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

产肠毒素大肠杆菌定植因子CS6受体结合机制的阐明

基本信息

批准号：
22K15466
负责人：
アイビエケアラファテ
金额：
$ 3万
依托单位：
National Center for Global Health and Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Early-Career Scientists
财政年份：
2022
资助国家：
日本
起止时间：
2022-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-22K15466/
关键词：
Enterotoxigenic E. coli Colonization factor CS6 Receptor Cell receptor

项目摘要

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.

先前的研究已提出粘蛋白是 ETEC 定植因子 CS6 的潜在受体。我们对 INT407 和 Caco-2 细胞系进行了 RNA 测序和随后的差异基因表达分析，这两个细胞系分别表现出高和低的 CS6 结合亲和力。根据基因表达谱和之前的发现，两种跨膜糖蛋白 Mucin 1 和 Mucin 16 被选为潜在的候选受体。通过慢病毒传递使用 shRNA 干扰，我们在 INT407 细胞系中创建了这些候选者的敲低菌株。我们使用 RT-qPCR 和 ELISA 评估了 RNA 和蛋白质水平的敲低效率。我们分析了 ETEC CS6 对敲低细胞的粘附，并将其与对照和非靶敲低细胞进行比较，发现粘附细菌的数量没有显着减少。此外，我们对表达 EYFP 的 ETEC 细菌与 INT407 细胞的粘附进行了免疫荧光测定，以观察 Mucin 1 和 Mucin 16 与表达 CS6 的细菌的共定位。结果表明，Mucin 1 不与 ETEC 4266 共定位，并且各种抗体无法在 INT407 细胞中明显标记 Mucin 16。这些发现表明跨膜蛋白 Mucin 1 和 Mucin 16 可能不是 CS6 的潜在受体。