lactis ssp. cremoris SMBI198, a strain derived from NZ9000, knocked out in the chromosomal htrA gene (Poquet et al., 2000; Rigoulay et al., 2004). The resulting strain produced only a surface-associated see more form of the recombinant flagellin (Fig. 1b). Interestingly, two bands showed homology with B. cereus flagellin, one of around 45 and the other one of around 63 kDa. It is known that, in certain cases, protein aggregates are difficult to disassociate, producing this kind of artefact
in SDS-PAGE (Kankainen et al., 2009). This tendency to aggregation may lead to bacterial autoaggregation, and the physical–chemical dynamics of this process are currently under investigation. This could reflect the trend to auto-assembly that flagellins display in vivo (Hueck, 1998). In addition, it reinforces the role of HtrA as the major housekeeping protease on the L. lactis surface as, in the absence of it, the aggregated flagellin cannot be proteolyzed and thus shed into the bacterial surroundings. Lactococcuslactis ssp. cremoris CH showed a better ability to adhere to mucin when flagellin production was induced with nisin (Fig. 2). Adhesion of both
L. lactis ssp. cremoris SMBI198 and L. lactis ssp. cremoris SMBI198 (pNZ8110) strains was similar to uninduced L. lactis ssp. cremoris CH cultures (data not shown). After gene induction, the adhesion was increased by a factor CHIR-99021 manufacturer of 4.7. Nisin-induced L. lactis CH cultures inhibited the adhesion to mucin of the two enteropathogens used in this study in a dose-dependent manner (Fig. 2). A lower inhibition was also observed when uninduced L. lactis ssp. cremoris
CH cultures were used. This is not surprising as L. lactis is also able to bind to mucin; an interference with enteropathogen adhesion to mucin is thus expected. The adhesion data, corrected by the inhibitory effect observed for the uninduced L. lactis CH strain cultures, showed that L. lactis ssp. cremoris CH expressing the Bacillus flagellin was able to inhibit the adhesion of E. coli 4.4 times (1.8 times in the case of uninduced cultures), and 3.9 times in the case of S. enterica (1.6 times in the case of uninduced cultures), when the E. coli/L. lactis ratio was 1 : 10. In our previous work, we showed that adhesion of B. cereus CH to mucin could be explained, Dichloromethane dehalogenase to a large extent, by the presence of a flagellin on its surface (Sánchez et al., 2009a). Flagella have been proposed as important factors for bacterial adhesion to mucosal surfaces (Rumbo et al., 2006), and are glycosylated in several microorganisms (Ewing et al., 2009; Hayakawa et al., 2009; Konishi et al., 2009; Logan et al., 2009). One B. cereus strain lacking the flhA gene, which results in the absence of flagella, presented a lower adhesion to both Caco-2 and HeLa cell lines (Ramarao & Lereclus, 2006). In addition, monomeric flagellins detached from the cell surface have been proposed as the soluble probiotic factor secreted by the strain E. coli Nissle 1917.