Abstract
SummarySubtilase cytotoxin (SubAB) is a virulence factor produced by locus of 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/Cas9 knockout 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-glycans but also O-glycans serve 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-glycans and core 1 O-glycans, and the O-glycan reduction was attributed to the reduction of core 1 synthase (C1GalT1). These results provide insights into the post-transcriptional regulation of glycosyltransferases by SLC39A9, as well as sialoglycan species as SubAB receptors.
Highlights
Shiga-toxigenic Escherichia coli (STEC) causes various gastrointestinal symptoms in humans, including severe bloody diarrhea, hemorrhagic colitis, and life-threatening hemolytic-uremic syndrome (HUS) (Kaper et al, 2004)
After binding to the cell surface, the toxin is retrogradely transported to the endoplasmic reticulum (ER) through the Golgi apparatus; the transport is dependent on the conserved oligomeric Golgi (COG) complex (Zolov and Lupashin, 2005; Smith et al, 2009)
We focused on genes that affected sialoglycan receptors and revealed that N-glycans and O-glycans of glycoproteins serve as SubAB receptors
Summary
Shiga-toxigenic Escherichia coli (STEC) causes various gastrointestinal symptoms in humans, including severe bloody diarrhea, hemorrhagic colitis, and life-threatening hemolytic-uremic syndrome (HUS) (Kaper et al, 2004). SubAB is lethal to mice, causing microvascular damage and HUS-like symptoms (Wang et al, 2007, 2011; Furukawa et al, 2011), indicating that the toxin increases the virulence of STEC. SubAB binds to several glycoproteins, including integrin and L1 cell adhesion molecule (L1CAM) (Yahiro et al, 2006, 2011). It is still unclear which type of glycan is used by SubAB as a functional receptor in cells and which host factors, including glycan-regulating factors, are critical for SubAB to kill cells
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