Speaking the host language: how Salmonella effector proteins manipulate the host

Abstract

Salmonella injects over 40 virulence factors, termed effectors, into host cells to subvert diverse host cellular processes. Of these 40 Salmonella effectors, at least 25 have been described as mediating eukaryotic-like, biochemical post-translational modifications (PTMs) of host proteins, altering the outcome of infection. The downstream changes mediated by an effector's enzymatic activity range from highly specific to multifunctional, and altogether their combined action impacts the function of an impressive array of host cellular processes, including signal transduction, membrane trafficking, and both innate and adaptive immune responses. Salmonella and related Gram-negative pathogens have been a rich resource for the discovery of unique enzymatic activities, expanding our understanding of host signalling networks, bacterial pathogenesis as well as basic biochemistry. In this review, we provide an up-to-date assessment of host manipulation mediated by the Salmonella type III secretion system injectosome, exploring the cellular effects of diverse effector activities with a particular focus on PTMs and the implications for infection outcomes. We also highlight activities and functions of numerous effectors that remain poorly characterized.

Keywords: Salmonella pathogenesis; biochemical mechanism; intracellular pathogen; post-translational modification; type III effector protein.

Fig. 1.

Summary of modifications mediated by Salmonella effectors. Salmonella effectors enact a range of host modifications, either enzymatically or through adaptor activity, to manipulate host cell processes, shown here in summary form. Effector proteins are categorized by colour corresponding to sections of the review, and demonstrated with their PDB structure in cartoon form (outside the circle) and surface form (in circle) using Pymol. Protein structures used here and in other figures were as follows: SptP (PDB: 1G4W, phosphatase and GAP domains used in corresponding parts of this figure, and phosphatase domain used in Fig. 2), SpvC (2P1W, here and Fig. 2), SopA (2QYU here and Fig. 3), SlrP (4PUF, Fig. 3), SspH2 (3G06, Fig. 3), SseL (5HAF, here and Fig. 3), SseK1 (5H60 here and Fig. 4), SseK2 (5HAF, Fig. 4), SseK3 (6EYR, Fig. 4), SpvB (2GWJ), AvrA (6BE0), GtgE (5KDG, here and Fig. 5), GtgA (6GGR) and SopE (1GZS). Where no relevant PDB structure is available, a generic protein symbol is used. A ‘?’ before the effector name refers to cases where the evidence of enzymatic activity is equivocal. Host substrates or co-factors are represented in grey. Symbols are either circular with atomic symbols (carbon [C], magnesium [Mg], nitrogen [N], oxygen [O] and zinc [Zn], with X referring to a variable atomic group), square signifying an amino acid by three-letter code (arginine [Arg]), skeleton chemical structures (UDP-GlcNAc in GlcNAcylation, glycerophospholipids in acylation, inositol hexakisphosphate in acetylation) and rectangles (ATP, ADP, GDP and GTP). The cartoon protein ‘Ub’ refers to ubiquitin. For a complete list of the Salmonella effector proteins see Table 1. Image created with BioRender.com.

Fig. 2.

Enzymatic reactions involving protein phosphorylation. (a) SteC is a Salmonella effector kinase that has not been structurally characterized. It has three identified host substrates, MEK1, HSP27 and FMNL 1/2. Its kinase activity leads to actin polymerization in the host cytosol. (b) SptP is a phosphatase with four identified host substrates, VCP, NSF, Syk and vimentin. Its dephosphorylation of these substrates prevents F-actin severing and mast cell degranulation. (c) SpvC has a non-canonical phosphothreonine lyase activity meaning it is able to irreversibly dephosphorylate phosphothreonine residues by severing the Cβ–Oγ bond, requiring a double bond to form between the Cβ and Cα. This is in contrast to the O–P bond which is formed by kinases (a) or broken by phosphatases (b) and makes the threonine residue unable to reaccept a phosphate moiety. Single letters are used to represent carbon (C), oxygen (O) and phosphate (P). Image created with BioRender.com.

Fig. 3.

Mechanisms of host manipulation by Salmonella effectors through ubiquitylation. (a) SopA mimics the activity of a host HECT E3 ligase and ubiquitylates TRIM56 and TRIM65, leading to increased IFN-β gene expression. The ubiquitin molecule is transferred from a host E2 ligase (Ube2D1/Ube2D3/Ube2 L3) and a temporary bond is formed with SopA. (b) SlrP is a novel E3 ligase (NEL) that ubiquitylates thioredoxin to increase cell death and may ubiquitylate an unknown target to inhibit antigen presentation of dendritic cells. The E2 conjugating enzyme involved in the ubiquitin transfer is Ube2D2. (c) SspH1 is an NEL that facilitates binding of ubiquitin to PKN1 following transfer from the E2 Ube2D leading to suppression of NF-κB signalling. (d) SspH2 is an NEL and following transfer of a ubiquitin molecule from the E2 Ube2D, interacts with SGT1 and NOD1 resulting in the ubiquitylation of NOD1, causing an increase in IL-8 production. (e) SseL is a deubiquitylating enzyme (DUB) which removes ubiquitin from IκBα and RPS3 and causes a reduction in NF-κB signalling and IL-8 production respectively. Ubiquitin molecules are depicted as light blue circles containing the text ‘Ub’. Curved arrows demonstrate a transfer of ubiquitin molecules. Bar-headed lines demonstrate inhibition while arrows indicate facilitation/increase in a downstream effect. Human names for the E2 conjugating enzymes have been used throughout. Image created with BioRender.com.

Fig. 4.

The molecular basis of arginine GlcNAcylation. (a) Salmonella effectors SseK1 and SseK3 utilize UDP-GlcNAc to catalyse Arg-GlcNAcylation on host proteins (grey). (b) During Salmonella infection, SseK1 localizes to the host cell cytosol and modifies the mammalian signalling protein TRADD, while Golgi-localized SseK3 modifies TNFR1 and TRAILR. Thereby, SseK1 and SseK3 target the TNF and TRAIL signalling pathways in host cells. SseK3 also targets Rab1, which mediates host protein transport from the endoplasmic reticulum to the Golgi, thus interfering with host protein secretion. SseK2 localizes to the Golgi, though a host substrate is yet to be identified. Image created with BioRender.com.

Fig. 5.

Structural alignment of cysteine proteases GtgE, SseI and SpvD with papain-like folds. (a) Structure of papain (pdb: 1ppn) compared to that of Salmonella effectors GtgE (pdb: 5kdg), SseI (pdb: 4g29) and SpvD (pdb: 5lq6). Structures are aligned to the catalytic cysteine of papain (Cys25). The papain-fold motif is highlighted in dark pink and comprises an α-helix containing the catalytic cysteine residue and an anti-parallel β-sheet. The arrow indicates the location of the catalytic cleft/active centre. The structure of SpvD was rotated by −45° along the xy-axis for better comparability. (b) Zoom into the active centre of the cysteine proteases shown in (a). Residues of the catalytic triad are shown as sticks and atoms were coloured as follows: carbon – grey, oxygen – red, nitrogen – blue, sulphur – yellow. In contrast to the catalytic triad of GtgE, SseI and SpvD, which is made up of cysteine, histidine and aspartate, that of papain contains an asparagine residue instead of aspartate.

Fig. 6.

Adaptor proteins that reprogramme host enzymes to new substrates SteE (a) and SteD (b) (both in brown) are (i) translocated to the cytosol through a type III secretion system. (ii) After translocation they interact with host proteins; SteE forms a complex with GSK3 in the cytosol and SteD binds to TMEM127 in the trans-Golgi network (TGN), from where it is transported to an endosome called the MHCII compartment. (iii) GSK3 phosphorylates SteE and E3 ligase WWP2 is recruited to the TMEM127–SteD complex. (iv) phosphorylated SteE recruits STAT3 to the complex and GSK3 phosphorylates the non-canonical substrate STAT3 on tyrosine 705. SteD recruits MHCII or CD97 to the complex and WWP2 ubiquitylates the targets. (v) Phosphorylated STAT3 activates anti-inflammatory pathways and ubiquitylated MHCII and CD97 are targeted for degradation, reducing their cell surface levels and dampening T-cell activation. Image created with BioRender.com.