Abstract
Shiga toxin (STx) or Vero toxin is a virulence factor produced by enterohemorrhagic Escherichia coli. The toxin binds to the glycosphingolipid globotriaosylceramide (Gb3) for its entry, and causes cell death by inhibiting ribosome function. Previously, we performed a loss-of-function screen in HeLa cells using a human CRISPR knockout (KO) library and identified various host genes required for STx-induced cell death. To determine whether this library targeted to the human genome is applicable to non-human primate cells and to identify previously unrecognized factors crucial for STx-induced cell death, we herein performed a similar screen in the African green monkey kidney-derived Vero C1008 subline. Many genes relevant to metabolic enzymes and membrane trafficking were enriched, although the number of enriched genes was less than that obtained in the screening for HeLa cells. Of note, several genes that had not been enriched in the previous screening were enriched: one of these genes was SYS1, which encodes a multi-spanning membrane protein in the Golgi apparatus. In SYS1 KO Vero cells, expression of Gb3 and sphingomyelin was decreased, while that of glucosylceramide and lactosylceramide was increased. In addition, loss of SYS1 inhibited the biosynthesis of protein glycans, deformed the Golgi apparatus, and perturbed the localization of trans-Golgi network protein (TGN) 46. These results indicate that the human CRISPR KO library is applicable to Vero cell lines, and SYS1 has a widespread effect on glycan biosynthesis via regulation of intra-Golgi and endosome–TGN retrograde transports.
Highlights
Introduction ofSYS1 cDNA into SYS1-KO cells restored the GSL pattern including the amount of Gb3
We used a subline of Vero cells, Vero C1008, as the parent cells to determine whether the human lentivirus-based GeCKO v2 pooled library [32] could be applied to monkey-derived cells
Two independent pools of singleguide RNA (sgRNA)-expressed Vero C1008 cells were prepared by transducing with lentiviral libraries targeting 19,050 genes, and the cells were treated with Shiga toxin 1 (STx1)
Summary
Shiga toxin (STx) is a key virulence factor produced by enterohemorrhagic Escherichia coli (EHEC) as well as Shigella dysenteriae serotype 1 [1,2]. We and other groups have employed CRISPR genome-wide knockout (KO) screening to identify host factors required for STx-induced cell death [24,25,26] In these screens, many membrane trafficking genes were enriched including COG complex subunits and GARP complex subunits involved in endosomes and the TGN/Golgi apparatus, which are likely involved in both GSL glycosylation and STx retrograde transport [24,27]. Many membrane trafficking genes were enriched including COG complex subunits and GARP complex subunits involved in endosomes and the TGN/Golgi apparatus, which are likely involved in both GSL glycosylation and STx retrograde transport [24,27] These screens and another screen related to EHEC infection demonstrated that LAPTM4A and TM9SF2 are critical factors for the biosynthesis of Gb3 [24,25,27]. We performed STx-resistance screening in Vero C1008 cells using the GeCKO v2 sgRNA library [32] and showed that SYS1, a Golgi trafficking protein, is crucial for the production of the STx receptor Gb3
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