Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation

Abstract

Glycosylation is a fundamental modification of proteins and membrane lipids. Toxins that utilize glycans as their receptors have served as powerful tools to identify key players in glycosylation processes. Here, we carried out Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9–mediated genome-wide loss-of-function screens using two related bacterial toxins, Shiga-like toxins (Stxs) 1 and 2, which use a specific glycolipid, globotriaosylceramide (Gb3), as receptors, and the plant toxin ricin, which recognizes a broad range of glycans. The Stxs screens identified major glycosyltransferases (GTs) and transporters involved in Gb3 biosynthesis, while the ricin screen identified GTs and transporters involved in N-linked protein glycosylation and fucosylation. The screens also identified lysosomal-associated protein transmembrane 4 alpha (LAPTM4A), a poorly characterized four-pass membrane protein, as a factor specifically required for Stxs. Mass spectrometry analysis of glycolipids and their precursors demonstrates that LAPTM4A knockout (KO) cells lack Gb3 biosynthesis. This requirement of LAPTM4A for Gb3 synthesis is not shared by its homolog lysosomal-associated protein transmembrane 4 beta (LAPTM4B), and switching the domains between them determined that the second luminal domain of LAPTM4A is required, potentially acting as a specific “activator” for the GT that synthesizes Gb3. These screens also revealed two Golgi proteins, Transmembrane protein 165 (TMEM165) and Transmembrane 9 superfamily member 2 (TM9SF2), as shared factors required for both Stxs and ricin. TMEM165 KO and TM9SF2 KO cells both showed a reduction in not only Gb3 but also other glycosphingolipids, suggesting that they are required for maintaining proper levels of glycosylation in general in the Golgi. In addition, TM9SF2 KO cells also showed defective endosomal trafficking. These studies reveal key Golgi proteins critical for regulating glycosylation and glycolipid synthesis and provide novel therapeutic targets for blocking Stxs and ricin toxicity.

A genome-wide CRISPR/Cas9 screen reveals that the aryl hydrocarbon receptor stimulates sphingolipid levels

“…1B). LAPTM4A and TM9SF2, which have been correlated with Shiga toxin resistance (25)(26)(27), were also found to be highly enriched and among the 20 top-ranking genes in our genetic screen. Surprisingly, AHR, a transcription factor with broad physiological functions (14)(15)(16)(17)(18)(19), also showed high enrichment comparable with that observed for the SL biosynthetic genes (Fig.…”

“…Because the level of cell-surface Gb3 on HeLa cells correlates with their sensitivity to the toxin (23,24), we reasoned that selection of Shiga toxin-resistant HeLa cells after transduction of a genome-scale knockout (KO) library could yield not only the biosynthetic genes required for Gb3 synthesis as has been previously shown (25)(26)(27), but also identify novel positive regulatory genes that control membrane SL levels.…”

Cross-talks of glycosylphosphatidylinositol biosynthesis with glycosphingolipid biosynthesis and ER-associated degradation

“…GS28 and STX5 are components of the Golgi target-membrane-bound (t)-SNARE-complex 40 , and UNC50 is a factor involved in intra-cellular trafficking 41, 42 . These genes were previously found in several CRISPR screens for host factors required for actions of ricin 43 , Shiga toxin 44 , and for regulators of the cell surface localization of galectin-3 45 . Their defects induced mildly positive T5 mAb staining, decreased CTxB staining, and slightly increased levels of staining by GS-II lectin.…”

Mammalian membrane trafficking as seen through the lens of bacterial toxins

“…What is the relevance of multiple phenotypes targeted by pore-forming toxins, namely, how do pore-forming toxins lead to the fission or vacuolation of the ER (Brito, Cabanes, Sarmento Mesquita, & Sousa, 2019;Gonzalez et al, 2018;Mesquita et al, 2017)? Or how further can we exploit toxins to screen for regulators of organelle function and biosynthetic pathways (Tian et al, 2018)? Combined with model organisms, tailored pharmacological treatments, and emerging fine-tuned genetic manipulations, bacterial toxins will continue to reveal clever strategies that help us to dissect and identify fundamental, and exceedingly precise, properties of membrane trafficking.…”