A CRISPR Screen Using Subtilase Cytotoxin Identifies SLC39A9 as a Glycan-Regulating Factor

Summary Subtilasecytotoxin (SubAB) is a virulence factor produced by locusof enterocyte effacement-negative Shiga-toxigenic Escherichia coli strains. The toxin recognizes sialoglycans for entry and cleaves an endoplasmic reticulum chaperon, binding immunoglobulin protein, to cause cell death. However, no systematic screening has yet been performed to identify critical host factors. Here, we performed a genome-wide CRISPR/ Cas9knockout screen for SubAB-induced cell death and identified various sialoglycan-related and membrane-trafficking genes. Analysis of glycan-deficient cells demonstrated that not only N- glycansbut also O- glycansserve as SubAB receptors. In addition, SLC39A9, which is a predicted zinc transporter, as well as KDELRs and JTB, were required for SubAB to induce maximal cell death. Disruption of the SLC39A9 gene markedly reduced both complex-type N- glycansand core 1 O- glycans, and the O- glycanreduction was attributed to the reduction of core 1 synthase ( C1GalT1). These results provide insights into the post-transcriptional regulationof glycosyltransferasesby SLC39A9, as well as sialoglycan species as SubAB receptors.