Vibrio cholerae toxin co-regulated pilus provokes inflammatory responses in co-culture model of Caco-2 and Peripheral Blood Mononuclear Cells (PBMC) leading to increased colonization.

The aim of this study was to assess the modulatory effect of TcpA in the expression of CEACAM1 adhesin molecule and IL-1, IL-8 and TNFα pro-inflammatiory cytokines in the co-culture model of Caco-2/PBMC that can mimic the intestinal milieu. The tcpA gene from V.cholerae ATCC14035 was cloned in pET-28a and transformed into E.coli Bl-21. The recombinant TcpA-His6 protein was expressed and purified using the Ni-column chromatography. The sequencing of transformed plasmid and Western blotting of purified protein confirmed the identity of rTcp. Cytotoxicity of different concentrations of recombinant protein for human colon carcinoma cell line (Caco-2 cell) was assessed by MTT assay and showed viability of 92%, 82% and 70 %, for10 µg/mL of TcpA after 24, 48 and 72 h, respectively. Co-cultures of Caco-2 and peripheral blood mononuclear cells (PBMCs) were used to mimic the intestinal milieu and treated with different concentrations of rTcpA (1, 5, 10 and 50 µg/mL). Our data showed about 2.04, 3.37, 3.68 and 42.7-folds increase in CEACAM1 gene expression, respectively, compared with non-treated Caco-2/PBMC co-culture. Moreover, the expression of IL-1, IL-8 and TNFα genes was significantly increased up to 15.75, 7.04 and 80.95 folds, respectively. In conclusion, V.cholerae TcpA induces statistically significant dose-dependent stimulatory effect on TNFα, IL-1 and IL-8 pro-inflammatory cytokines expression. Among all, TNFα was much more affected which consequently, elevated CEACAM1 expression level in IECs. This suggests TcpA protein as critical effector as an inducer of increased adhesion potential of V. cholera as well as inflammatory responses of host intestinal tissue. This article is protected by copyright. All rights reserved.