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J M Ketley

Publications and source records attributed to J M Ketley.

At least 19 recordsLinked to original sources
PubMed1998

Iron-responsive gene regulation in a campylobacter jejuni fur mutant.

The expression of iron-regulated systems in gram-negative bacteria is generally controlled by the Fur protein, which represses the transcription of iron-regulated promoters by using Fe2+ as a cofactor. Mutational analysis of the Campylobacter jejuni fur gene was carried out by generation of a set of mutant copies of fur which had a kanamycin or chloramphenicol resistance gene introduced into the regions encoding the N and C termini of the Fur protein. The mutated genes were recombined into the C. jejuni NCTC 11168 chromosome, and putative mutants were confirmed by Southern hybridization. C. jejuni mutants were obtained only when the resistance genes were transcribed in the same orientation as the fur gene. The C. jejuni fur mutant grew slower than the parental strain. Comparison of protein profiles of fractionated C. jejuni cells grown in low- or high-iron medium indicated derepressed expression of three iron-regulated outer membrane proteins with molecular masses of 70, 75, and 80 kDa. Characterization by N-terminal amino acid sequencing showed the 75-kDa protein to be identical to CfrA, a Campylobacter coli siderophore receptor homologue, whereas the 70-kDa protein was identified as a new siderophore receptor homologue. Periplasmic fractions contained four derepressed proteins with molecular masses of 19, 29, 32, and 36 kDa. The 19-kDa protein has been previously identified, but its function is unknown. The cytoplasmic fraction contained two iron-repressed and two iron-induced proteins with molecular masses of 26, 55, 31, and 40 kDa, respectively. The two iron-repressed proteins have been previously identified as the oxidative stress defense proteins catalase (KatA) and alkyl hydroperoxide reductase (AhpC). AhpC and KatA were still iron regulated in the fur mutant, suggesting the presence of Fur-independent iron regulation. Further analysis of the C. jejuni iron and Fur regulons by using two-dimensional gel electrophoresis demonstrated the total number of iron- and Fur-regulated proteins to be lower than for other bacterial pathogens.

Amino Acid Sequence↗
PubMed1997

Campylobacter-host cell interactions.

The enteric pathogens Campylobacter jejuni and Campylobacter coli are a major cause of infectious diarrhoea. Their ability to adhere to human epithelial cells is ubiquitous and their propensity to invade cells is also well documented and requires motility and de novo protein synthesis, as well as several host factors. The molecular basis of the interaction between campylobacters and host cells is only beginning to be elucidate. The characteristics of this interaction promise to be interesting and may provide new insights into host-pathogen interactions in other enteric diseases.

Campylobacter Infections↗
PubMed1996

Host signal transduction and endocytosis of Campylobacter jejuni.

Caveolae are plasma membrane invaginations found in a variety of mammalian cells and are implicated in clathrin-independent endocytosis and signal transduction. Here we show that pretreatment of Caco-2 cell monolayers with filipin III, which disrupts caveolae by chelating cholesterol, significantly reduces the ability of Campylobacter jejuni to enter these cells. Furthermore inhibitors of host protein tyrosine phosphorylation, the phosphatidylinositol-3 kinase (Pl 3-kinase) inhibitor wortmannin, and cholera toxin, all significantly reduced invasion of Caco-2 cells by C. jejuni.

Androstadienes↗
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

Iron-responsive genetic regulation in Campylobacter jejuni: cloning and characterization of a fur homolog.

The Fur protein of Escherichia coli represses transcription from Fur-responsive genes in an iron-dependent manner. We have demonstrated a Fur-like iron-responsive genetic regulatory activity operating in Campylobacter jejuni by using a chloramphenicol acetyl transferase reporter gene separated from its promoter by a synthetic Fur-responsive operator. A fur-like gene has been cloned from C. jejuni by partial functional complementation of an E. coli fur mutation. Sequence analysis has shown that, at the amino acid level, the C. jejuni Fur protein is 35% identical with its E. coli counterpart.

Amino Acid Sequence↗
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

Pathological changes in the rabbit ileal loop model caused by Campylobacter jejuni from human colitis.

Four strains of Campylobacter jejuni isolated from children with inflammatory diarrhoea were assayed in the rabbit ileal loop model of infectious diarrhoea. All caused inflammatory reactions with severe macroscopic and microscopic damage in infected rabbit ileal tissue similar to that observed in the patients by endoscopy and histological analysis of colonic biopsies. Haemoglobin and other proteins were observed in loop fluids, consistent with leakage of serum from damaged mucosa. Loop fluids also contained significant bicarbonate concentrations, indicative of an active secretory component similar to that in control loops inoculated with cholera toxin. However, although three of the four clinical strains produced small amounts of a protein immunologically related to cholera toxin in vitro, none such was detected in either tissues or fluids of infected ileal loops. We propose instead that host-derived mediators of secretion may be important in pathogenesis. A mutant strain of C. jejuni with impaired motility, obtained from the National Collection of Type Cultures, did not induce tissue damage or fluid secretion in rabbit ileal loops.

Animals↗
PubMed1992

Differentiated Caco-2 cells as a model for enteric invasion by Campylobacter jejuni and C. coli.

A collection of 44 Campylobacter isolates (37 C. jejuni and seven C. coli) from children with colitis (21 strains) or watery diarrhoea (23 strains) was analysed for toxin production, association with HeLa cells, and invasion of differentiated Caco-2 cell cultures. There was no obvious association of clinical symptoms with species, biotype or enterotoxin production. All colitis strains and most of the isolates from watery diarrhoea were cytotoxic for Chinese hamster ovary cells. Measurements of bacterial association indices with HeLa cells varied with time, and were considered to be unreliable for discriminating between isolates from the two diagnostic groups. Statistically significant differences were observed between the two groups (all colitis strains and 65% of strains from non-inflammatory diarrhoea) with respect to invasion of both HeLa and Caco-2 cell monolayers. However, among the strains from non-inflammatory diarrhoea that did invade, numbers of internalised bacteria were similar to the range observed for colitis strains. Of the colitis strains, 86% were able to transcytose through polarised Caco-2 monolayers grown on filters, compared with 48% of isolates from non-inflammatory disease. We propose the use of Caco-2 cells as a model for studying invasion of intestinal epithelia by C. jejuni and C. coli.

Animals↗
PubMed1992

Role of Vibrio cholerae neuraminidase in the function of cholera toxin.

Vibrio cholerae neuraminidase (NANase) is hypothesized to act synergistically with cholera toxin (CT) and increase the severity of a secretory response by increasing the binding and penetration of CT to enterocytes. To test this hypothesis, the NANase gene (nanH) from V. cholerae Ogawa 395 was first cloned and sequenced. Isogenic wild-type and NANase- V. cholerae 395 strains were then constructed by using suicide vector-mediated mutagenesis. The influence of NANase on CT binding and penetration was examined in vitro by using culture filtrates from these isogenic strains. Fluorescence due to binding of fluorescein-conjugated CT to C57BL/6 and C3H mouse fibroblasts exposed to NANase+ filtrates increased five- and eightfold, respectively, relative to that with NANase- filtrates. In addition, NANase+ filtrates increased the short-circuit current measured in Ussing chambers 65% relative to that with NANase- filtrates, although this difference decreased as production of CT increased. The role of NANase in V. cholerae pathogenesis was examined in vivo by intragastric inoculation of the isogenic strains into CD1 suckling mice. No difference in fluid accumulation ratios was seen at doses of 10(4) to 10(8) CFU, but NANase+ strains produced 18% higher fluid accumulation ratios at 10(9) CFU than NANase- strains when inoculated into nonfasted suckling mice. It is concluded that NANase plays a subtle but significant role in the binding and uptake of CT by susceptible cells under defined conditions.

Amino Acid Sequence↗
PubMed1991

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.

Animals↗
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

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↗
PubMed1987

The effects of Clostridium difficile crude toxins and purified toxin A on stripped rabbit ileal mucosa in Ussing chambers.

Clostridium difficile crude toxins and purified toxin A had similar effects on stripped rabbit ileal mucosa in Ussing chambers. Both toxin preparations caused secretion of sodium and chloride ions by increasing serosa to mucosa (s----m) fluxes. Transmural potential difference and resistance decreased after toxin treatment. Onset of changes in electrical measurements and ion fluxes coincided with onset of histological changes. The response to theophylline was greatly reduced in toxin-treated tissue compared with control tissue.

Animals↗