Exploitation of the host cell ubiquitin machinery by microbial effector proteins

Abstract

Pathogenic bacteria are in a constant battle for survival with their host. In order to gain a competitive edge, they employ a variety of sophisticated strategies that allow them to modify conserved host cell processes in ways that favor bacterial survival and growth. Ubiquitylation, the covalent attachment of the small modifier ubiquitin to target proteins, is such a pathway. Ubiquitylation profoundly alters the fate of a myriad of cellular proteins by inducing changes in their stability or function, subcellular localization or interaction with other proteins. Given the importance of ubiquitylation in cell development, protein homeostasis and innate immunity, it is not surprising that this post-translational modification is exploited by a variety of effector proteins from microbial pathogens. Here, we highlight recent advances in our understanding of the many ways microbes take advantage of host ubiquitylation, along with some surprising deviations from the canonical theme. The lessons learned from the in-depth analyses of these host-pathogen interactions provide a fresh perspective on an ancient post-translational modification that we thought was well understood.This article is part of a Minifocus on Ubiquitin Regulation and Function. For further reading, please see related articles: 'Mechanisms of regulation and diversification of deubiquitylating enzyme function' by Pawel Leznicki and Yogesh Kulathu (J. Cell Sci.130, 1997-2006). 'Cell scientist to watch - Mads Gyrd-Hansen' (J. Cell Sci.130, 1981-1983).

Keywords: Bacterial effector; Microbial virulence; Ubiquitin.

Fig. 1.

Enzymes of the ubiquitylation cascade. The ubiquitylation reaction can be divided into three steps, each of which is catalyzed by a different class of enzyme. The first step is mediated by ubiquitin-activating enzymes (E1) that use the energy from hydrolyzing ATP (to AMP and PPi) to form a covalent thioester linkage with the C-terminus of ubiquitin (Ub). Ubiquitin is then passed on to a cysteine residue within the Ub-conjugating enzyme (E2). In the final step, the E2 forms a complex with an E3 ubiquitin ligase that coordinates the final transfer of ubiquitin (or preformed ubiquitin chains) onto the substrate protein. A depiction of the detailed mechanisms of how ubiquitin is transferred from the E2 to substrate proteins by different types of E3s can be found in Fig. 2. The type of ubiquitylation determines the outcome for the substrate protein (Komander and Rape, 2012). The attachment of a single ubiquitin molecule (mono-ubiquitylation) typically affects the trafficking behavior or activity of the protein (Jura et al., 2006; Mukhopadhyay and Riezman, 2007), while the formation of linear or branched chains of ubiquitin consisting of two or more molecules (polyubiquitylation) alters processes such as signal transduction (K63-linked ubiquitin chains) (Erpapazoglou et al., 2014; Sun and Chen, 2004), protein half-life (K48-linked or branched ubiquitin chains) (Finley and Chau, 1991) or NF-κB signaling and protein trafficking (mixed linkages) (Iwai, 2012). Effects of the most common types of ubiquitylation on the substrate protein are listed in the white box. Reversal of the ubiquitylation reaction is catalyzed by a class of proteases called DUBs that remove ubiquitin or ubiquitin chains from substrate proteins. Bacterial effector proteins that target the ubiquitylation pathway by functioning as E3 ligases or DUBs are shown in the blue and pink box, respectively, and are detailed in Table S1.

Fig. 2.

Categories of bacterial E3 ubiquitin ligases. Shown here is a schematic illustration of the domain organization and mode of action of selected bacterial E3 ligases. For simplicity, the substrate protein is shown with only one ubiquitin molecule attached. The HECT-type E3 ligase is composed of an N- and C-terminal lobe. Ubiquitin is transferred from the E2 to a cysteine residue in the C-lobe before its final attachment to the substrate protein, which is bound by the substrate-binding domain (SBD). Unlike HECT E3s, RING/U-box-type E3 ligases do not form a stable intermediate with ubiquitin. Instead, they function as scaffolds that mediate ubiquitin transfer from the E2 directly to the substrate protein. F-box (Fbx) domain-containing proteins in the SCF complex (formed by Skp1, Cul1 and the E3 ligase Rbx1) bind to the substrate protein and facilitate ubiquitin transfer. NEL-type E3 ligases, although structurally unrelated to HECT E3s, use a conceptually similar mode of action that involves the formation of a transient ubiquitin intermediate. The N-terminal leucine-rich repeat (LRR) domain mediates substrate binding, while ubiquitin is first transferred onto a cysteine residue in the NEL domain before its final attachment to the substrate protein. The XL-box-containing E3 ligase, which is similar to the RING/U-box-type E3 ligase, facilitates substrate ubiquitylation without the formation of a stable intermediate. Substrate binding of the XL-box-containing E3 ligase is mediated by the N-terminal LRR domain, a mechanism that parallels that of NEL-type E3 ligases. The L. pneumophila effector SidC contains an N-terminal ubiquitin ligase (SNL) domain that lacks structural similarity to other classes of E3s. This domain needs to bind to host cell E2s to mediate substrate ubiquitylation. The C-terminal domain (P4C) regulates SidC localization to PI(4)P-containing membranes such as the Legionella-containing vacuole.

Fig. 3.

E1- and E2-independent ubiquitylation by SidE. (A) SidE is a multi-domain-containing protein composed of a deubiquitylation domain (DUB), a nucleotidase-phosphohydrolase domain (NP) and a mono-ADP-ribosyltransferase domain (mART). The residues required for activity are shown above each of the functional domains. (B) The reaction mechanism of SidE is shown at the molecular level. The mART domain of SidE hydrolyses NAD and transfers ADP-ribose to the Arg42 residue of ubiquitin, generating ADP-ribosylated ubiquitin (ADPr-Ub). Next, the NP domain cleaves the phosphodiester bond in ADPr-Ub, releasing an adenosine monophosphate (AMP) molecule and creating phospho-ribosylated ubiquitin (Pr-Ub), which is then conjugated to substrate proteins such as Rtn4 or Rab33.

Fig. 4.

Post-translational modification of host proteins. Illustrated here are bacterial effector proteins that modify host proteins with different chemical groups to regulate or interfere with ubiquitin binding or ubiquitylation. (A) The methyltransferase NleE from E. coli covalently attaches a methyl group to TAB2 and TAB3, thereby decreasing their binding to polyubiquitin chains and preventing downstream signaling. (B) E. coli NleB conjugates N-acetyl-D-glucosamine (O-GlcNAc) onto the host cell enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), thereby preventing its association with TRAF2 and reducing TRAF2 ubiquitylation. (C) Shigella OspI deamidates the Gln100 residue in Ubc13 to glutamic acid, which disrupts Ubc13-mediated polyubiquitylation of TRAF6.

Fig. 5.

Limiting the half-life of effector proteins through ubiquitylation. Illustrated here are bacterial effector proteins whose stability and biological activity are controlled through host-mediated ubiquitylation. (A) Salmonella effector proteins SopE and SptP control actin cytoskeleton rearrangements by functioning as an activator and inactivator, respectively, of the small GTPases Cdc42 and Rac1. Although SopE and SptP are simultaneously delivered into infected host cells, SopE is much more rapidly ubiquitylated and degraded than SptP, resulting in the SptP-mediated downregulation of actin rearrangements induced by Cdc42 and Rac1. (B) Yersinia YopE functions as GAP protein that de-activates host cell Rho GTPases. The cellular level of YopE is tightly controlled through host-mediated ubiquitylation on Lys62 and Lys75, which targets YopE for proteasomal degradation. (C) Unlike SopE/SptP and YopE, which the pathogen deliberately targets for degradation, the downregulation of ExoT is a host-induced countermeasure aimed at obstructing Pseudomonas infection. To accomplish this, ExoT is polyubiquitylated by the host E3 ligase Cbl-b and targeted for proteasomal degradation.

Fig. 6.

Effect of ubiquitin on the localization and function of bacterial effector proteins. Summarized here are bacterial effector proteins that utilize ubiquitin as a cue for their subcellular distribution or activity. By coupling catalytic activity to ubiquitylation, pathogens can ensure that the activities of the translocated effectors are specifically directed towards a particular membrane compartment or host cell type. (A) At the early stage of Salmonella infection, the phosphoinositide phosphatase SopB localizes to the host cell plasma membrane, where it contributes to actin-mediated bacterial internalization. Later on, SopB is mono-ubiquitylated by the host E3 ligase TRAF6 at multiple lysine residues, which causes the effector to relocate to the Salmonella-containing vacuole, where it contributes to intracellular bacterial growth. (B) Pseudomonas ExoU is a T3SS effector with PLA₂ activity. Upon delivery into host cells, ExoU forms a complex with ubiquitin or ubiquitin chains, which triggers its phospholipase activity. (C) Like ExoU, the Shigella effector OspG is delivered into host cells in its inactive form, and binding to ubiquitin-conjugated UbcH5 or UbcH7 triggers its kinase activity.