SummaryQuorum sensing (QS) in Yersinia pseudotuberculosis involves two pairs of LuxRI orthologues (YpsRI and YtbRI) and multiple N-acylhomoserine lactones (AHLs). In a ypsI/ytbI mutant, AHL synthesis was abolished, unaffected in a ypsR/ytbR double mutant and substantially reduced in a ypsI/ytbR mutant, indicating that neither YpsR nor YtbR is essential for AHL synthesis. To determine the interrelationship between YpsRI and YtbRI we constructed chromosomal luxpromoter fusions to ypsR, ypsI, ytbR and ytbI and examined their expression in each of the QS mutant backgrounds. The YpsRI system negatively autoregulates itself but positively regulates the expression of the ytbRI system whereas the ytbRI system is positively autoregulated but only at the level of ytbI expression. YtbRI does not control expression of ypsR or ypsI. This hierarchical QS system controls swimming motility via regulation of flhDC and fliA. The AHLs synthesized via YtbI play a dual role, activating flhDC, in conjunction with YpsR but repressing fliA in conjunction with YtbR and YpsR. In liquid and plate assays, the early onset of motility observed in ypsR and ypsI mutants was abolished in ytbI, ytbR ypsI/ytbI, ypsR/ytbR mutants, indicating that QS regulates motility both positively (via YtbRI) and negatively (via YpsRI).

Yersinia pseudotuberculosis forms biofilms on Caenorhabditis elegans which block nematode feeding. This genetically amenable host-pathogen model has important implications for biofilm development on living, motile surfaces. Here we show that Y. pseudotuberculosis biofilm development on C. elegans is governed by N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS) since (i) AHLs are produced in nematode associated biofilms and (ii) Y. pseudotuberculosis strains expressing an AHL-degrading enzyme or in which the AHL synthase (ypsI and ytbI) or response regulator (ypsR and ytbR) genes have been mutated, are attenuated. Although biofilm formation is also attenuated in Y. pseudotuberculosis strains carrying mutations in the QS-controlled motility regulator genes, flhDC and fliA, and the flagellin export gene, flhA, flagella are not required since fliC mutants form normal biofilms. However, in contrast to the parent and fliC mutant, Yop virulon proteins are up-regulated in flhDC, fliA and flhA mutants in a temperature and calcium independent manner. Similar observations were found for the Y. pseudotuberculosis QS mutants, indicating that the Yop virulon is repressed by QS via the master motility regulator, flhDC. By curing the pYV virulence plasmid from the ypsI/ytbI mutant, by growing YpIII under conditions permissive for type III needle formation but not Yop secretion and by mutating the type III secretion apparatus gene, yscJ, we show that biofilm formation can be restored in flhDC and ypsI/ytbI mutants. These data demonstrate that type III secretion blocks biofilm formation and is reciprocally regulated with motility via QS.

BackgroundIn order to identify new virulence determinants in Y. pseudotuberculosis a comparison between its genome and that of Yersinia pestis was undertaken. This reveals dozens of pseudogenes in Y. pestis, which are still putatively functional in Y. pseudotuberculosis and may be important in the enteric lifestyle. One such gene, YPTB1572 in the Y. pseudotuberculosis IP32953 genome sequence, encodes a protein with similarity to invasin, a classic adhesion/invasion protein, and to intimin, the attaching and effacing protein from enteropathogenic (EPEC) and enterohaemorraghic (EHEC) Escherichia coli.ResultsWe termed YPTB1572 Ifp (Intimin family protein) and show that it is able to bind directly to human HEp-2 epithelial cells. Cysteine and tryptophan residues in the C-terminal region of intimin that are essential for function in EPEC and EHEC are conserved in Ifp. Protein binding occurred at distinct foci on the HEp-2 cell surface and can be disrupted by mutation of a single cysteine residue at the C-terminus of the protein. Temporal expression analysis using lux reporter constructs revealed that ifp is expressed at late log phase at 37°C in contrast to invasin, suggesting that Ifp is a late stage adhesin. An ifp defined mutant showed a reduction in adhesion to HEp-2 cells and was attenuated in the Galleria mellonella infection model.ConclusionA new Y. pseudotuberculosis adhesin has been identified and characterised. This Ifp is a new member in the family of invasin/intimin outer membrane adhesins.

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