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Modulatory effect of Vibrio cholerae toxin co-regulated pilus on mucins, toll-like receptors and NOD genes expression in co-culture model of Caco-2 and peripheral blood mononuclear cells (PBMC)
Microbial Pathogenesis ( IF 3.3 ) Pub Date : 2020-10-12 , DOI: 10.1016/j.micpath.2020.104566
Maryam Ghasemi 1 , Bita Bakhshi 1 , Reza Khashei 2 , Sara Soudi 3
Affiliation  

Vibrio cholerae, the causative agent of cholera, tend to colonize the small intestine as a Gram-negative pathogen. The intestinal mucus layer forms mucin physical barrier, consisted of high molecular weight proteins. Regarding the role of toxin–coregulated pilus (TCP) as one of the most important colonization factors of V. cholerae, this experimental study was designed to determine the role of TcpA in induction of mucin production and its regulatory effect on innate immunity molecules including toll like receptors (TLRs) and Nucleotide-binding oligomerization domain-containing proteins (NODs) using Caco2- PBMC co-cultures as an interactive model. The rTcpA protein of V. cholerae was expressed in BL21 Escherichia coli, purified using Ni-column chromatography and confirmed by western blotting. Nontoxic doses of rTcpA was determined on Caco-2 cell lines and different concentrations of rTcpA (1, 5, 10 and 50 μg/mL) showed a statistically significant effect on the expression of muc genes (MUC3 and MUC4) in a dose-dependent manner. This finding is supposed to facilitate physical adhesion and colonization of V. cholerae in intestinal lumen. The rTcpA moderately stimulated the expression of tlr4 and overexpressed tlr1, both of which are supposed to induce a mucosal protective response against bacterial infection. NOD2 was significantly increased which suggests that it may contribute in pro-inflammatory responses observed in cholera disease. No change in NOD1 expression was seen which might be attributed to the non-invasive nature of V. cholerae as an intestinal pathogen. In conclusion, the rTcpA protein of V. cholerae showed a statistically significant modulatory effect on the human gut epithelium gene expression which would help promising protection in prophylaxis applications.



中文翻译:

的调节作用霍乱弧菌对粘蛋白毒素共调节菌毛,toll样受体和NOD基因的Caco-2和外周血单核细胞共培养模型表达式(PBMC)

霍乱弧菌是霍乱的病原体,它容易在小肠内定殖,成为革兰氏阴性菌。肠粘液层形成粘蛋白物理屏障,由高分子量蛋白质组成。关于毒素核心菌毛(TCP)作为霍乱弧菌最重要的定居因子之一的作用,本实验研究旨在确定TcpA在诱导粘蛋白产生中的作用及其对先天免疫分子(包括通行费)的调节作用使用Caco2-PBMC共培养作为交互模型,例如受体(TLR)和含有核苷酸结合的寡聚化域的蛋白质(NOD)。霍乱弧菌的rTcpA蛋白在BL21大肠杆菌中表达,使用镍柱色谱法纯化,并通过蛋白质印迹证实。在Caco-2细胞系上确定了rTcpA的无毒剂量,不同浓度的rTcpA(1、5、10和50μg/ mL)对muc基因(MUC3和MUC4)的表达具有统计学意义的剂量依赖性。方式。该发现被认为有助于霍乱弧菌的物理粘附和定植在肠腔中。rTcpA适度刺激了tlr4的表达和tlr1的过表达,两者均被认为诱导了针对细菌感染的粘膜保护反应。NOD2显着增加,表明它可能参与了霍乱疾病中观察到的促炎反应。NOD1表达未见变化,这可能归因于霍乱弧菌作为肠道病原体的非侵入性。总之,V的rTcpA蛋白。霍乱弧菌对人肠上皮基因的表达具有统计学上的显着调节作用,有助于预防应用中的保护作用。

更新日期:2020-10-16
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