The goal of this study was to characterize the Yersinia pestis KIM OmpX protein. Yersinia spp. provide a model for studying several virulence processes including attachment to, and internalization by, host cells. For Yersinia enterocolitica and Yersinia pseudotuberculosis, Ail, YadA and Inv, have been implicated in these processes. In Y. pestis, YadA and Inv are inactivated. Genomic analysis of two Y. pestis strains revealed four loci with sequence homology to Ail. One of these genes, designated y1324 in the Y. pestis KIM database, encodes a protein designated OmpX. The mature protein has a predicted molecular mass of 17.47 kDa, shares approximately 70 % sequence identity with Y. enterocolitica Ail, and has an identical homologue, designated Ail, in the Y. pestis CO92 database. The present study compared the Y. pestis KIM6 + parental strain with a mutant derivative having an engineered disruption of the OmpX structural gene. The parental strain (and a merodiploid control strain) expressed OmpX at 28 and 37 6C, and the protein was detectable throughout all phases of growth. OmpX was required for efficient adherence to, and internalization by, cultured HEp-2 cell monolayers and conferred resistance to the bactericidal effect of human serum. Deletion of ompX resulted in a significantly reduced autoaggregation phenotype and loss of pellicle formation in vitro. These results suggest that Y. pestis OmpX shares functional homology with Y. enterocolitica Ail in adherence, internalization into epithelial cells and serum resistance.

Yersinia pestis, the causative agent of plague, is one of the most virulent microorganisms known. The outer membrane protein X (OmpX) in Y. pestis KIM is required for efficient bacterial adherence to and internalization by cultured HEp-2 cells and confers resistance to human serum. Here, we tested the contribution of OmpX to disease progression in the fully virulent Y. pestis CO92 strain by engineering a deletion mutant and comparing its ability in mediating pneumonic plague to that of the wild type in two animal models. The deletion of OmpX delayed the time to death up to 48 h in a mouse model and completely attenuated virulence in a rat model of disease. All rats challenged with 1 ؋ 10 8 CFU of the ompX mutant survived, compared to the 50% lethal dose (LD 50 ) of 1.2 ؋ 10 3 CFU for the wild-type strain. Because murine serum is not bactericidal for the ompX mutant, the mechanism underlying the delay in time to death in mice was attributed to loss of adhesion/internalization properties but not serum resistance. The rat model, which is most similar to humans, highlighted the critical role of serum resistance in disease. To resolve conflicting evidence for the role of Y. pestis lipopolysaccharide (LPS) and OmpX in serum resistance, ompX was cloned into Escherichia coli D21 and three isogenic derivatives engineered to have progressively truncated LPS core saccharides. OmpX-mediated serum resistance, adhesiveness, and invasiveness, although dependent on LPS core length, displayed these functions in E. coli, independently of other Yersinia proteins and/or LPS. Also, autoaggregation was required for efficient OmpX-mediated adhesiveness and internalization but not serum resistance.

A comprehensive TnphoA mutant library was constructed in Yersinia pestis KIM6 to identify surface proteins involved in Y. pestis host cell invasion and bacterial virulence. Insertion site analysis of the library repeatedly identified a 9,042-bp chromosomal gene (YPO3944), intimin/invasin-like protein (Ilp), similar to the Gram-negative intimin/invasin family of surface proteins. Deletion mutants of ilp were generated in Y. pestis strains KIM5(pCD1 ؉ ) Pgm ؊ (pigmentation negative)/, KIM6(pCD1 ؊ ) Pgm ؉ , and CO92. Comparative analyses were done with the deletions and the parental wild type for bacterial adhesion to and internalization by HEp-2 cells in vitro, infectivity and maintenance in the flea vector, and lethality in murine models of systemic and pneumonic plague. Deletion of ilp had no effect on bacterial blockage of flea blood feeding or colonization. The Y. pestis KIM5 ⌬ilp strain had reduced adhesion to and internalization by HEp-2 cells compared to the parental wild-type strain (P < 0.05). T he genus Yersinia is comprised of 12 species, three of which, Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis, are pathogenic for humans and rodents (5). Y. pestis, the causative agent of plague, is typically transmitted subcutaneously to humans by the bite of an infected flea, causing either bubonic or septicemic plague, but can also be transmitted by aerosols, causing pneumonic plague (25). Pathogenesis of Y. pestis is dependent on the presence of three plasmids: pCD1 encoding a type III secretion system, pPCP1 encoding plasminogen activator (Pla), and pMT1 encoding the capsular antigen fraction 1 (Caf1) (25). Y. pestis has been considered an extracellular pathogen because it contains mutations in two major invasin and adhesin homologues (Inv and YadA) found in invasive Y. pseudotuberculosis (24, 29). However, recent studies demonstrate that Y. pestis is able to invade eukaryotic cells mediated in part by Pla, capsular F1 antigen, OmpX (Ail), and Psa fimbriae (3,18,20,21). None of these factors alone confers the full invasion phenotype.To identify additional Y. pestis surface proteins that may contribute to invasion and virulence, a comprehensive TnphoA insertion library of avirulent Y. pestis KIM6(pCD1 Ϫ ) Pgm ϩ (pigmentation positive) was generated. This avirulent strain was used because it lacks the well-characterized type III secretion system and effector Yop genes on pCD1, and therefore the phoA fusions would be biased toward uncharacterized chromosomal loci. The DNA sequence of transposon insertion junctions was determined, and insertionally inactivated gene DNA sequences were compared to bacterial DNA sequence databases. In this screen, DNA sequence analysis repeatedly identified insertions in a large gene of 9,042 bp (YPO3944) which we designated ilp (intimin/invasinlike protein), with predicted similarity to the class of Gram-negative bacterial intimin/invasin/autotransporter proteins. Because computer analysis of Ilp predicted a three-dimensional structure similar to the C-typ...