First there is localised destruction (effacement) of the microvilli, which leads to intimate attachment of the bacterium to the host cell [20]. EPEC and EHEC encode a specific intimin receptor, translocated intimin receptor (Tir). This receptor is translocated directly into the host cells via a type III secretion system, where it becomes expressed on the cell surface [21, 22]. Intimin binds to Tir leading to its activation, which results in actin polymerisation within the host cell and the formation of a pedestal, facilitating

tighter adherence between the host cell and the bacterium [17, 23]. Other eukaryotic receptors have been suggested for intimin, including nucleolin and some β1 integrins, but as yet it is unknown if these interactions have a role in vivo [24, 25]. There is considerable sequence variation between the intimins from different E. coli strains and they have been categorised into different subtypes, each with a high affinity for its own cognate BAY 80-6946 Tir [26]. However, despite this diversity, it has been found that within the C-terminal binding domain there are four tryptophan residues and two cysteine residues, which are conserved between all subtypes [27, 28]. The two cysteines are also conserved in similar locations within the Y. pseudotuberculosis invasin. In both invasin and intimin a disulphide bond is formed, which is essential for the structure of the C-terminal binding

domain and therefore required for full functionality [29, 30]. In the instance of invasin, disruption of either cysteine results in an inability to bind to integrin, GF120918 in vitro and therefore is defective for invasion [29]. Analysis of Y. pseudotuberculosis

strain IP32953 sequence data identified a gene encoding a protein with significant amino acid similarity to invasin and intimin, which has not been previously investigated. We have termed this protein Ifp (intimin family protein) and intriguingly it has been mutated to a pseudogene in all seven Y. Casein kinase 1 pestis genomes sequenced to date. Examination of the amino acid sequence of Ifp revealed that three of the four tryptophans and both of the cysteine residues that are important in intimin function are conserved. However, no Tir orthologue can be identified in the IP32953 genome sequence. Given the amino acid similarity of Ifp to both invasin and intimin, coupled with it being putatively non-functional in Y. pestis, we postulated that Ifp may be an adhesin. We demonstrate that Ifp is a functional adhesin that binds to distinct foci on host cells. Expression occurs in late log or early stationary phase at 37°C only and coincides with a decline in the expression of invasin at this temperature. Methods Strains used and culture conditions All Y. pseudotuberculosis strains were cultured in Luria-Bertani (LB) broth Miller (BD Biosciences, Oxford, UK) or on LB agar (Novagen, Nottingham, UK) at 28°C unless otherwise click here stated. The retention of the virulence plasmid (pYV) was screened by plating Y.