The S. Typhi effector StoD is an E3/E4 ubiquitin ligase which binds K48- and K63-linked diubiquitin

Abstract

Salmonella enterica (e.g., serovars Typhi and Typhimurium) relies on translocation of effectors via type III secretion systems (T3SS). Specialization of typhoidal serovars is thought to be mediated via pseudogenesis. Here, we show that the Salmonella Typhi STY1076/t1865 protein, named StoD, a homologue of the enteropathogenic Escherichia coli/enterohemorrhagic E. coli/Citrobacter rodentium NleG, is a T3SS effector. The StoD C terminus (StoD-C) is a U-box E3 ubiquitin ligase, capable of autoubiquitination in the presence of multiple E2s. The crystal structure of the StoD N terminus (StoD-N) at 2.5 Å resolution revealed a ubiquitin-like fold. In HeLa cells expressing StoD, ubiquitin is redistributed into puncta that colocalize with StoD. Binding assays showed that StoD-N and StoD-C bind the same exposed surface of the β-sheet of ubiquitin, suggesting that StoD could simultaneously interact with two ubiquitin molecules. Consistently, StoD interacted with both K63- (KD = 5.6 ± 1 μM) and K48-linked diubiquitin (KD = 15 ± 4 μM). Accordingly, we report the first S. Typhi–specific T3SS effector. We suggest that StoD recognizes and ubiquitinates pre-ubiquitinated targets, thus subverting intracellular signaling by functioning as an E4 enzyme.

Detection of Salmonella Typhi in Bile by Quantitative Real-Time PCR

“…The stoD gene (STY1076) has not previously been targeted for a diagnostic. StoD ( S almonella T yphi o uter protein D ) is a Salmonella Pathogenicity Island 1 (SPI-1) type III secretion system effector containing an active C-terminal RING-type E3 ubiquitin-ligase domain ( 15 ). The stoD locus is highly conserved among S. Typhi strains.…”

Single cell analyses reveal distinct adaptation of typhoidal and non-typhoidal Salmonella enterica serovars to intracellular lifestyle

“…Additional effector proteins that are specific to STY and SPA may be present and mediate serovar-specific properties. One example is StoD, an E3/E4 ubiquitin ligase that is translocated by the SPI1-T3SS [ 42 ]. A systematic screen by bioinformatics tools and experimental validation may identify TS-specific effector proteins of SPI2-T3SS.…”

Structural biology of the invasion arsenal of Gram‐negative bacterial pathogens

“…Several effector E3 ligases contain U‐box‐like domains with fold similar to their eukaryotic counterparts. These include the C‐terminal domain of E. coli NleG [84], Salmonella StoD [85] as well as Legionella RavN [86] and LubX [87] (Fig. 3A) E. coli contains several NleG paralogs which ubiquitinate different host proteins.…”

“…3A). The C‐terminal domain is predicted to have a U‐box fold and was shown to recognize and ubiquitinate pre‐ubiquitinated host proteins, recognizing K48‐ and K63‐linkages and extending the ubiquitin chain [85]. LubX contains two U‐box domains and the structure of LubX in complex with the E2 ubiquitin‐conjugating enzyme UbcH5 (aka Ube2D2; PDB ID https://doi.org/10.2210/pdb4WZ3/pdb; Fig.…”