Role of a New Intimin/Invasin-Like Protein in Yersinia pestis Virulence

Abstract

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...

BsaB, a Novel Adherence Factor of Group B Streptococcus

“…Sal0825 harbors a bacterial im- munoglobulin-like domain (BID1) spanning aa 210 to 291. BID1 domains are found in bacterial surface proteins such as intimin-like proteins and cell adhesion molecules of Gram-negative pathogens that mediate bacterial adhesion and/or invasion into host cells (31,32). The upstream ORF, sal0824, is transcribed in the same orientation as sal0825 (Fig.…”

“…As mentioned above, Sal0825 harbors a BID1 domain at aa 210 to 291, which is also present in other adhesion proteins (31,32). To understand the role of the BID1 domain and other regions of the protein in adherence, we constructed recombinant pNZ plasmids corresponding to various regions of Sal0825 and expressed the recombinant peptides in L. lactis (Fig.…”

Physiological Levels of Glucose Induce Membrane Vesicle Secretion and Affect the Lipid and Protein Composition of Yersinia pestis Cell Surfaces

“…This protein was selected as it encompassed a major constituent of the extracellular proteins and has been shown to have very important biological significance for Y. pestis transmission and pathogenesis (25). Two Y. pestis KIM6 ϩ miniTnphoA mutants (AS-C-12 and MF-73) (38) with insertions in the murine toxin gene were grown with and without 11 mM glucose for 24 h as described above. Alkaline phosphatase was then assayed in the permeabilized cultures as described previously (39) with a small modification of 1 M Tris buffer, pH 8.5.…”

Adhesines of the Plague Agent

“…в клетках возбудителя чумы ген inv инактивирован вставкой IS200, но присутствует его гомолог [30]. экспериментами R.Seo et al [42] в клетках возбу-дителя чумы выявлен белок, принадлежащий к се-мейству интиминов и получивший обозначение Ilp (от англ. intimin/invasin-like protein) из-за исклю-чительно плотного контакта бактерий с клетками-мишенями.…”

“…клетки Y. pestis, мутантные по гену ilp, обладают сниженной способности к адгезии и проникновению в клетки HEp-2 [42]. между тем, от-сутствие белка Ilp не влияет на блокообразование и колонизацию блох X. cheopis.…”