Screening of gene function in cell intoxication by CNF1 links Sec61 translocon to Rac1 GTPase activity

Area of Science:

• Cell Biology
• Microbiology
• Molecular Biology

Background:

• The CNF1 toxin from pathogenic Escherichia coli (ExPEC) modifies Rho GTPases like Rac1, RhoA, and Cdc42, enhancing signal transduction but sensitizing Rac1 to degradation.
• This modification leads to Rac1 depletion, impacting cellular processes regulated by the actin cytoskeleton.

Purpose of the Study:

• To identify host factors involved in CNF1-mediated Rac1 depletion using a genome-wide siRNA screen.
• To elucidate the role of protein biosynthesis and N-glycosylation in Rac1 regulation by CNF1.

Main Methods:

• Quantitative genome-wide siRNA screen in primary human cells to identify inhibitors of CNF1-mediated Rac1 depletion.
• Functional studies involving chemical and genetic inhibition of Sec61A1 and N-glycosylation.
• Analysis of Rac1 GTP-loading and membrane association.

Main Results:

• The screen identified siRNAs targeting Sec61A1 (a translocon subunit), HACE1, and the Lu/BCAM receptor as key hits.
• Inhibition of Sec61A1 reduced Rac1 GTP-loading and membrane association in intoxicated cells.
• Inhibition of N-glycosylation in the ER abrogated Rac1 GTP-loading in CNF1-treated cells.

Conclusions:

• Rac1 signaling is controlled by protein biosynthesis and N-glycosylation occurring at the endoplasmic reticulum.
• The Sec61 translocon plays a crucial role in regulating Rac1 activity following CNF1 intoxication.
• These findings link ER protein processing pathways to the modulation of Rho GTPase signaling by bacterial toxins.