It is likely that other OmpR-dependent adherence factors are missing in the ompR mutant. It has previously been shown that cells of the ompR mutant AR4 lack YompF and YompC porins in the outer membrane (Brzostek et al., 2007), and the latter protein may play a role in microbial attachment to eukaryotic cells (Brzostek & Raczkowska, 2007). These observations suggest that YompC might partially mediate the adhesion of Y. enterocolitica to HEp-2 cells. The results of invasion assays performed without the centrifugation step demonstrated that the ability of ompR, flhDC and inv mutants to invade HEp-2 cells was decreased to different extents (Fig. 3b).

However, the invasiveness of the ompR mutant was higher than that of the flhDC mutant. When Y. enterocolitica cells were centrifuged onto the monolayer to ensure bacterial PI3K inhibitor contact with the host cells, the invasiveness of all applied mutants increased, but that of the ompR strain AR4, unlike the flhDC and inv mutants, actually exceeded the wild-type level (Fig. 3c). This suggests that upregulation

of invasin expression was responsible for the higher level of invasiveness of the ompR strain, although motility appeared to play a crucial role in the overall invasion of HEp-2 cells by the Y. enterocolitica strains. This is consistent with the results of previous studies, which showed that motility of Y. enterocolitica is required to initiate host cell invasion (Young et al., 2000). Complementation of the AR4 ompR LY2109761 manufacturer mutation with the coding sequence of ompR cloned in vector pBBR1 MCS-3 (plasmid pBR3) restored the wild-type outer membrane porin profiles and inv expression (Brzostek et al., 2007). When tested for its ability to invade HEp-2 cells, the strain AR4/pBR3 exhibited increased invasion compared with the noncomplemented ompR mutant

(Fig. 3c). This was probably the effect of overproduction of OmpR and the increased expression of other OmpR-dependent adhesion–invasion factors. Forced contact between bacteria and host cells through centrifugation was found not to fully restore the adhesion–invasion defect of the nonmotile flhDC mutant DN1, which suggests the involvement of the FlhDC flagellar regulator in the modulation of virulence–invasion determinants other than flagella. Recently, it has 4��8C been shown that FlhDC, besides its regulatory function in motility, may also act as a global regulator of Y. enterocolitica metabolism (Kapatral et al., 2004) and promote the secretion of virulence factors via the flagellar export apparatus (Young et al., 1999). Thus, apart from its effect on motility, the modulation of flhDC expression by OmpR is likely to have considerable implications for Y. enterocolitica physiology, including its adherent–invasive abilities. Biofilm formation is a feature of enteropathogenic yersiniae that is likely to play a role in pathogenesis. As reported previously for Yersinia species, several genes are potentially involved in biofilm formation (Hinnebusch, 2008; Kim et al.