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

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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.