Salmonella Type III Secretion Effector SlrP Is an E3 Ubiquitin Ligase for Mammalian Thioredoxin

Abstract

Salmonella enterica encodes two virulence-related type III secretion systems in Salmonella pathogenicity islands 1 and 2, respectively. These systems mediate the translocation of protein effectors into the eukaryotic host cell, where they alter cell signaling and manipulate host cell functions. However, the precise role of most effectors remains unknown. Using a genetic screen, we identified the small, reduction/oxidation-regulatory protein thioredoxin as a mammalian binding partner of the Salmonella effector SlrP. The interaction was confirmed by affinity chromatography and coimmunoprecipitation. In vitro, SlrP was able to mediate ubiquitination of ubiquitin and thioredoxin. A Cys residue conserved in other effectors of the same family that also possess E3 ubiquitin ligase activity was essential for this catalytic function. Stable expression of SlrP in HeLa cells resulted in a significant decrease of thioredoxin activity and in an increase of cell death. The physiological significance of these results was strengthened by the finding that Salmonella was able to trigger cell death and inhibit thioredoxin activity in HeLa cells several hours post-infection. This study assigns a functional role to the Salmonella effector SlrP as a binding partner and an E3 ubiquitin ligase for mammalian thioredoxin.

The structure of the Slrp–Trx1 complex sheds light on the autoinhibition mechanism of the type III secretion system effectors of the NEL family

“…The same production and purification protocols were used for all proteins, except that 5mM β-mercapto-ethanol was added in all Trx buffers to stabilize the reduced form of the protein that has been shown to interact with SlrP [24]. Bacteria were grown in LB medium containing 100 µg/ml of ampicillin and 50µg/ml of kanamycin at 37°C.…”

“…Derivatives of pGEX-4T-1 and pGEX-4T-3 (GE Healthcare) were used for production of GST fusion proteins. To generate point mutations in slrP and trx1, the previously described plasmids [24] pIZ1635 (pGBKT7-SlrP), pIZ1710 (pGADT7-Trx1) and pIZ1712 (pGEX-4T-1-Trx1) were used as templates. Amplification reactions were carried out in a T100 thermal cycler (BioRad) using KAPA HiFi DNA polymerase (Kapa Biosystems) and the primers listed in Table S1.…”

“…In a previous study [24] we showed that one of the members of this LRR-NEL family of T3SS effectors, the Salmonella secreted protein SlrP, specifically recognizes the reduced form of human thioredoxin-1 (Trx1). Ubiquitination of this essential redox protein resulted in an increased susceptibility of the host cells to death.…”

Global impact of Salmonella type III secretion effector SteA on host cells

“…93021013) were cultured, transfected, and lysed as previously described [20]. For stable transfection, HeLa cells were electroporated with pBABEpuro or its derivative and selection was started 24 h after electroporation in medium containing 1 µg/ml puromycin (InvivoGen).…”

“…Cell death was measured using a previously described protocol based on lactate dehydrogenase (LDH) release in the cultures [20]. Cell adhesion was determined as previously described [23].…”