Elucidation of receptor binding mechanism of Enterotoxigenic Escherichia coli colonization factor CS6

先前的研究已经提出粘蛋白是ETEC定植因子CS6的潜在受体。我们对INT407和Caco-2细胞株进行了RNA测序和随后的差异基因表达分析，这两株细胞分别显示出高和低的CS6结合亲和力。根据基因表达谱和以前的发现，粘蛋白1和粘蛋白16这两个跨膜糖蛋白被选为潜在的受体候选。利用慢病毒传递的shRNA干扰，我们在INT407细胞系中创建了这些候选基因的击倒菌株。我们使用RT-qPCR和ELISA法在RNA和蛋白质水平上评估了基因敲除效率。我们分析了ETEC CS6对基因敲除细胞的黏附，并将其与对照组和非靶标基因敲除细胞进行了比较，发现黏附细菌的数量没有明显减少。此外，我们还对表达EYFP的ETEC细菌与INT407细胞的黏附进行了免疫荧光检测，以观察粘蛋白1和粘蛋白16与表达CS6的细菌的共定位。结果表明，粘蛋白1不与ETEC 4266共定位，各种抗体不能明显标记INT407细胞中的粘蛋白16。这些发现表明，跨膜蛋白粘蛋白1和粘蛋白16可能不是CS6的潜在受体。

Prior research has proposed mucin as a potential receptor for the ETEC colonization factor CS6. We performed RNA sequencing and subsequent differential gene expression analysis on INT407 and Caco-2 cell lines, which display high and low CS6 binding affinity, respectively. Based on gene expression profiles and previous findings, Mucin 1 and Mucin 16, two trans-membrane glycoproteins, were selected as potential receptor candidates. Using shRNA interference through lentiviral delivery, we created knockdown strains of these candidates in the INT407 cell line. We assessed knockdown efficiencies at both RNA and protein levels employing RT-qPCR and ELISA. We analyzed the adhesion of ETEC CS6 to the knockdown cells and compared it with control and non-target knockdown cells, finding no significant decrease in the number of adhesive bacteria. Additionally, we conducted immuno-fluorescent assays on the adhesion of EYFP-expressing ETEC bacteria to INT407 cells to observe co-localization of Mucin 1 and Mucin 16 with the CS6-expressing bacteria. The results demonstrated that Mucin 1 did not co-localize with ETEC 4266, and various antibodies were unable to distinctly label Mucin 16 in INT407 cells. These findings suggest that the transmembrane proteins Mucin 1 and Mucin 16 may not be potential receptors for CS6.