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

Abstract

ABSTRACTA comprehensive TnphoAmutant library was constructed inYersinia pestisKIM6 to identify surface proteins involved inY. pestishost 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 ofilpwere generated inY. pestisstrains 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 cellsin vitro, infectivity and maintenance in the flea vector, and lethality in murine models of systemic and pneumonic plague. Deletion ofilphad no effect on bacterial blockage of flea blood feeding or colonization. TheY. pestisKIM5 Δilpstrain had reduced adhesion to and internalization by HEp-2 cells compared to the parental wild-type strain (P< 0.05). Following intravenous challenge withY. pestisKIM5 Δilp, mice had a delayed time to death and reduced dissemination to the lungs, livers, and kidneys as monitored byin vivoimaging using aluxreporter system (in vivoimaging system [IVIS]) and bacterial counts. Intranasal challenge in mice withY. pestisCO92 Δilphad a 55-fold increase in the 50% lethal dose ([LD50] 1.64 × 104CFU) compared to the parental wild-type strain LD50(2.98 × 102CFU). These findings identified Ilp as a novel virulence factor ofY. pestis.