(B) Attachment of E. coli XL2/pPGL1 to immobilized
SBA lectin (1) is inhibited by GalNAc at 5 mM (2). No binding of the recipient strain E. coli XL2 was detected (3). Expression of PEB3 is required for binding of C. jejuni cells to immobilised SBA lectin Previous 3-deazaneplanocin A studies suggested a possible location of PEB3 protein on a bacterial cell surface [25, 26]. The purified PEB3 protein was able to bind SBA lectin due to the presence of a GalNAc-containing glycan moiety . In order to confirm that attachment of C. jejuni cells to immobilised SBA in our experiments is mediated by PEB3, we constructed and investigated the binding properties of the respective mutant. The results demonstrated significant reduction of attachment of 11168H/peb3::kan buy EPZ5676 r , which was restored after complementation (Figure 5). Figure 5 Insertional inactivation of gene peb3 reduced the ability of strain 11168H to bind immobilised lectin. 1, recipient (11168H); 2, mutant (11168H/peb3::kan r ); 3, complementation derivative (11168H/peb3::kan r /peb3+). The results of this experiment also showed that peb3 mutation did not completely eliminate binding, suggesting that other glycoprotein(s) may be involved in specific interactions with this analogue of a host cell receptor. This hypothesis was supported by reduction of the residual binding of 11168H/peb3::kan r mutant in the presence of soluble lectin (Figure 5). One of the other cell surface-located
proteins of C. jejuni is JlpA, which was found to be an PRIMA-1MET concentration adhesin specifically binding to heat shock protein 90 . As JlpA was also
predicted to be an N-link glycosylated protein , there was a possibility that it might be responsible for residual binding of 11168H/peb3::kan r mutant. To verify this hypothesis, we constructed a jlpA mutant and tested the effect of this mutation on attachment. Surprisingly, none of the three independent clonal isolates showed any difference when compared with the control recipient strain 11168H (data not Atezolizumab clinical trial shown) suggesting the presence of other GalNAc-containing adhesins. Production of capsule has a negative effect on binding The results shown in Figure 3 also have demonstrated a significantly higher efficiency of binding of the non-capsular mutant of strain 11168H. These results, confirmed by analysis of three independent clonal isolates of this mutant (data not shown), revealed significant increase in binding upon inactivation of bacterial ability to produce capsule, suggesting an interfering effect of the later on the bacterial interaction with host cell receptors. Peb3 and capsule-related genes are differentially expressed Due to antagonistic effects of capsule and PEB3 adhesin on bacterial attachment, we hypothesized that these structures might be differentially expressed. To test this hypothesis we conducted a comparative analysis of the dynamics of kpsM and peb3 gene expression at different growth stages in a liquid culture using real time PCR (RT-PCR).