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 TNF-α pro-inflammatory cytokines in the Coculture model of Caco-2/PBMC (peripheral blood mononuclear cell) that can mimic the intestinal milieu. The TcpA gene from Vibrio cholerae ATCC14035 was cloned in pET-28a and transformed into Escherichia coli Bl-21. The recombinant TcpA-His6 protein was expressed and purified using Ni-column chromatography. The sequencing of transformed plasmid and Western blot analysis of purified protein confirmed the identity of rTcp. The cytotoxicity of different concentrations of recombinant protein for human colon carcinoma cell line (human colorectal adenocarcinoma cell [Caco-2 cell]) was assessed by MTT assay and showed viability of 92%, 82%, and 70%, for 10 µg/mL of TcpA after 24, 48, and 72 h, respectively. Co-cultures of Caco-2 and 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-fold increase in CEACAM1 gene expression, respectively, compared with the nontreated Caco-2/PBMC Coculture. Moreover, the expression of IL-1, IL-8, and TNF-α 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 TNF-α, IL-,1, and IL-8 pro-inflammatory cytokines expression. Of these, TNF-α was much more affected which, consequently, elevated the CEACAM1 expression level in IECs. This suggests that TcpA protein is a critical effector as an inducer of increased adhesion potential of V. cholera as well as inflammatory responses of host intestinal tissue.

中文翻译：

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霍乱弧菌毒素共调节菌毛在 Caco-2 和外周血单核细胞 (PBMC) 共培养模型中引发炎症反应，导致定植增加

本研究的目的是评估在表达TCPA的调节作用CEACAM1粘附分子和IL-1，IL-8 ，和TNF-α在的Caco-2 / PBMC的共培养模型中的促炎细胞因子（外周血单核细胞），可以模拟肠道环境。将霍乱弧菌ATCC14035的TcpA基因克隆到pET-28a并转化大肠杆菌BL-21。使用镍柱层析表达和纯化重组的 TcpA-His6 蛋白。转化质粒的测序和纯化蛋白的蛋白质印迹分析证实了 rTcp 的身份。不同浓度的重组蛋白对人结肠癌细胞系（人结肠直肠腺癌细胞 [Caco-2 细胞]）的细胞毒性通过 MTT 测定进行评估，并显示 92%、82% 和 70% 的活力，对于 10 µg/mL分别在 24、48 和 72 小时后的 TcpA。Caco-2 和 PBMC 的共培养物用于模拟肠道环境，并用不同浓度的 rTcpA（1、5、10 和 50 µg/mL）处理。我们的数据显示CEACAM1增加了约 2.04、3.37、3.68 和 42.7 倍分别与未处理的 Caco-2/PBMC 共培养进行比较。此外，IL-1、IL-8和TNF-α基因的表达分别显着增加了 15.75、7.04 和 80.95 倍。总之，霍乱弧菌TcpA 对TNF-α、IL-,1和IL-8促炎细胞因子表达诱导具有统计学意义的剂量依赖性刺激作用。其中，TNF-α受到的影响更大，从而提高了 IEC 中 CEACAM1 的表达水平。这表明 TcpA 蛋白是霍乱弧菌粘附潜能增加的诱导剂的关键效应物 以及宿主肠道组织的炎症反应。