ALBERT

Notes: A TnphoA-generated mutant C5060, attenuated for virulence, was derived from the mouse-virulent Salmonella typhimurium strain C5. This mutation, designated hns-112::TnphoA, harbours the transposon in the 3 end of hns, with the alkaline phosphatase open reading frame in the opposite orientation to that of hns. Bacterial strains harbouring hns-112::TnphoA were mucoid and had altered levels of DNA supercoiling, as monitored using pUC18 as a reporter plasmid. Transduction of hns-112::TnphoA into mouse virulent strains, including S. typhimurium SL1344 and Salmonella enteritidis Se795, resulted in attenuation. When an independent hns mutation, harbouring a kanamycin-resistance cassette inserted into the Kpnl site at base pair 237 of the hns gene, was introduced into S. typhimurium C5, the isolates were also attenuated. S. typhimurium C5 isolates harbouring the multicopy plasmid pGB651, which encodes the Escherichia coli hns gene, were partially attenuated in mice. Transductional analysis, using Tn10 insertions located close to the hns gene, showed that virulence could be restored In genetic crosses that eliminated the resident hns mutations. However, some hns+ transductants were stilt attenuated, suggesting that secondary attenuating lesions can accumulate in hns-deficient strains. These studies show that the hns locus plays a role in Salmonella virulence.

Notes: Citrobacter rodentium is used as an in vivo model system for clinically significant enteric pathogens such as enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens all colonize the lumen side of the host gastrointestinal tract via attaching and effacing (A/E) lesion formation. In order to identify genes required for the colonization of A/E-forming pathogens, a library of signature-tagged transposon mutants of C. rodentium was constructed and screened in mice. Of the 576 mutants tested, 14 were attenuated in their ability to colonize the descending colon. Of these, eight mapped to the locus of enterocyte effacement (LEE), which is required for the formation of A/E lesions, underlying the importance of this mechanism for pathogenesis. Another mutant, P5H2, was found to have a transposon insertion in an open reading frame that has strong similarity to type IV pilus nucleotide-binding proteins. The region flanking the transposon insertion was sequenced, identifying a cluster of 12 genes that encode the first described pilus of C. rodentium (named colonization factor Citrobacter, CFC). The proteins encoded by cfc genes have identity to proteins of the type IV COF pilus of enterotoxigenic E. coli (ETEC), the toxin co-regulated pilus of Vibrio cholerae and the bundle-forming pilus of EPEC. A non-polar mutation in cfcI, complementation of this strain with wild-type cfcI and complementation of strain P5H2 with wild-type cfcH confirmed that these genes are required for colonization of the gastrointestinal tract by C. rodentium. Thus, CFC provides a convenient model to study type IV pilus-mediated pathogen–host interactions under physiological conditions in the natural colonic environment.

Notes: Mice harbouring a null deletion mutation in the IFNγ receptor gene were used to study the role of IFNγ responsiveness during experimental systemic candidiasis of mucosal or haematogenous origin. After intravenous (i.v.) or intranasal (i.n.) challenge with Candida albicans the progression of infection and concomitant cellular and antibody anti-C. albicans immune responses were analysed. During the week following i.v. challenge, the rate of C. albicans multiplication in kidneys, liver and spleen was faster in IFNγR (−/−) than IFNγR (+/+) mice. As a result, IFNγR (−/−) mice perished earlier than IFNγR (+/+) mice when challenged with equal numbers of live yeast cells. However, the overall susceptibility of the two mouse strains, in terms of survival against different C. albicans challenge doses over a 60-day period, was similar. No differences were found in the cellular anti-C. albicans response generated by i.v. challenge in both mouse strains. In contrast the kinetics and strength of the serum anti-C. albicans antibody responses were markedly different. Significantly stronger, predominantly IgG2a antibody responses accompanied the eventual control of C. albicans infection in IFNγR (−/−) mice. Following intranasal challenge, there was no difference in the rate of C. albicans clearance from the lungs of IFNγR (−/−) and IFNγR (+/+) mice. However, 48 h after challenge, large, conspicuous abscesses appeared in the lungs, liver, kidneys and spleen of IFNγR (−/−) mice. These abscesses were characterised by the presence of C. albicans and abundant neutrophilic infiltrates, but very few macrophages. No such abscesses developed in i.n. challenged IFNγR (+/+) mice. In both mouse strains, i.n. challenge induced strong systemic anti-C. albicans cellular responses, but relatively low titre systemic antibody responses. Mucosal anti-C. albicans antibody responses were detected in IFNγR (+/+), but not IFNγR (−/−) mice. Splenic adherent macrophages obtained from IFNγR (−/−) mice exhibited a significantly lower candidacidal activity than those of IFNγR (+/+) mice, and as expected, were not responsive to IFNγ. In summary, these data suggest that IFNγ has a role in limiting C. albicans multiplication during the early stages of infection, as well as in preventing the development of C. albicans-associated abscesses. Activation of macrophages by IFNγ might be pivotal in mediating this role.

Notes: Enteropathogenic Escherichia coli (EPEC) encode a type III secretion system located on a pathogenicity island known as the locus for enterocyte effacement. Four proteins are known to be exported by this type III secretion system – EspA, EspB and EspD required for subversion of host cell signal transduction pathways and a translocated intimin receptor protein (Tir) required for intimin-mediated intimate attachment and attaching and effacing lesion formation. The espA gene is located within the locus for enterocyte effacement and the EspA polypeptide from the prototype EPEC strain E2348/69 (O127:H6) has recently been shown to be a component of a filamentous structure involved in bacteria-host cell interaction and locus for enterocyte effacement-encoded protein translocation involved in attaching and effacing lesion formation. In this study we have extended our investigation of EspA to strains belonging to other classical EPEC serotypes. DNA sequencing demonstrated that the espA gene from the different EPEC strains share at least 65% DNA identity. In addition, we detected morphologically and antigenically similar EspA filaments in all but one of the bacterial strains examined including recombinant, non-pathogenic E. coli expressing espA from a cloned locus for enterocyte effacement region (HB101(pCVD462)).

Notes: 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.

Notes: Interaction of two clinical Edwardsiella tarda isolates with HEp-2 cells was investigated. By electron microscopy we observed at 1 h post infection that E. tarda induced formation of extensive plasma membrane projections resembling membrane ruffles. The ruffles did not coincide with adhering bacteria. Only few invading bacteria were seen. Vacuolated nuclear membrane was occasionally observed. Three hours post infection, E. tarda induced a contact-dependent cell lysis, revealing the host cell cytoskeleton and nucleus. Only one of the E. tarda strains was seen residing within the host cell remains. The results indicate that E. tarda-induced membrane ruffles may involve a distinct mechanism of bacterial pathogenesis.

Notes: Abstract Attenuated Salmonella strains are currently being evaluated as live vectors for the delivery of heterologous antigens to the mammalian mucosal and systemic immune systems. An approach to improving the stability of heterologous antigen expression during vaccination is to drive expression of the foreign protein from promoters e.g. nirB, that become activated when Salmonella enter the host. Salmonella strains were constructed that harboured similar multicopy plasmids encoding the lacZ gene. In each strain, lacZ expression was driven from either the nirB, htrA or groE promoters. Expression of LacZ increased in all vaccine strains as they were shifted from conditions of low to high temperature. In addition, expression of lacZ driven from the htrA and nirB promoters significantly increased when the Salmonella entered eukaryotic cells, including macrophages. Expression of lacZ from the groE promoter was significantly elevated in macrophages but not in cells derived from epithelia. These promoters may be useful for optimising heterologous antigen expression within immune cells of the host.

Notes: Abstract As part of a study of genes whose transcription is maintained in stationary phase, cloned segments of DNA were selected from a Lambda ZAP II library of Campylobacter jejuni NCTC 11168. One such clone was found to encode a homologue of the Escherichia coli cell division gene ftsA. Examination of mRNA by transcription mapping revealed that the Campylobacter gene has one major and three minor transcription start sites. There were several significant differences in the structure and organisation of the C. jejuni ftsA promoter compared to that of E. coli.

Notes: Abstract Plasmid pKT274 encoding a determinant for the Escherichia coli K1 polysaccharide was introduced into the Salmonella typhimurium aroA vaccine strain SL3261 and cells harbouring the plasmid were shown to express K1 polysaccharide at their cell surface. SL3261 (pKT274) could be detected in the livers and spleens of BALB/c mice infected by the intravenous route and viable organisms persisted for several weeks. SL3261 (pKT274) was cleared from the livers more rapidly and from the spleens more slowly than SL3261. Unlike mice infected with SL3261 those infected with SL3261 (pKT274) did not exhibit gross splenomegaly during the first three weeks after infection. Mice vaccinated with viable SL3261 (pKT274) were protected against challenge with virulent S. typhimurium but failed to produce detectable levels of humoral anti-K1 polysaccharide antibodies.