Construction and characterisation of a Yersinia enterocolitica O:8 ompR mutant.

The ompR-envZ two-component regulatory system has been shown to contribute to virulence in a number of enteric bacterial pathogens. A Yersinia enterocolitica O:8 ompR homologue was amplified, cloned and sequenced, showing 99.2% homology to the Escherichia coli OmpR. An isogenic ompR mutant was constructed by reverse genetics-based methodology. The mutant was shown to have increased sensitivity to high osmolarity, high temperature and low pH stresses in vitro. In the murine yersiniosis model, the mutant was attenuated and offered partial protection against wild-type challenge.

Construction and characterization of a Yersinia enterocolitica O:8 high-temperature requirement (htrA) isogenic mutant.

The high-temperature requirement (HtrA) family of stress response proteins are induced by different environmental stress conditions in a variety of bacteria and have been shown to contribute to the pathogenicity of some of these species. In this study, the htrA gene from Yersinia enterocolitica O:8 was amplified, cloned, and sequenced. Analysis of the deduced amino acid sequence predicted that the putative HtrA homolog contains a serine protease active site and a catalytic triad characteristic of trypsin-like serine proteases, structural features characteristic of previously described HtrA proteins. In order to evaluate the biological functions of Y. enterocolitica HtrA, an isogenic mutant was constructed by a reverse-genetics PCR-based approach. Characterization of the mutant provided evidence supporting a stress response function for the Y. enterocolitica htrA gene product. In contrast to the parent strain, the mutant showed increased sensitivity to killing by H2O2, O2- and temperature stress (50 degrees C). The mutant was avirulent in the murine yersiniosis injection model and offered partial protection to mice challenged with the parent strain. Further studies with the Y. enterocolitica htrA mutant should increase our knowledge of the host-pathogen interactions which occur during Yersinia infections.

Roles of leukotriene B4, prostaglandin E2, and cyclic AMP in Campylobacter jejuni-induced intestinal fluid secretion.

Infection of rabbit ileal loops with inflammatory Campylobacter jejuni strains caused elevation of cyclic AMP, prostaglandin E2, and leukotriene B4 levels in tissue and fluids. Incubation of cultured Caco-2 cells with loop fluids caused elevated cellular cyclic AMP levels, an effect which was inhibited by antiserum against prostaglandin E2.

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.

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.