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

## TL;DR

The study shows how a bacterial toxin affects cell signaling by linking protein production in the endoplasmic reticulum to the activity of a key cell protein called Rac1.

## Contribution

The study reveals a novel connection between the Sec61 translocon and Rac1 GTPase activity through a genome-wide siRNA screen in CNF1-intoxicated cells.

## Key findings

- siRNAs targeting Sec61A1, HACE1, and Lu/BCAM significantly inhibit CNF1-mediated depletion of Rac1.
- Inhibition of Sec61A1 reduces GTP-loading and membrane association of Rac1 in intoxicated cells.
- Blocking N-glycosylation in the ER prevents Rac1 GTP-loading in CNF1-treated cells.

## Abstract

The CNF1 toxin from extraintestinal pathogenic Escherichia coli (ExPEC) deamidates glutamine 61 of Rac1 small GTPase, as well as its equivalents in RhoA and Cdc42 into glutamic acid. This post-translational modification of Rho proteins abrogates the hydrolysis of GTP into GDP, thereby enhancing signal transduction. Meanwhile, the sustained GTP-loading of Rac1 Q61E sensitizes it to ubiquitin-mediated proteasomal degradation catalyzed by the HACE1 E3 ligase rate-limiting factor, leading to a cellular depletion of Rac1 over time. We report data from a quantitative genome-wide screen of siRNAs inhibiting CNF1-mediated cellular depletion of Rac1 in primary human cells. As best hits, we identified a group of three siRNAs targeting the Sec61A1 subunit of the Sec61 translocon, as well as HACE1 and the Lu/BCAM host cell receptor of CNF1. We extend these findings by identifying a group of siRNAs targeting genes involved in ER and Golgi homeostasis and trafficking. Functional studies showed that both chemical and genetic inhibition of Sec61A1 dampens GTP-loading and membrane association of Rac1 in CNF1-intoxicated cells, while the proper deamidation of RhoA provides a control of CNF1 cytosolic action. Finally, we extend these findings by showing that inhibition of N-glycosylation of neo-synthesized proteins in the ER abrogates Rac1 GTP-loading in CNF1-treated cells. Collectively, these data point to a control of Rac1 signaling operated by protein biosynthesis and N-glycosylation in the ER.

The remarkable evolutionary convergence of bacterial effectors from pathogens toward the host small GTPase Rac1, the master regulator of the actin cytoskeleton, confers to these microbes an enhanced capacity to invade host cells and tissues. The CNF1 toxin, a colonization factor of the gastrointestinal tract produced by pathogenic strains of Escherichia coli, has been instrumental in deciphering the regulation and function of Rac1. By performing a whole-genome screen based on CNF1 action, we establish the key requirement of Sec61 translocon-dependent protein biosynthesis and N-glycosylation at the endoplasmic reticulum for proper activation of Rac1 in intoxicated cells. Our data connect the Sec61 translocon and N-glycosylation of neo-synthesized proteins at the endoplasmic reticulum in the control of the activity of Rac1 and other Rho GTPases.