Identification of a conjunctivitis-associated gene locus from the virulence plasmid of Yersinia enterocolitica

Abstract

The virulence plasmid (pYV) of Yersinia enterocolitica is necessary for production of conjunctivitis in guinea pigs and for mouse lethality. To identify the genes responsible for production of conjunctivitis in guinea pigs, we subcloned the BamHI and SalI restriction fragments of the virulence plasmid of Y. enterocolitica A2635 (serotype O:8) into derivatives of the broad-host-range plasmid pRK290 and introduced the constructions into plasmid-negative Y. enterocolitica strains. A mild, transient conjunctivitis was evident 24 h after inoculation with strains containing a 2.8-kilobase (kb) BamHI fragment of pYV. These strains were cytotoxic to HEp-2 cells but did not cause death in iron-loaded adult mice. When the 2.8- and adjacent 0.5-kb BamHI fragments were deleted from the virulence plasmid of a fully virulent Y. enterocolitica isolate, the resultant strain did not cause conjunctivitis in guinea pigs and was not cytotoxic to HEp-2 cells. However, the strain with the deletion appeared to be more virulent for mice, with more rapid dissemination after orogastric inoculation, compared with that of the parent strain. When the deletion was complemented by introduction of a plasmid containing the 2.8-kb BamHI fragment, the strain again caused conjunctivitis but had decreased virulence for mice.

Detection of pathogenic Yersinia enterocolitica by polymerase chain reaction and digoxigenin-labeled polynucleotide probes

“…Several nucleic acid-based hybridization techniques have been evaluated for the detection of virulent strains of Y enterocolitica (13,15,22,27,33). A further improvement of the sensitivity of DNA probes is the amplification of the target DNA by the PCR, which offers the advantages of maximum sensitivity, specificity, and rapidity (37).…”

Contribution of urease to acid tolerance in Yersinia enterocolitica

“…This gene was chosen for the site of insertion because it contains a unique BglII site into which the kanamycin resistance cassette could be cloned and because disruption of this gene in p19EPv in E. coli renders the bacteria urease negative. The mutagenesis procedure required cloning the 6.6-kb fragment containing the urease gene complex from p19EPv into pRK404, a plasmid which can easily be conjugated into Y. enterocolitica and is stably maintained in this host (17,26). pRK404 was digested with HindIII, and the overhang was filled in to create a blunt end.…”

Pathogenicity ofYersinia enterocoliticabiotype 1A

“…Studies to identify the essential virulence determinants of pYV‐bearing Y. enterocolitica have been facilitated by access to animal models of infection, such as guinea pigs or mice pre‐treated with iron and desferrioxamine [65,95]. The latter is a microbial siderophore that permits pYV‐bearing strains of Y. enterocolitica , which lack the high‐pathogenicity island [39], to grow under iron‐limiting conditions in vitro and in vivo [65].…”