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

Area of Science:

• Microbiology
• Cell Biology
• Molecular Biology

Background:

• Subtilase cytotoxin (SubAB) is a key virulence factor from certain E. coli strains.
• SubAB enters cells by binding sialoglycans and disrupts endoplasmic reticulum function, leading to cell death.
• Critical host factors mediating SubAB toxicity remain largely uncharacterized.

Purpose of the Study:

• To conduct a genome-wide screen to identify host factors essential for SubAB-induced cell death.
• To elucidate the role of sialoglycans and other host factors in SubAB pathogenesis.
• To investigate the function of identified host factors, particularly SLC39A9, in SubAB toxicity.

Main Methods:

• Genome-wide CRISPR/Cas9 knockout screen to identify genes involved in SubAB-induced cell death.
• Analysis of glycan expression in knockout cells to determine receptor requirements.
• Functional assays to assess the role of SLC39A9, KDELRs, and JTB in SubAB toxicity.

Main Results:

• The screen identified sialoglycan-related and membrane-trafficking genes critical for SubAB activity.
• Both N-glycans and O-glycans were confirmed as SubAB receptors.
• SLC39A9, KDELRs, and JTB were found to be essential for maximal SubAB-induced cell death.
• Disruption of SLC39A9 reduced N-glycans and O-glycans, impacting core 1 synthase (C1GalT1).

Conclusions:

• Sialoglycans are crucial receptors for SubAB entry and toxicity.
• SLC39A9 plays a significant role in SubAB pathogenesis by regulating glycosyltransferase activity and glycan synthesis.
• These findings offer new insights into SubAB-host interactions and potential therapeutic targets.