Identification of regions of Ail required for the invasion and serum resistance phenotypes.

Yersinia enterocolitica is an enteric pathogen that has served as a model system for the study of microbial pathogenesis. Numerous virulence gene have been identified both on the virulence plasmid and on the chromosome. One of the chromosomal genes that is highly correlated with virulence is ail, a gene identified along with inv in a screen for Y. enterocolitica genes that could confer an invasive phenotype to Escherichia coli. Ail also promotes serum resistance in both E. coli and Y. enterocolitica. Several virulence factors homologous to Ail have been identified in other pathogens, yet very little is known about what constitutes the functional domain(s) of these proteins. Proteins in this family are predicted to consist of eight transmembrane beta-sheets and four cell surface-exposed loops. We constructed and characterized a number of insertion, deletion and point mutations in the regions of ail predicted to encode the cell surface loops. The results from the analysis of these mutants indicate that cell surface loops one and four do not directly promote invasion or serum resistance, whereas mutations in loop three appear to modulate both phenotypes. Analysis of mutations in loop 2 suggests that this surface-exposed loop contains sequences required for serum resistance and invasion. In addition, a peptide derived from the sequence of loop 2 was able specifically to inhibit Ail-mediated invasion in a dose-dependent manner. These results suggest that Ail directly promotes invasion and that loop 2 contains an active site, perhaps a receptor-binding domain. Analyses of the mutations also suggest that the serum resistance and invasion phenotypes may be separable, because there are numerous mutations that affect one phenotype but not the other.

Detection on surfaces and in Caco-2 cells of Campylobacter jejuni cells transformed with new gfp, yfp, and cfp marker plasmids.

We have developed two sets of Campylobacter shuttle vectors containing either the gfp (green fluorescent protein), yfp (yellow fluorescent protein), or cfp (cyan fluorescent protein) reporter gene. In one set, the reporter gene is fused to a consensus Campylobacter promoter sequence (P(c)). The other set contains a pUC18 multicloning site upstream of the reporter gene, allowing the construction of transcriptional fusions using known promoters or random genomic fragments. C. jejuni cells transformed with the P(c) fusion plasmids are strongly fluorescent and easily visualized on chicken skin, on plant tissue, and within infected Caco-2 cells. In each C. jejuni strain tested, these plasmids were maintained over several passages in the absence of antibiotic selection. Also, in many C. jejuni strains, >91% of the cells transformed with the P(c) fusion plasmids remained fluorescent after several days. Experiments with yellow fluorescent and cyan fluorescent C. jejuni transformants suggest that aggregates containing two or more strains of C. jejuni may be present in an enrichment broth culture. Colonies arising from these aggregates would be heterologous in nature; therefore, isolation of a pure culture of C. jejuni, by selecting single colonies, from an environmental sample may not always yield a single strain.

Decreased adherence of enterohemorrhagic Escherichia coli to HEp-2 cells in the presence of antibodies that recognize the C-terminal region of intimin.

Antiserum raised against intimin from enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain 86-24 has been shown previously by our laboratory to inhibit adherence of this strain to HEp-2 cells. In the present study, we sought to identify the region(s) of intimin important for the effect of anti-intimin antisera on EHEC adherence and to determine whether antisera raised against intimin from an O157:H7 strain could reduce adherence of other strains. Compared to preimmune serum controls, polyclonal sera raised against the histidine-tagged intimin protein RIHisEae (intimin(O157)) or against His-tagged C-terminal fragments of intimin from strain 86-24 reduced adherence of this strain. Furthermore, an antibody fraction purified from the anti-RIHisEae serum that contained antibodies to the C-terminal third of intimin, the putative receptor-binding domain, also reduced adherence of strain 86-24, but a purified fraction containing antibodies to the N-terminal two-thirds of intimin did not inhibit adherence. The polyclonal anti-intimin(O157) serum raised against RIHisEae inhibited, to different degrees, the adherence of another O157:H7 strain, an EHEC O55:H7 strain, one of two independent EHEC O111:NM isolates tested, and one of two EHEC O26:H11 strains tested. Adherence of the other O26:H11 and O111:NM strains and an EPEC O127:H6 strain was not reduced. Finally, immunoblot analysis indicated a correlation between the antigenic divergence in the C-terminal third of intimins from different strains and the capacity of anti-intimin(O157) antiserum to reduce adherence of heterologous strains. Taken together, these data suggest that intimin(O157) could be used as an immunogen to elicit adherence-blocking antibodies against O157:H7 strains and closely-related EHEC.

Novel detection techniques for human pathogens that contaminate poultry.

Poultry products are presumed to be a major contributor to human foodborne illness due to their high frequency of contamination with pathogens Salmonella spp. and Campylobacter spp. This has stimulated the development of more sensitive and rapid methods for identifying pathogens present in poultry. These new methods include immunomagnetic separation of pathogen, PCR amplification of pathogen-specific sequences, pathogen-specific DNA and RNA probes, and identification of pathogen-specific ions by mass spectrometry.

Pathogenesis of defined invasion mutants of Yersinia enterocolitica in a BALB/c mouse model of infection.

It has been hypothesized for many years that the ability of Yersinia spp. to invade tissue culture cells is reflective of their ability to penetrate the intestinal epithelium and that this capacity is an important aspect of the disease process. Three different genes from Yersinia spp. that are involved in the tissue culture invasion phenotype have been identified: inv, ail, and yadA. It was previously shown that inv is necessary for efficient penetration of the intestinal epithelium by Yersinia enterocolitica. The present study was initiated to determine whether other known Yersinia invasion factors could promote uptake of the bacteria by mice in the absence of invasion. In addition, the roles of these three invasion factors in the survival of the bacteria, lethality for mice, and development of pathology were compared. We found that YadA is necessary for persistence of Y. enterocolitica in Peyer's patches, and consistent with this observation, the yadA mutant was avirulent for mice infected either orally or intraperitoneally. In addition, the inv yadA double mutant was avirulent. Histological and immunohistological examination of the Peyer's patches of infected mice indicated that despite the presence of large numbers of CFU at 24 h the yadA and ail yadA mutants cause only minimal pathology and recruitment of macrophages. At 42 h postinfection, Peyer's patches from mice infected with the inv mutant showed no pathology, despite the prediction that some of the mice by this time would be colonized. However, at 72 h, inflammation and necrosis were evident in some Peyer's patches. Together, these observations suggest that for visible pathology to develop, a threshold number of bacteria (> 10(5)) is needed and the bacteria need to persist for more than 24 h. Lastly, YadA but not Ail may play a role in the less efficient, delayed invasion of the intestinal epithelium observed for the inv mutant.

In vitro and in vivo characterization of an ail mutant of Yersinia enterocolitica.

Ail is a 17-kDa protein of Yersinia enterocolitica previously identified on the basis of its ability to confer upon Escherichia coli the phenotype of attachment and invasion of cultured epithelial cells. Here we report an examination of the contribution of ail to the pathogenicity of Y. enterocolitica. A low-copy-number ail plasmid that promoted serum resistance in E. coli HB101 was constructed. The serum resistance phenotype conferred by ail to E. coli was affected by the growth phase of the culture as well as by the gene copy number. In contrast, the copy number of ail (and the relative quantity of Ail) was found to have little effect on the amount of Ail-promoted invasion of cultured epithelial cells. An ail mutant of Y. enterocolitica was constructed and characterized in vitro. This mutant produced no detectable Ail and had a reduced ability to invade CHO cells. Serum resistance of Y. enterocolitica was Ail dependent and was affected by growth phase and ail copy number. The phenotype of the ail mutant was examined in vivo by using a murine model for infection. The ail mutant phenotype was identical to that of the wild-type strain in oral 50% lethal dose studies and early colonization of Peyer's patches as well as in kinetic studies. Western blot (immunoblot) analysis of Ail produced by bacteria growing in vivo at 48 h postinfection indicated that ail was expressed at this time point. Thus, our findings confirm that Ail contributes to the serum resistance and invasion phenotypes of Y. enterocolitica in vitro and indicate that Ail is not required to establish an infection or to cause systemic infection of BALB/c or DBA/2 mice.