent EPEC.

The enteropathogenic Escherichia coli adherence factor (EAF) probe detects isolates of enteropathogenic E. coli that exhibit localized adherence to HEp-2 cells. A 21-base oligonucleotide probe was constructed on the basis of a sequence from within the 1-kb EAF probe and was shown to have greater sensitivity and specificity than the EAF fragment probe in detecting localized adherent E. coli.

Adhesins, Escherichia coli↗