Exploitation of the host ubiquitin system by human...

Post-translational protein modifications, such as ubiquit-ylation, phosphorylation and acetylation, modulate many biological processes, enabling cells to react rapidly to environmental changes. During bacterial infection, host cells engage the ubiquitin system as part of their defence programme1–4. Ubiquitylation is involved in such dispa-rate processes as the sensing of intruding bacteria, the activation of innate immune responses and antigen pres-entation. For example, ubiquitylation functions as a signal for nuclear factor-κB (NF-κB) activation, which triggers a broad range of host inflammatory responses that prevent bacterial proliferation (BOX 1). In addition, ubiquitylation can also trigger the host cell to kill bacterial pathogens via proteasome-, phagolysosome- and autophagosome-mediated degradation pathways (BOX 2). To disarm the host response, many bacterial pathogens disrupt and exploit the host ubiquitin system using several distinct types of virulence factors, such as secreted effector proteins, tox-ins and surface proteins. Effector proteins are typically delivered to the host cell by the type III and type IV secretion systems (T3SSs and T4SSs) and are particularly efficient at targeting host ubiquitin systems. These effectors enable pathogens to evade host defence systems (for example, by interfering with the host inflammatory response and pre-venting autophagic activation (BOX 2)), to rearrange the host cell cytoskeleton, to obtain nutrients and to promote intracellular multiplication3–6. In this Review, we high-light some selected examples that illustrate how human bacterial pathogens exploit host ubiquitin systems and manipulate host protein-modification pathways for their own benefit during infection, with a particular emphasis on the roles of secreted effector proteins. A discussion of

plant pathogens is beyond the scope of the current article, so we refer the interested reader to other recent reviews7,8.

Ubiquitylation: the basicsUbiquitin is a small (76 amino acids), highly conserved eukaryotic protein that can be covalently attached to one or more lysine residues of substrate proteins in a process known as ubiquitylation. This common protein modi-fication regulates several key cellular functions, includ-ing signal transduction, protein degradation, endocytosis and sorting, as well as the trafficking of transmembrane proteins9–11. Ubiquitylation is a sequential multi-enzyme cascade that involves E1 (ubiquitin-activation enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) proteins (FIG. 1a). The cascade begins with the activation of ubiquitin in an ATP-dependent manner by the formation of a thioester linkage between the carboxy-terminal glycine of ubiquitin and a specific cysteine residue of the E1 protein. The activated ubiqui-tin molecule is then transferred to the E2 protein, after which the E3 ligase mediates the transfer of ubiquitin from E2 to a lysine residue on the substrate. Eukaryotic E3 ubiquitin ligases are classified into two major groups: HECT-type and the superfamily of ligases RING finger domain and RING finger-like, such as U-box proteins, which are referred to collectively here as RING–U-box-type E3s. For HECT-type enzymes, ubiquitin is transferred from an E2 protein to E3 (by forming an intermediate thioester bond with a catalytic cysteine residue of E3) before it is transferred to the substrate. By contrast, RING–U-box-type enzymes function as scaf-fold molecules that recruit the E2–ubiquitin complex

ProteasomeA large complex of proteases that degrades ubiquitylated proteins in eukaryotic cells.

PhagolysosomeAn organelle that is formed by the fusion of a phagosome and a lysosome. Within it, phagocytosed material is degraded by lysosomal hydrolytic enzymes.

Exploitation of the host ubiquitin system by human bacterial pathogensHiroshi Ashida1, Minsoo Kim1 and Chihiro Sasakawa1–3

Abstract | Ubiquitylation is a crucial post-translational protein modification that regulates several cellular processes in eukaryotes, including inflammatory responses, endocytic trafficking and the cell cycle. Importantly, ubiquitylation also has a central role in modulating eukaryotic defence systems; however, accumulating evidence shows that many bacterial pathogens exploit host ubiquitin systems for their own benefit. In this Review, we highlight the ways in which human bacterial pathogens target ubiquitylation to subvert and manipulate host defence systems, with a focus on the role of molecular mimicry and secreted bacterial effector proteins. These strategies enable bacterial pathogens to maximize effector function and obtain nutrients, thereby promoting bacterial proliferation.

1Division of Bacterial Infection Biology, Institute of Medical Science, University of Tokyo, 4‑6‑1, Shirokanedai, Minato‑ku, Tokyo 108–8639, Japan. 2Nippon Institute for Biological Science, 9‑2221‑1 Shinmachi, Ome, Tokyo 198–0024, Japan. 3Medical Mycology Research Center, Chiba University, 1‑8‑1 Inohana, Chuo‑ku, Chiba, 260–8673 Japan.Correspondence to H.A. and C.S. e‑mails: [email protected]‑tokyo.ac.jp; [email protected]‑tokyo.ac.jpdoi:10.1038/nrmicro3259Published online 7 May 2014

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Nature Reviews | Microbiology

TRAF6UEV1A

UBC13

TAB2 and TAB3

UbUbUb

UbUbUb

UbUbUb

Unanchoredpolyubiquitin

UbUbUb

UbUbUb

UbUbUbUbUb

Proteasomal degradation

Peptidoglycan

TLR or IL-1RTNFR

Pro-inflammatory cytokines

IKKα IKKβ

TAK1

NOD1

TAB1

RIP2

NEMO

IκBIκB

SCFβTRCP

p50 p65 NF-κB

UbUb

Ub

UbUbUb

p50 p65Transcription

RIP1

TRADD TRAF2 and TRAF5 MYD88

IRAK

IKK complex

P

PP

c-IAP1 andc-IAP2

AutophagosomeA double-membrane organelle that engulfs cellular components and delivers them to lysosomes.

Type III and type IV secretion systems (T3SSs and T4SSs). Multicomponent machines that deliver bacterial effectors into host cells. T3SSs are evolutionarily and structurally related to flagellar export systems, whereas T4SSs are related to bacterial conjugation systems that translocate DNA.

and catalyse the direct transfer of ubiquitin from E2 to E3-bound substrates9,11 (FIG. 1a).

Ubiquitin can be conjugated as a monomer to a sin-gle lysine residue (known as monoubiquitylation) or to multiple lysine residues of the substrate (known as mul-tiubiquitylation). The ubiquitin molecule itself contains seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) and each of these residues, as well as the amino-terminal methionine residue (Met1) (FIG. 1b), can be covalently attached to the C terminus of other ubiquitin molecules via the same enzymatic cascade. Attachment of a ubiquitin chain to a substrate results in polyubiquitylation9–11. Although internal lysine residues (and occasionally Met1) are the most frequent sites of ubiquitylation, there are a few examples that document

ubiquitin conjugation to residues other than lysine, including cysteine residues, by thioester bonds, and serine or threonine residues, by oxyester bonds12,13. The different forms of ubiquitylation determine the fate of the ubiquity-lated substrate (FIG. 1c). Among the best characterized link-ages are Lys48-linked polyubiquitin chains, which target substrate proteins for proteasomal degradation. By con-trast, Lys63-linked polyubiquitin chains are involved in signal transduction cascades, such as the NF-κB pathway. In addition, polyubiquitylation, monoubiquitylation and multiubiquitylation also have functional consequences, such as changes in endocytosis and subcellular localization signalling14. Furthermore, recent studies have found that unanchored (that is, substrate free) Lys63-linked poly-ubiquitin chains have an important role in inflammatory

Box 1 | The NF‑κB signalling pathway

The nuclear factor-κB (NF-κB) transcription factor family has a pivotal role in many biological processes, such as inflammation and cell survival118,119, and comprises five members: p50, p52, p65 (also known as RelA), RelB and proto-oncogene protein c-Rel. In a quiescent (that is, unstimulated or uninfected) cell, NF-κB is retained in the cytoplasm by a direct interaction with the inhibitor of NF-κB (IκB proteins, including IκBα, IκBβ and IκBε). Following activation by extracellular or intracellular stimuli, including cytokines, reactive oxygen species (ROS), pathogen-associated molecular patterns (PAMPs) and DAMPs, NF-κB is translocated into the nucleus, where it promotes expression of its target genes (see the figure). Many host cell sensors, such as Toll-like receptor (TLR), interleukin-1 receptor (IL-1R), tumour necrosis factor receptor (TNFR) and NOD-like receptors (such as NOD1), sense bacterial infection by the recognition of PAMPs, DAMPs and cytokines, and trigger signal transduction pathways, ultimately resulting in activation of the inhibitor of NF-κB kinase (IKK) complex (which comprises IKKα, IKKβ and NF-κB essential modulator (NEMO; also known as IKKγ)) and subsequent NF-κB activation. For example, MYD88 and TNF receptor-associated factor 6 (TRAF6) are downstream signalling factors of TLR and IL-1R, TRAF2 and TRAF5 are downstream signalling factors of TNFR and RIP2 is a downstream signalling factor for NOD1. In the TLR and IL-1R pathways, TRAF6 functions as a RING-type E3 ligase that cooperates with E2 enzymes (such as UBC13 and UEV1A), which are required for Lys63-linked TRAF6 self- polyubiquitylation (see the figure). In the TNFR signalling pathway, stimulation of TNFR leads to the binding of TNFR type 1-associated DEATH domain protein (TRADD), which recruits TRAF2 and TRAF5 (which are RING-type E3 ligases), c-IAP1 and c-IAP2 (which are RING-type E3 ligases) to form a complex that results in the polyubiquitylation of RIP1 (see the figure). In the cytoplasm, NOD1 senses bacterial peptidoglycan, which is followed by the recruitment and polyubiquitylation of RIP2 (REF. 120). These polyubiquitin chains, which are linked to TRAF6, RIP1 and RIP2, function as scaffolds that recruit the TGFβ-activating kinase 1 (TAK1)–TAK1-binding protein 1 (TAB1) complex via the ubiquitin-binding adaptors TAB2 and TAB3. This interaction activates TAK1, which phosphorylates IKKβ, leading to activation of the IKK complex. Furthermore, recent studies have identified that unanchored (that is, substrate free) Lys63-linked polyubiquitin chains directly activate TAK1 by binding to TAB2 or TAB3 in vitro16,17. The activated IKK complex phosphorylates IκB, which is subsequently ubiquitylated by the SCFβTRCP ubiquitin ligase complex and targeted for proteasomal degradation.

NF-κB is now free to translocate into the nucleus to activate the transcription of genes encoding pro-inflammatory cytokines118,119.

Uncontrolled NF-κB activation is extremely detrimental to cell homeostasis, and cells have developed specific mechanisms to regulate NF-κB activity via the modification of ubiquitin systems. Ubiquitylation can be reversed by deubiquitylating enzymes (DUBs), such as A20 and CYLD, which negatively regulate essential signalling molecules to terminate NF-κB signalling and resolve inflammation. DUBs remove ubiquitin in a substrate-specific manner, which is essential for signal transduction, as it is required to terminate NF-κB activation121,122. In the figure, green ubiquitin (Ub) molecules are involved in signal transduction, whereas orange ubiquitin molecules target proteins for degradation.

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Box 2 | Bacterial circumvention of ubiquitin‑dependent autophagy

?

Autophagosome maturation

Actin

Autophagosome

Lysosome

Ubiquitylated aggregates or ALIS

LCV Membranedisruption

SCV

S. Typhimurium L. pneumophila L. monocytogenes

L. monocytogenesLC3

RavZ

InlK

MVP

p62

SseL

Ub

UbUb

UbUb Ub

Ub

Ub

ActA

ARP2/3

VASP

Autolysosome

ALIS

LC3


UbUb

UbUbUb

Ub

UbUb

UbUbUb

Ub

SCV

EndocytosisA process by which a cell imports molecules from the external environment by engulfing a volume of extracellular space within its membrane.

signalling15,16 (BOX 1) and that Met1-linked linear poly-ubiquitin chains, which are formed by the linear ubiq-uitin chain assembly complex (LUBAC), are involved in NF-κB activation17 (FIG. 1c). By contrast, the functions of other types of linkages (such as Lys6-, Lys11-, Lys27-, Lys29- and Lys33-linkages) are less well understood, but it is becoming increasingly clear that they are involved in multiple biological processes, including protein degradation, DNA repair and cell cycle regulation18.

Importantly, ubiquitylation is reversible by the action of deubiquitylating enzymes (DUBs). These enzymes have substrate specificity and hydrolyse either the iso-peptide bond that links individual ubiquitin monomers in chains or the link between ubiquitin and its substrate (FIG. 1a). As such, DUBs recycle ubiquitin and antago-nize the function of ubiquitylated substrate proteins19. In addition to eukaryotic DUBs, bacterial pathogens also encode their own DUBs (see below).

Autophagy is a process in which intruding bacteria are engulfed in autophagosomes, which fuse with lysosomes (to form an autolysosome), ultimately resulting in bacterial destruction2,123. Although autophagosomes can also nonspecifically sequester cytosolic material, autophagy exploits the ubiquitin system to tag unwanted cellular components, such as protein aggregates and intruding pathogens; collectively, these processes are known as selective autophagy. Ubiquitin-dependent selective autophagy uses adaptor proteins, such as p62, NDP52 (nuclear dot protein 52 kDa) and optineurin, which bind to unwanted ubiquitin-tagged components (via ubiquitin-binding domains (UBDs)) and to the autophagosome-associated ubiquitin-like protein (UBL) LC3 via LC3-interacting regions124–126.

Although ubiquitin-dependent selective autophagy functions as an antibacterial defence, many bacterial pathogens have evolved to have mechanisms to circumvent autophagic recognition by targeting host ubiquitin systems for intracellular growth and survival127,128. For example, intracellular Salmonella enterica subsp. enterica serovar Typhimurium infection causes the formation of ubiquitylated protein aggregates or aggresome-like induced structures (ALISs) around the Salmonella-containing vacuole (SCV) (see the figure). ALISs are ubiquitylated by host E3 ligases, which triggers autophagy of the SCV and destroys S. Typhimurium129,130. However, the S. Typhimurium deubiquitylating enzyme (DUB) SseL54,131 deubiquitylates ubiquitin aggregates and ALISs, thereby decreasing the recruitment of ubiquitin, p62 and LC3 to the SCV (only LC3 is shown in the figure), which promotes escape from autophagy131. Infection of macrophages with an S. Typhimurium sseL-null mutant (ΔsseL) results in elevated levels of ubiquitin aggregates and autophagic flux, ultimately reducing the level of bacterial colonization compared with wild-type infection. Thus, SseL DUB activity is important for bacterial replication within the SCV, as it prevents autophagy131. Furthermore, SseL DUB activity can alter lipid metabolism to prevent the accumulation of lipid droplets in infected cells132.

In the process of autophagosome maturation, the UBL LC3 is conjugated to the lipid phosphatidylethanolamine (PE) on the autophagosome membrane. Conjugation of LC3 to PE is essential for the autophagosome membrane to enclose proteins and organelles in a double-membrane vacuole. A recent study showed that Legionella pneumophila can interfere with autophagy at the stage of autophagosome maturation by delivering the RavZ effector via the type IV secretion system (T4SS)133. RavZ functions as a cysteine protease and specifically targets PE-conjugated LC3 by irreversibly cleaving off the carboxy-terminal glycine residue that is required for LC3 conjugation (see the figure). As a result of RavZ-mediated deconjugation of LC3, the pathogen prevents autophagosome formation and evades autophagy133.

Listeria monocytogenes escapes autophagic recognition by expressing the bacterial surface protein ActA, which is essential for actin-based intracellular and intercellular

motility134. An actA-null mutant (ΔactA), which is defective in motility, is recognized by ubiquitin and p62, resulting in the recruitment of the autophagic machinery. By contrast, L. monocytogenes that escape into and move around the cytosol cannot be recognized by ubiquitin, p62 and the LC3 autophagic pathway, as surface-expressed ActA recruits the actin-related protein 2/3 (ARP2/3) complex, VASP and actin. By recruiting these host proteins to the bacterial surface, the bacterium masks itself from autophagic recognition134. In addition, a recent study showed that the L. monocytogenes surface protein internalin K (InlK) — a protein of the internalin family — also participates in masking the bacteria from ubiquitylation and facilitating the evasion of autophagy by interacting with host major vault protein (MVP) on the bacterial surface135. LCV, Legionella-containing vacuole; Ub, ubiquitin.

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Reactive oxygen species (ROS). Reactive chemical molecules that contain oxygen. They are generated by various cellular processes.

Pathogen-associated molecular patterns (PAMPs). Pathogen-specific molecules, such as peptidoglycan, flagellin or viral RNA, that are sensed as foreign material by the innate immune system.


Ub E1

E1

E1AMP + PPi

a Ubiquitylation cascade

b Ubiquitin molecule

c Different forms of ubiquitylation

ATP+

E2

E2

E2E2E2

C OHO

Ub Ub

Ub

CS

OSH

SH SH

CS

O

Ub

UbCS

O

UbCS

O

S NH2 NH

C O C O

Ub

NHC O

SubstrateRING–U-box-E3 RING–U-box-E3

SubstrateSubstrate Substrate

E2

NEL-E3

Ub

NH2 NHC O

Substrate

NH2

HECT-E3

Substrate

Cys

Cys

E2

HECT-E3

Substrate

LRR

E2

NEL-E3

Substrate

LRR

DUB

UbUbUb

Ub Ub

Ub

M K K K K K K K -COOHNH2-

76 amino acids

1 6 11 27 29 33 48 63

Monoubiquitylation

• Trafficking of membrane proteins • Protein regulation

Lys48-ubiquitylation


Lys63-ubiquitylation

Signal transdution

Unanchored Lys63-polyubiquitin

Met1-ubiquitylation(linear)

NF-κB activation Inflammatory signalling

Ub

Substrate

Ub Ub Ub

Substrate

UbUbUb

Substrate

UbUbUb

UbUbUb

Substrate

Figure 1 | The ubiquitylation cascade. a | The ubiquitylation pathway comprises sequential enzymatic steps that result in the conjugation of ubiquitin (Ub) usually to internal lysine residues in the substrate protein. The process involves E1, E2 and E3 enzymes. E1 activates ubiquitin in an ATP-dependent manner and subsequently transfers it to E2, which then transfers the activated ubiquitin to the substrate proteins via the activity of E3 ligases. E3 ligases are classified into three groups according to their structures and functions: homologous to the E6-associated protein C terminus (HECT)-type, RING–U-box-type and novel E3 ligase (NEL)-type. HECT-type E3s (such as SopA from Salmonella enterica subsp. enterica serovar Typhimurium and NleL from enterohaemorrhagic Escherichia coli (EHEC)) transfer ubiquitin indirectly; the activated ubiquitin is first transferred from E2 to the catalytic cysteine residue of HECT-E3 before it is transferred to the substrate. By contrast, RING–U-box-type E3 ligases (such as NleG from EHEC and enteropathogenic E. coli (EPEC) and LubX from Legionella pneumophila) transfer ubiquitin directly from E2 to the substrate that is bound to the E3 ligase. NEL-type E3 ligases (such as the Shigella flexneri IpaH family of enzymes and the S. Typhimurium SspH1 and SspH2) enzymes have no structural similarity to HECT-type and RING–U-box-type E3 ligases, but, similarly to HECT-type E3 ligases, they rely on a conserved cysteine residue for E3 activity. Ubiquitylation is reversible by the action of deubiquitylating enzymes (DUBs), which hydrolyse the isopeptide bond of the ubiquitin-chain linkage between individual ubiquitin moieties or between ubiquitin and its substrate. b | The ubiquitin monomer contains seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys63), and each of these residues, as well as the N-terminal methionine residue (Met1), can be covalently attached to ubiquitin molecules. c | There are different forms of ubiquitylation, which determine the fate of the modified substrate. The attachment of a single ubiquitin molecule to a single residue of the substrate protein is known as monoubiquitylation. Additional ubiquitin molecules can subsequently be attached to the remaining lysine residues or the Met1 residue (multiubiquitylation) or a chain of ubiquitin molecules is formed when ubiquitin molecules are conjugated to each other (polyubiquitylation). The form of ubiquitylation determines the fate of modified proteins, such as proteasome degradation, signal transduction, trafficking of membrane proteins and endocytosis. Unanchored polyubiquitin chains, which are not conjugated to any cellular proteins, have recently been shown to have an important role in inflammatory signalling. LRR, leucine-rich repeat; PPi, inorganic pyrophosphate.

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Prokaryotic ubiquitin-like protein system(Pup system). A functional homologue of the eukaryotic ubiquitin system; it targets prokaryotic substrates for proteasomal degradation.

Attaching and effacing lesionsLesions that form by intimate bacterial attachment, resulting in the effacement of the brush-border microvilli of epithelial cells and the formation of an actin pedestal on the cell surface.

Molecular mimicryAlthough archaea and some bacterial species (for exam-ple, Mycobacterium tuberculosis) have a functionally analogous system, which is known as the prokaryotic ubiquitin-like protein system (Pup system)20, the prototypical ubiquitylation pathway is absent in bacteria. However, accumulating evidence has shown that several bacte-rial pathogens encode T3SS or T4SS effectors that have E3 ligase activity. These E3 effectors are delivered into host cells and can ‘hijack’ the host ubiquitylation path-way, enabling the bacterium to subvert host defences, usurp host cellular functions and manipulate host signalling for their own benefit21 (TABLE 1).

HECT-type and RING–U-box-type E3 ligases. The facultative intracellular pathogen Salmonella enterica subsp. enterica serovar Typhimurium invades macro-phages and epithelial cells, where it can survive and replicate within a host-derived vacuole known as the Salmonella-containing vacuole (SCV). This is achieved by delivering a subset of effectors through two differ-ent T3SSs: T3SS1 is essential for bacterial invasion of intestinal epithelial cells, whereas T3SS2 is required for intracellular replication and survival within macro-phages22. The T3SS1 effector SopA is structurally similar to eukaryotic HECT-type E3 ligases23 (FIG. 1a). Although the target molecules in the host remain unclear, it has been shown that, compared with infection with the wild-type strain, reduced polymorphonuclear (PMN) transepithelial migration (which is a hallmark of S. Typhimurium pathogenesis) occurs in polarized T84 monolayer cells that have been infected with a mutant S. Typhimurium strain that expresses a catalytically inac-tive SopA24. This suggests that SopA-mediated ubiquity-lation of as-yet-unidentified host proteins contributes to S. Typhimurium-induced enteritis.

Other producers of HECT-type and RING–U-box-type E3 ligases include enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC). These bacteria adhere to the surface of intestinal epithe-lial cells, form an actin pedestal and induce the efface-ment of brush-border microvilli. The resulting attaching and effacing lesions are a hallmark of EPEC- and EHEC-induced inflammatory colitis. EHEC delivers HECT-type E3 effectors (such as NleL) and RING-type E3 effectors (such as NleG) into host cells. The C-terminal domain of NleL shares sequence similarity with S. Typhi-murium SopA, and its E3 ligase domain is functionally and structurally similar to that of eukaryotic HECT-type E3 ligases. NleL has been shown to catalyse the forma-tion of free Lys6- and Lys48-linked polyubiquitin chains in vitro25,26. Although the role of NleL in infection is still unclear, it is known that its E3 activity modulates the rate of EHEC-induced actin-pedestal formation27, which suggests that NleL E3 ligase activity contributes to bacte-rial adherence to intestinal epithelial cells. More than 20 NleG homologues have been identified in pathogenic E. coli and Salmonella spp. strains. Although they are not homologous to any other proteins28,29, their conserved C-terminal domains are structurally similar to the eukaryotic RING–U-box motif and they exert E3 ligase

activity in vitro30. However, the target molecules of NleL and NleG remain unknown; identification of these fac-tors should be considered as an important priority for future work.

Finally, Legionella pneumophila, which is the causative agent of a severe form of pneumonia, proliferates in envi-ronmental amoebae and human cells within a membrane-derived compartment known as the Legionella-containing vacuole (LCV). Intracellular growth of L. pneumophila requires the Dot–Icm T4SS, which delivers a subset of effectors into host cells31. In particular, L. pneumophila delivers the E3 effector LubX, which contains two U-box domains: one of these U-box domains is cru-cial for E3 ligase activity and the other is required for substrate binding32,33. Using its U-box domains, LubX interacts with and ubiquitylates the host cell kinase CDC2-like kinase 1 (CLK1). Although the exact role of LubX-mediated CLK1 ubiquitylation during infec-tion has not been completely elucidated, a recent study showed that the kinase activity of CLK1 is essential for bacterial growth within mouse macro phages32. Thus, LubX-mediated CLK1 ubiquitylation might be an essential modification that promotes its kinase activity.

Novel E3 ligases. In addition to HECT-type and RING–U-box-type E3 ligases, a third class of bacterial E3 effec-tors, which are known as novel E3 ligases (NELs), have recently been described21,34. The first identified members of this class belong to the IpaH–SspH family, which have an N-terminal leucine-rich repeat (LRR) domain for sub-strate recognition and a conserved C-terminal domain (CTD), which encodes E3 ligase activity. This family of effectors is widely conserved among bacterial pathogens that infect humans, animals, fish and plants, including Shigella flexneri, Salmonella enterica, Edwardsiella ictluri, Bradyrhizobium japonica, Rhizobium sp. strain NGR234 and some Pseudomonas species (such as Pseudomonas putida, Pseudomonas entomophila, Pseudomonas fluores-cens and Pseudomonas syringae). Similarly to HECT-type E3 ligases, the CTD of these effectors contains a con-served cysteine residue that is essential for E3 activity and is involved in the formation of the cysteine–ubiq-uitin intermediate34–37 (FIG. 1a). However, crystal struc-tures of IpaH and SspH2 have supported the idea that the IpaH–SspH family represents a new class of E3 enzymes that are distinct from the typical HECT-type and RING–U-box-type E3 ligases35–37. Furthermore, the crystal structure of SspH2 showed that the LRR domain sequesters the catalytic cysteine residue in the CTD — this has been termed autoinhibition. Following the bind-ing of a substrate to the LRR, a conformational change occurs, and this seems to be important for releasing the autoinhibition of E3 activity37. Indeed, removal of the LRR domain from Shigella flexneri IpaH9.8 or S. Typh-imurium SspH2 promotes self-ubiquitylation, and the inhibitory effect of the LRR domain is suppressed when it interacts with a substrate34–38. Furthermore, a recent structural analysis of S. Typhimurium SspH1 and its substrate serine/threonine protein kinase N1 (PKN1) has shown that SspH1 LRR–PKN1 interactions release an inhibitory interaction between the LRR domain

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and the catalytic CTD, which indicates that interac-tion of the substrate with the LRR domain is coupled to activation of E3 (REF. 39).

Several functions of S. flexneri and S. Typhimurium NEL effectors have been characterized. S. flexneri, which is the causative agent of bacillary dysentery, delivers a subset of T3SS effectors into host cells40–42. The IpaH9.8 effector is translocated to the nucleus of epithe-lial cells43, where it inhibits the U2AF35 (also known as U2AF1)-dependent splicing reaction via its E3 ligase activity (by an unknown mechanism), thereby reducing

the expression of several genes, including genes that encode pro-inflammatory cytokines and chemokines44,45 (FIG. 2a). In addition, IpaH9.8 also interferes with the NF-κB-mediated inflammatory response (BOX 1) by interacting with and polyubiquitylating NF-κB essen-tial modulator (NEMO; also known as IKKγ), which is an essential component of the inhibitor of NF-κB kinase (IKK) complex46. IpaH9.8 also interacts with A20-binding inhibitor of NF-κB activation 1 (ABIN-1; also known as TNIP1) — a ubiquitin-binding adap-tor protein — to further promote polyubiquitylation

Table 1 | Summary of mechanisms used by bacterial pathogens to interfere with and manipulate the host ubiquitin system

Mechanism Bacteria Factor Biochemical activity Target References

E3 ubiquitin ligase

Formation of actin pedestal EHEC NleL HECT-type E3 ligase Unknown 25–27

Unknown EPEC and EHEC NleG RING-type E3 ligase Unknown 30

Promotion of growth in macrophages

Legionella pneumophilla

LubX U-box type E3 ligase CLK1 32

Regulation of host inflammation Salmonella enterica subsp. enterica serovar Typhimurium

SopA HECT-type E3 ligase Unknown 23,24

Promotion of IL-8 secretion S. Typhimurium SspH2 NEL-type E3 ligase NOD1, SGT1 53

Induction of host cell death S. Typhimurium SlrP NEL-type E3 ligase TRX 49

Inhibition of U2AF35-mediated splicing and inhibition of NF-κB activation

Shigella flexneri IpaH9.8 NEL-type E3 ligase U2AF35 and NEMO 44–46

Inhibition of NF-κB activation S. flexneri IpaH0722 NEL-type E3 ligase TRAF2 47

S. flexneri IpaH4.5 NEL-type E3 ligase p65 48

S. Typhimurium SspH1 NEL-type E3 ligase PKN1 51

Deubiquitylation enzyme

Inhibition of NF-κB activation and type I IFN activation

Burkholderia pseudomallei

TssM DUB TRAF3, TRAF6 and IκBα 63

Inhibition of NF-κB activation Chlamydia trachomatis

ChlaDub1 DUB IκBα 65

Yersinia enterocolitica

YopP DUB TAK1, TAB1, NEMO and TRAF6

59,60

Yersinia pseudotuberculosis

YopJ DUB TRAF2, TRAF6 and IκBα 58

Deubiquitylation of ubiquitin aggregates

S. Typhimurium SseL DUB Ubiquitin aggregates and ALISs

131

Interference with signalling cascades

Cell cycle arrest B. pseudomallei CHBP Deamidase Primarily NEDD8 but also ubiquitin

90

EPEC and EHEC Cif Deamidase Primarily NEDD8 but also ubiquitin

90–92

Inhibition of sumoylation Listeria monocytogenes

LLO Pore-forming toxin UBC9 88

Inhibition of NF-κB activation S. flexneri OspG Serine/Threonine kinase E2 and ubiquitin 75-77

S. flexneri OspI Deamidase UBC13 72–74

EPEC and EHEC Tir Unknown SHP-1 and SHP-2 82,83

EPEC and EHEC NleE Methyltransferase TAB2 and TAB3 80

Citrobacter rodentium

NleB O-GlcNAc transferase GAPDH 81

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Danger-associated molecular patterns (DAMPs). Molecules that are released by damaged or stressed cells, either in the presence or absence of bacterial infection, and trigger immune responses.

Thioredoxin(TRX). A small redox protein that is involved in several functions related to defence against oxidative stress and apoptosis.

of NEMO. Polyubiquitylated NEMO undergoes pro-teasome-dependent degradation, thereby inhibiting NF-κB activation46 (FIG. 2a). Indeed, in mice that have been infected with an S. flexneri mutant that expresses a catalytically inactive IpaH9.8 (owing to a C337A mutation), the inflammatory response is augmented compared with that in mice infected with the wild-type strain46. Furthermore, the E3 ligase activity of another S. flexneri effector, IpaH0722, also inhibits NF-κB activation. IpaH0722 preferentially targets the protein kinase C (PKC)−NF-κB pathway, which is activated by vacuolar membrane rupture when the bacterium escapes into the cytoplasm and generates signals that are recognized as danger-associated molecular patterns (DAMPs)47. IpaH0722 targets tumour necrosis factor (TNF) receptor-associated factor 2 (TRAF2; a factor downstream of PKC) for ubiquitylation and promotes the proteasome-dependent degradation of TRAF2, thereby inhibiting NF-κB activation47 (FIG. 2a). In addi-tion to IpaH9.8 and IpaH0722, S. flexneri delivers the E3 effector IpaH4.5, which inhibits NF-κB activity by targeting the NF-κB subunit p65 for ubiquitylation48 (FIG. 2a). IpaH9.8, IpaH0722 and IpaH4.5 all contri-bute to S. flexneri colonization in mouse lung-infection models44,46–48.

S. Typhimurium delivers several NEL effectors into the host cell, including SlrP, SspH1 and SspH2. SlrP

targets mammalian thioredoxin (TRX) — which func-tions as a cell death regulator — for ubiquitylation, lead-ing to a decrease in TRX activity and, ultimately, host cell death49. An S. Typhimurium mutant that lacks the slrP gene is less virulent than the wild type, which sug-gests that SlrP activity is important for S. Typhimurium pathogenesis50. Using its LRR domain, SspH1 interacts with PKN1 and catalyses the ubiquitylation of PKN1. Ubiquitylation of PKN1 increases its activity in mam-malian cells and causes a decrease in NF-κB activity, which suggests that the interaction between SspH1 and PKN1 is probably important for modulation of the host inflammatory response34,51. A recent report showed that SspH2, which localizes to the host cell membrane after host-mediated S-palmitoylation of its N-terminal cysteine residue, subverts the innate immune response in a manner that is dependent on its E3 ligase activity52,53. SspH2 forms a trimeric complex with the NOD-like receptors SGT1 and NOD1, and the interactions between SGT1 and SspH2 in this complex increase SspH2 sta-bility and E3 activity. SspH2 targets NOD1 for mono-ubiquitylation, which increases the NOD1-dependent secretion of interleukin-8 (IL-8)53. Although the impor-tance of SspH2-mediated subversion of the immune response during infection has not yet been resolved, these functional roles of SspH2 have been confirmed in mammalian and plant cell infection models53.

Temporal regulation

Promotion of bacterial uptake by the host cell

S. Typhimurium SopE GEF RAC1 and CDC42 106

Termination of bacterial invasion S. Typhimurium SptP GAP RAC1 and CDC42 106

Regulation of another effector function

L. pneumophilla LubX U-box-type E3 ligase SidH 33

Spatial regulation

Promotion of bacterial uptake and intracellular survival

S. Typhimurium SopB Phosphoinositide phosphatase

SGEF and AKT 108,109

Nutrient acquisition

Generation of amino acids for bacterial proliferation

L. pneumophilla AnkB F-box protein Lys48-linked polyubiquitylated proteins

111

Evasion of autophagy

Inhibition of autophagosome maturation

L. pneumophilla RavZ Protease LC3 133

Masking of bacteria from autophagic recognition

Listeria monocytogenes

ActA Unknown ARP2/3 complex and VASP 134

Listeria monocytogenes

InlK Unknown MVP 135

Deubiquitylation of ubiquitin aggregates

S. Typhimurium SseL DUB Ubiquitin aggregate and ALISs

131

AKT, RAC-alpha serine/threonine-protein kinase; ALISs, aggresome-like induced structures; ARP, actin-related protein complex; CLK1, Cdc2-like kinase 1; DUB, deubiquitylating enzyme; EHEC, enterohaemorrhagic Escherichia coli; EPEC, enteropathogenic E. coli; GAP, GTPase-activating protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GEF, guanine nucleotide exchange factor; HECT, homologous to the E6-associated protein C-terminus; IκBα, NF-κB inhibitor-α; IL-8, interleukin-8; LC3, light chain 3; LLO, listeriolysin O; MVP, major vault protein; NEL, novel E3 ligase; NEMO, NF-κB essential modulator; NF-κB, nuclear factor-κB; O-GlcNAc, O-linked N-acetyl-d-glucosamine; PKN1, Protein kinase N1; SGEF, SRC homology 3 domain–containing guaninenucleotide exchange factor; SGT1, suppressor of the G2 allele of Skp1; SUMO, small ubiquitin-like modifier; TAB, TAK1-binding protein; TAK1, TGFβ activating kinase 1; TRAF, TNF receptor-associated factor; TRX, thioredoxin; U2AF35, U2 snRNP auxiliary factor 35 kDa.

Table 1 (cont.) | Summary of mechanisms used by bacterial pathogens to interfere with and manipulate the host ubiquitin system

Mechanism Bacteria Factor Biochemical activity Target References

R E V I E W S



Bacterial DUBs. Some T3SS effectors are DUBs that subvert host signal transduction, resulting in the down-regulation of innate immune responses3–5. For example, S. Typhimurium delivers the DUB SseL, which promotes pathogenesis in a mouse infection model54 (BOX 2). In addition, the DUB YopJ of Yersinia pseudo tuberculosis destroys macrophages by inducing apoptosis, thereby

promoting systemic infection55. YopJ exerts multiple activities during infection, including the modulation of the NF-κB pathway and the mitogen-activated protein kinase (MAPK) pathway (FIG. 2b). Secondary-structure analysis revealed that YopJ has a cysteine-protease-like catalytic triad56,57, and mutation of the catalytic cysteine residue abolishes the inhibitory activity that YopJ exerts

Ub

a b

c

Membrane disruption

ActinMembrane ruffling

Shigellaflexneri

• NF-κB pathway• MAPK pathway

CBMPKC

TRAF6

UBC13

TRAF2

TRAF2, TRAF6 or IκBα

Y. pseudotuberculosis B. pseudomallei C. trachomatis

IpaH0722

TRAF2

DAG

UEV1A

IKKα

UBC13

UbUb

UbUb

UbUbUb

EQOspI

IKKβNEMO NEMO

ABIN1

IκBα

p50 p65NF-κB

p50 p65

P

P

IpaH9.8

IpaH4.5

IpaH9.8

UbUb


Ub

IKK complex

Transcription

Nucleus

Pro-inflammatory cytokines

Ub

E2OspG

p65

SCFβTRCP

U2AF35

Ub

EHEC and EPEC EHEC and EPEC C. rodentium

YopJ ChlaDub1

UbUb

UbUb

Ub

TRAF3, TRAF6 or IκBα

UbUb

Ub

TssM

Ub

NF-κB pathway

UbUb

Ub

TAB1TAK1

Ub

CH3

TAB2 and TAB3

NIeE

Ub TRAF6

SHP-1 and SHP-2

UbUb

GAPDH TRAF2NIeB

O-GlcNAcUbUb


• NF-κB pathway• Type I IFN activation

TirP

IκBα

IκBα

Figure 2 | Bacterial interference with ubiquitin-mediated innate immune signalling. Ubiquitylation has a central role in activating signal transduction in innate immune-response pathways, such as the nuclear factor κB (NF-κB) pathway. However, some bacterial pathogens counteract NF-κB-mediated host inflammatory responses by delivering type III secretion system (T3SS) effectors. a | Shigella flexneri delivers a subset of effector proteins, including OspG, OspI and IpaH proteins, into host cells; these proteins target ubiquitylation and inhibit NF-κB activation. OspG binds to ubiquitylated E2 proteins and prevents inhibitor of NF-κB α (IκBα) ubiquitylation and proteasomal degradation, thereby blocking NF-κB activation. OspI deamidates and inactivates UBC13, resulting in the inhibition of TRAF6 (tumour necrosis factor-associated factor 6) polyubiquitylation and the DAG–CBM–TRAF6–NF-κB (diacylglycerol–CARMA-BCL10- MALTI–TRAF6–NF-κB) pathway is subsequently inactivated. IpaH9.8, IpaH0722 and IpaH4.5 target NF-κB essential modulator (NEMO; also known as IKKγ), TRAF2 and NF-κB subunit p65 for ubiquitylation, respectively, and some of the resultant modified proteins are degraded by the proteasome. IpaH9.8 also inhibits U2AF35-dependent splicing, which results in the inhibition of transcription and the downregulation of pro-inflammatory cytokines. b | Some bacterial pathogens deliver deubiquitylating enzymes (DUBs) as T3SS effectors, which counteract ubiquitylation and subvert host signal transduction. YopJ of Yersinia pseudotuberculosis deubiquitylates TRAF2, TRAF6 and IκBα, thereby inhibiting NF-κB activation and the mitogen-activated protein kinase (MAPK) pathway. Burkholderia pseudomallei TssM deubiquitylates TRAF3, TRAF6 and IκBα, resulting in inhibition of type I interferon (IFN) and NF‑κB activation. Chlamydia trachomatis ChlaDub1 targets and deubiquitylates IκBα, leading to inhibition of the NF-κB pathway. c | Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) deliver NleE and Tir, which inhibit NF-κB activation by targeting ubiquitylation. The methyltransferase NleE adds a methyl group to Tak1-binding protein 2 (TAB2) and TAB3, which prevents TAB2 and TAB3 from binding to ubiquitin chains, resulting in the loss of NF-κB activation. Phosphorylated Tir binds to protein-tyrosine phosphatases SHP-1 and SHP-2, which leads to interactions between SHP-1 and SHP-2 and TRAF6 and impairs the ubiquitylation of TRAF6. NleB of Citrobacter rodentium mediates O-linked N-acetyl-d-glucosamine modification (O-GlcNAcylation) of gyceraldehyde-3-phosphate dehydrogenase (GAPDH). This modification prevents the interaction between GAPDH and TRAF2, thereby inhibiting self-ubiquitylation of TRAF2. Ubiquitin molecules shown in green are involved in signal transduction, whereas ubiquitin molecules shown in orange target proteins for degradation. ABIN1, A20-binding inhibitor of NF-κB activation 1; PKC, protein kinase C; UEV1A, ubiquitin-conjugating enzyme E2 variant 1; U2AF35, splicing factor U2AF 35 kDa subunit.

R E V I E W S



on NF-κB, which indicates that protease activity is cru-cial for YopJ function56,57. YopJ cleaves ubiquitin mol-ecules from various substrates, including inhibitor of NF-κB α (IκBα; a member of the IκB proteins), TRAF2 and TRAF6; in all cases, the deubiquitylation activity of YopJ is dependent on the catalytic cysteine residue58 (FIG. 2b; TABLE 1). Similarly, Yersinia enterocolitica YopP, which is a homologue of YopJ, cleaves ubiquitin moieties from TRAF6, NEMO, transforming growth factor β (TGFβ)-activating kinase 1 (TAK1) and TAK1-binding protein (TAB1)59,60 (TABLE 1). In addition, YopJ functions as an acetyltransferase and uses acetyl-CoA to acetylate crucial serine and threonine residues in the activation loop of target proteins MAPK kinase (MAPKK) and IKK61,62. Together, these YopJ activities prevent the host from mounting a sufficient innate immune response against Y. pseudotuberculosis.

Other examples of DUBs include the Burkholderia pseudomallei and Chlamydia spp. T3SS effectors. B. pseu-domallei — which is the causative agent of melioidosis — delivers the effector TssM, which has DUB activity and subverts host inflammatory signalling63. TssM tar-gets and cleaves polyubiquitylated chains from TRAF3, TRAF6 and IκBα, which inhibits the activation of type I interferon (IFN) and NF-κB 63 (FIG. 2b; TABLE 1). The obligate intracellular bacterium Chlamydia trachomatis has two T3SS DUB effectors, ChlaDub1 and ChlaDub2 (REF. 64). ChlaDub1 inhibits NF-κB activation via the deubiquitylation of IκBα65 (FIG. 2b; TABLE 1). A recent study showed that Chlamydia caviae has another T3SS DUB effector — ChlaOTU. Although the importance of this effector is still unclear, ChlaOTU targets both ubiq-uitin and the ubiquitin-adaptor protein NDP52 (nuclear dot protein 52 kDa) and removes ubiquitin from bacterial invasion sites66.

Interfering with ubiquitin‑mediated signallingFollowing bacterial invasion and replication within host cells, the innate immune system quickly senses bacte-rial components and transmits various alarm signals to the rest of the immune system67,68. During bacte-rial infection, NF-κB has a crucial role in triggering a broad range of host inflammatory responses (BOX 1). The ubiquitylation and deubiquitylation of signalling factors, such as those that are involved in the NF-κB pathway, is required to ensure that these pathways are appropriately regulated; therefore, bacterial pathogens such as S. flexneri, Salmonella spp., EPEC and EHEC use distinct mechanisms to interfere with ubiquityla-tion. Although the bacterial effectors involved con-sist of a highly divergent array of functional proteins, they all have biochemical activities that enable them to target host ubiquitin systems and modulate NF-κB activation5,69,70 (FIG. 2; TABLE 1).

Inhibition of NF-κB signalling by S. flexneri. S. flexneri induces the formation of membrane ruffles following contact with epithelial cells by remodelling the actin cytoskeleton at the site of bacterial entry. Immediately after bacterial invasion, the bacterium is surrounded by a vacuolar membrane; however, subsequent disruption

of the membrane enables bacteria to disseminate into the cytoplasm40,71. Aberrant membrane ruffles, which protrude from the bacterial entry site and are accom-panied by diacylglycerol (DAG) production, are sensed as DAMPs by the innate immune system and trigger the activation of the DAG–CBM–TRAF6–NF-κB (diacyl-glycerol–CARMA-BCL10-MALTI–TRAF6–NF-κB) pathway72 (FIG. 2a). Activation of this pathway requires the E2 activity of UBC13, which mediates Lys63-linked polyubiquitylation and activation of the E3 enzyme TRAF6; this then activates the NF-κB pathway. However, S. flexneri delivers OspI into the cell via its T3SS; OspI functions as a glutamine deamidase for UBC13 and con-verts Gln100 to Glu100 (Q100E), thereby inactivating UBC13 and interfering with TRAF6–NF-κB activation72 (FIG. 2a). Structural analyses of an OspI–UBC13 complex have revealed that OspI and TRAF6 bind to the same site on UBC13 (REFS 73,74). Thus, in addition to deamida-tion, OspI also prevents TRAF6 from accessing UBC13 by masking the TRAF6 binding site.

Furthermore, the effector OspG, which shares sequence similarity with mammalian serine/threonine kinases, also inhibits NF-κB activation. OspG binds to ubiquitylated E2 enzymes, such as UbcH5 and UbcH7, and prevents E2 activity, thereby inhibiting E2-SCFβTRCP-mediated ubiquitylation and proteasomal degradation of phosphorylated IκBα, which is essen-tial for NF-κB activation75 (FIG. 2a). Recent co-crystal structural analysis of an OspG–E2–ubiquitin complex has shown that formation of this complex promotes the kinase activity of OspG and regulates the catalytic activ-ity of E2 (REF. 76). OspG also seems to directly bind to ubiquitin, which stimulates OspG activity and promotes the inhibition of NF-κB activation77.

Modulation of NF-κB signalling by EHEC and EPEC. Ubiquitin and polyubiquitin chains occasionally func-tion as signals and are recognized by various proteins that contain ubiquitin-binding domains (UBDs). As described in BOX 1, TAB2 and TAB3 have UBDs and bind to TRAF6 polyubiquitin chains or unanchored Lys63-linked polyubiquitin chains; this activates TAK1 and subsequently activates IKK and NF-κB signalling. NleE, which is delivered by the T3SSs of EHEC and EPEC, directly targets TAB2 and TAB3 and inhibits their ubiquitin-binding activity78–80. Mass spectrometry analysis has shown that NleE has methyltransferase activity and that it methylates a specific zinc-finger cysteine residue of TAB2 and TAB3. Cysteine-methyl-ated TAB2 and TAB3 lose the ability to bind to ubiq-uitin chains, which results in inhibition of NF-κB80 (FIG. 2c). Furthermore, in Citrobacter rodentium (which is a mouse pathogen that mimics EPEC pathogenesis), NleB functions as an O-linked N-acetyl-d-glucosamine (O-GlcNAc) transferase and targets glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which is a co-activator of TRAF2. NleB-mediated O-GlcNAcyla-tion of GAPDH prevents the interaction between GAPDH and TRAF2, thereby inhibiting self-ubiquityla-tion of TRAF2, which is essential for NF-κB activation81 (FIG. 2c). Tir — another effector that is secreted by EPEC

R E V I E W S



TGFβ signalling(Transforming growth factor β signalling). Members of the TGFβ family are cytokines that are involved in the regulation of cell proliferation, differentiation and apoptosis.

Papain-like hydrolytic foldCertain hydrolytic enzymes have a Cys-His-Gln catalytic triad fold, which is conserved in papain cysteine protease.

and EHEC — is essential for pedestal formation and also inhibits host innate immune responses by targeting ubiquitin-dependent signalling. Tir has two immuno-receptor tyrosine-based inhibition motifs (ITIMs) and, following phosphorylation, binds to protein-tyrosine phosphatases SHP-1 (also known as PTPN6) and SHP-2 (REFS 82,83), facilitating the recruitment of SHP-1 or SHP-2 to TRAF6. Direct interactions between SHP-1 or SHP-2 and TRAF6 prevent the ubiquitylation of TRAF6, which results in the inhibition of downstream signalling cascades, such as those that are controlled by NF-κB and MAPK82,83 (FIG. 2c).

Interfering with ubiquitin‑like signallingSeveral ubiquitin-like proteins (UBLs), which have similar sequences and three-dimensional structures to ubiquitin, also function as protein modifiers in eukary-otes, including small ubiquitin-like modifier (SUMO), NEDD8, ISG15, ATG8, ATG12 and LC3. Similarly to the ubiquitin system, UBLs are covalently attached to target proteins by an enzymatic cascade (which involves E1 or E1-like, E2 or E2-like and E3 enzymes) and affect many biological processes, such as transcription, sig-nal transduction, the cell cycle, autophagy and DNA repair10,84.

Targeting sumoylation. Sumoylation is a reversible and essential post-translational modification in eukaryotic cells, in which the ubiquitin-like protein SUMO is cova-lently attached to target proteins by a three-step enzy-matic cascade that involves E1, E2 and E3 enzymes. In contrast to ubiquitin systems, in which several dozen E2 enzymes have been identified, UBC9 is the only E2 enzyme that has been identified for the sumoylation pathway so far10,85. Although some viral factors target or mimic sumoylation components and increase or decrease the sumoylation of host proteins86,87, the role of this modification in bacterial infection remained elusive until a recent study in Listeria monocytogenes88 showed the possible involvement of sumoylation in host defence. During L. monocytogenes infection, the level of sumoylated host proteins decreases, owing to the activity of the pore-forming toxin listeriolysin O (LLO) (FIG. 3a). LLO targets UBC9, resulting in its degradation in a proteasome-independent manner. The levels of sumoylated host proteins decline further as the loss of UBC9 indirectly promotes desumoylation, as the activity of host desumoylases is unperturbed, and other sumoylated host proteins are subject to proteaso-mal degradation88 (FIG. 3a). LLO-mediated degradation of UBC9 impairs downstream TGFβ signalling, which is involved in host resistance to L. monocytogenes infec-tion88. Inhibition of the sumoylation system might also be widely used by some Gram-positive bacterial patho-gens as a mechanism to counteract host defence systems: pore-forming bacterial toxins, such as perfringolysin O (PFO) from Clostridium perfringens and pneumolysin (PLY) from Streptococcus pneumoniae, also trigger the degradation of UBC9 (REF. 88).

Targeting neddylation. Some bacterial pathogens also target neddylation for their own benefit. Similarly to ubiquitin, the UBL NEDD8 is conjugated to substrate proteins and modulates their function. NEDD8 can be deconjugated from its substrates by the action of dened-dylases, in a process known as deneddylation89. Recent reports have shown that the EPEC T3SS effector Cif tar-gets and deamidates NEDD8, thereby interfering with its function90–92 (FIG. 3b). Cif and CHBP (Cif homologue in B. pseudomallei) have a papain-like hydrolytic fold with a Cys-His-Gln catalytic triad; both proteins function as cyclomodulins that block cell cycle progression by caus-ing G1–S and G2–M arrest93–96. Cell cycle progression is modulated by several regulators, such as the multiprotein


LLO

UBC9

Cullin-RING E3 Cullin-RING E3

Protein Protein

UBC9


Proteasome-independent degradation

TGFβ signalling

Cell cycle arrest

SUMO SUMOSUMOE3 SUMO Desumoylase

Desumoylation

Substrate SubstrateUb

UbUb

UbUb

Ub

E2Ub

E2Ub

NEDD8

CRL

NEDD8 NEDD8

Deneddylation

NEDD8

Cif or CHBP

a

b

E

EQ

Q

Figure 3 | Bacterial hijacking of ubiquitin-like systems. a | Listeria monocytogenes prevents sumoylation of host proteins by secreting the pore-forming toxin listeriolysin O (LLO). LLO triggers degradation of the small ubiquitin-related modifier (SUMO) E2 enzyme UBC9, which downregulates the sumoylation of host proteins. LLO-mediated UBC9 degradation occurs in a proteasome-independent manner and leads to the inhibition of transforming growth factor β (TGFβ) signalling. LLO also has indirect effects on the levels of sumoylated proteins by promoting desumoylation indirectly (owing to the unperturbed activity of host desumoylases) and owing to the proteasomal degradation of other sumoylated host proteins. b | Cif, which is produced by enteropathogenic Escherichia coli (EPEC), and CHBP, which is produced by Burkholderia pseudomallei, both have deamidase activity, which prevents the neddylation of host proteins. These effectors target NEDD8 and convert its Gln40 residue to Glu40 (Q40E). Cif-mediated NEDD8 deamidation promotes the deneddylation of Cullin, thereby inhibiting the activity of NEDD8-conjugated cullin-RING E3 ligase (CRL). Inhibition of CRL complexes contributes to cell cycle arrest, thereby reducing the rate of epithelial turnover, which promotes bacterial colonization of the intestinal mucosa.

R E V I E W S



CyclomodulinsBacterial effectors and toxins that inhibit eukaryotic cell cycle progression.

Actin stress fibreBundles that are composed of approximately 10–30 actin filaments; they have important roles in morphological stability, adhesion and motility.

Guanine nucleotide exchange factors (GEFs). Proteins that activate GTPases by catalysing the conversion of the inactive GDP-bound form to the active GTP-bound form.

GTPase-activating proteins (GAPs). Proteins that inactivate GTPases by catalysing the hydrolysis of GTP to GDP.

complex cullin-RING E3 ligase (CRL). Neddylation of cullin subunit proteins is essential for CRL activity as it promotes the ubiquitylation and proteasome-depend-ent degradation of proteins that are targeted by CRL97. Cif selectively binds to NEDD8 and converts Gln40 to Glu40. The deamidated form of NEDD8 (NEDD8 Q40E mutant) dissociates more efficiently from CRL than wild-type NEDD8, which increases the cellular levels of deneddylated (that is, inactive) CRLs98 (FIG. 3b). Perturba-tion of CRL activity as a result of Cif-mediated deamida-tion causes the accumulation of CRL substrates, includ-ing p21, p27 and RHOA, leading to cell cycle arrest and inhibition of actin stress fibre formation in EPEC-infected cells90,91. Although NEDD8 is highly similar to ubiqui-tin (the two proteins share more than 50% amino acid sequence identity), Cif has substantially lower activity with ubiquitin as a substrate90–92. Like Cif, CHBP binds to and deamidates both ubiquitin and NEDD8 but has a preference for NEDD8. Recent crystallographic analysis of a CHBP–ubiquitin–NEDD8 complex has indicated that ubiquitin–NEDD8 recognition by CHBP resembles the recognition mechanism that is used by the ubiquitin enzyme E1 (REF. 99). Furthermore, molecular dynamics simulations have shown that Glu31 of NEDD8, which corresponds to Gln31 of ubiquitin, mediates electrostatic interactions that are crucial for determining the prefer-ence of CHBP for NEDD8 over ubiquitin. In support of this model, CHBP binds to and deamidates a ubiq-uitin Q31E mutant as efficiently as NEDD8 (REF. 99). The activities of Cif and CHBP downregulate host cell cycle progression, which reduces the rate of epithelial turnover, thereby promoting bacterial colonization of the intestinal mucosa100,101.

Manipulation of the host ubiquitin systemSome bacterial pathogens use the host ubiquitin sys-tem to direct the temporal regulation and subcellular localization of their effector proteins, as described below (TABLE 1).

Temporal regulation. S. Typhimurium enters non-phagocytic cells by delivering T3SS effector proteins, which trigger actin cytoskeleton remodelling and sub-sequent bacterial uptake22. To generate membrane ruffles that enable entry into the cell, S. Typhimurium targets RHO GTPases, which function as molecular switches to manipulate actin dynamics. During bacterial invasion, the activities of RHO GTPases are under stringent con-trol by two classes of regulatory proteins: guanine nucleo-tide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Some S. Typhimurium effectors mimic the activities of GEF and GAP proteins to facilitate efficient bacterial internalization. For example, SopE functions as a GEF for RAC1 and CDC42, promoting the GTP-bound forms of both proteins, which enables bacterial uptake by inducing membrane ruffling102–104; by contrast, SptP functions as a GAP to inactivate the RHO GTPases that are induced by SopE, thereby restoring the integrity of the actin cytoskeleton and terminating bacterial inva-sion105. The sequential activation of the opposing func-tions of SopE and SptP contributes to the correct timing

of membrane ruffling during bacterial invasion, and this temporal regulation of SopE and SptP is thought to be essential for efficient S. Typhimurium invasion. Indeed, when either SopE or SptP are mutated, the efficiency of invasion decreases102–105.

S. Typhimurium uses the host ubiquitin-proteas-ome system to regulate the half-lives of SopE and SptP (FIG. 4a). SopE is ubiquitylated and degraded earlier than SptP, which is essential for preventing the formation of membrane ruffles after bacterial invasion106. This results in longer-lived SptP, which predominates later in infec-tion. The GAP activity of SptP switches off CDC42 and RAC1 to terminate membrane ruffling following inter-nalization, resulting in efficient bacterial colonization (FIG. 4a). It is not known whether the E3 ligase for SopE and SptP ubiquitylation is provided by the bacterium or by the host cell. However, it is clear that S. Typhimurium hijacks the host ubiquitin–proteasome system to opti-mize the activities of each effector by temporal regula-tion; this arrangement seems to be an outcome of the co-evolution of the bacterium with its host106.

A recent report showed that the L. pneumophila T4SS effector LubX functions as a ‘meta-effector’ that tem-porally regulates the function of another effector, SidH, within host cells33 (FIG. 4b). LubX, which has U-box-type E3 activity, targets SidH for ubiquitylation and protea-somal degradation (FIG. 4b). Although the precise role of SidH in L. pneumophila infection remains unclear, LubX is expressed and delivered into host cells later in infec-tion than SidH, which results in LubX-dependent regu-lation of SidH function during the late stages of infec-tion. The importance of LubX meta-effector function has been confirmed in a Drosophila melanogaster infec-tion model, in which the lethality rate and the number of viable bacterial cells in flies that had been infected with the ΔsidH ΔlubX double mutant were lower than in flies that had been infected with the ΔlubX single mutant. Thus, L. pneumophila has evolved to use ubiquitylation as a strategy to block an effector function, which seems to be important for pathogenesis33.

Spatial regulation. In addition to temporal regulation, bacterial pathogens exploit host ubiquitin systems to achieve spatial regulation. For example, the SopB effec-tor of S. Typhimurium has phosphoinositide phos-phatase activity and has at least three distinct functions during S. Typhimurium infection that are determined by its subcellular localization. At the earliest stage of infec-tion, SopB localizes to the host plasma membrane, where it promotes bacterial invasion via the activation of SGEF (SRC homology 3 domain-containing guanine nucleo-tide exchange factor), which results in the formation of GTP-bound RHOG, leading to remodelling of the actin cytoskeleton and membrane protrusion107 (FIG. 4a). At the plasma membrane, SopB also activates AKT, thus pro-moting intracellular survival of the bacterium108. Later during infection, SopB localizes to the SCV, where it modulates membrane trafficking by altering phospho-inositide composition via the generation and mainte-nance of phosphatidylinositol 3-phosphate on the SCV membrane, thereby preventing the delivery of bacteria

R E V I E W S



Vesicular membranesComplex structures composed of a lipid bilayer that contains transmembrane proteins and encloses soluble hydrophilic components that are derived from the cytosol of a donor cell.

F-box domainA 42–48 amino acid domain that is involved in polyubiquitylation in eukaryotic cells.

to lysosomes. Trafficking of SopB from the plasma mem-brane to the SCV is mediated by ubiquitylation109; during the late stage of infection, SopB is monoubiquitylated at multiple lysine residues (that is, multimonoubiquityla-tion), which functions as a signal to relocate to the SCV (FIG. 4a). A ubiquitylation-deficient SopB mutant that is retained at the host plasma membrane can still stimulate actin cytoskeleton remodelling and AKT activation, but it fails to promote intracellular proliferation, owing to its inability to undergo ubiquitin-mediated SCV localiza-tion109. Thus, the differential localization of SopB dur-ing infection, which is achieved by exploiting the host ubiquitin system, enables S. Typhimurium to modu-late several distinct stages of infection using the same effector.

Subcellular targeting of bacterial effectors to the host cell plasma membrane, vesicular membranes, mitochon-dria, the Golgi and the nucleus, as well as their temporal

regulation during infection, has an important role in accurately targeting and optimizing the enzymatic activ-ities of effectors within the host cell110. Other targeting mechanisms, such as those that involve prenylation, myristoylation and palmitoylation, also contribute to the subcellular targeting of bacterial effectors110.

Nutrient acquisition. Bacteria hijack the host ubiquitin system for another reason that is distinct from temporal and spatial regulation: to obtain nutrients and promote intracellular bacterial replication. For example, L. pneu-mophila uses the host ubiquitin-proteasome system to generate amino acids111 (FIG. 4b). This is achieved by the T4SS effector AnkB; AnkB is essential for intracellular bacterial survival, and ankB-deficient mutants have severe defects in proliferation112,113. Within host cells, AnkB is anchored to the LCV membrane (via host-mediated farnesylation of the AnkB F-box domain of the


a b

Membrane ruffling

Actin

S. Typhimurium

RHOGRAC1

Ub

GTP

CDC42

GTPGTP

RAC1

GDP GDP

GDP

CDC42

SopB

SCV

SopE

SopB

UbUb

UbUb



Lys48-ubiquitylated proteinBacterialproliferation

L. pneumophila

Amino acidsEarly ininfection

Late ininfection

RHOG SGEF

Ub

Intracellular survival

AnkB

SidH LubX

UbUb

UbUbUb

SptP

Lysosome

Figure 4 | Temporal and spatial regulation of ubiquitin pathways by bacterial pathogens. Salmonella enterica subsp. enterica serovar Typhimurium and Legionella pneumophila hijack the host ubiquitin-proteasome system in order to achieve temporal and spatial regulation of type III secretion system (T3SS) and type IV secretion system (T4SS) effectors. a | S. Typhimurium delivers SopE and SptP, which mimic the roles of guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) enzymes, respectively, by targeting the RHO GTPases RAC1 and CDC42. The activity of these GTPases is crucial for triggering actin cytoskeleton remodelling and subsequent bacterial uptake. SopE promotes the GTP-bound form of both proteins, which triggers membrane ruffling, whereas SptP induces the GDP-bound form of both proteins and aborts membrane ruffling. Although SopE and SptP undergo ubiquitylation and proteasomal degradation, SopE is degraded earlier than SptP. The differential timing of SopE and SptP ubiquitylation and subsequent proteasomal degradation contributes to proper actin cytoskeleton remodelling and efficient bacterial invasion. Another S. Typhimurium effector, SopB, localizes to the host cell plasma membrane and promotes membrane protrusion via the activation of SRC homology 3 domain–containing guanine nucleotide exchange factor (SGEF) — an exchange factor for RHOG — which is involved in actin cytoskeleton remodelling. Later in the infection, SopB is monoubiquitylated, and this modification functions as a signal that is required for the removal of SopB from the plasma membrane. SopB relocates to the Salmonella-containing vacuole (SCV), where it prevents phagosome–lysosome fusion and acidification of the SCV, thereby promoting intracellular bacterial survival. b | The L. pneumophila E3 effector LubX temporally regulates another effector protein, SidH. Although the function of SidH is currently unclear, the delayed delivery of LubX targets SidH for ubiquitylation and proteasomal degradation late in infection. L. pneumophila proliferates within the Legionella-containing vacuole (LCV), and the effector AnkB is anchored onto the LCV membrane via host-mediated farnesylation. This results in the recruitment of Lys48-linked polyubiquitylated proteins to the LCV membrane, leading to their proteasomal degradation, which generates amino acids to promote intracellular bacterial replication. Ubiquitin (Ub) molecules shown in green are involved in signal transduction, whereas ubiquitin molecules shown in orange target proteins for degradation.

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effector) and recruits Lys48-linked polyubiquitylated proteins to the LCV111,114. The polyubiquitylated pro-teins that are recruited by AnkB are degraded by the proteasome, resulting in the generation of amino acids, which are a major source of carbon and energy for use in bacterial proliferation (FIG. 4b). Importantly, the intracel-lular growth deficiency of an L. pneumophila ankB-null (ΔankB) mutant can be rescued in vivo by supplementa-tion with amino acids. Together, these findings indicate that L. pneumophila has evolved a strategy for promo-ting intracellular proliferation by exploiting the host ubiquitin-proteasome system111.

Summary and outlookUbiquitin (and UBL)-dependent post-translational modification systems have important roles in several aspects of bacterial pathogenesis as well as in host defence responses. As described in this Review — and in excellent previous articles1–8 — although bacterial patho-gens lack their own ubiquitin systems, they use special-ized mechanisms to modulate host ubiquitin systems for their own benefit — for example, by interfering with host ubiquitin-mediated cell signalling, deubiquitylation of host proteins and spatial and temporal exploitation of ubiquitin to maximize T3SS and T4SS effector func-tions. These mechanisms result in the modulation of cell cycle progression, the downregulation of inflammatory responses and the circumvention of ubiquitin-dependent (and ubiquitin-independent) autophagy (BOX 2).

Clearly, our understanding of the molecular basis that governs the interplay between bacterial effectors and host ubiquitin systems has advanced greatly during the past decade. However, we still have much to learn about the details of the mechanisms involved and the outcome during infections in vivo. For example, identification of the form of ubiquitylation (that is, whether it is mon-oubiquitylation, polyubiquitylation or an unanchored linkage) that is catalysed by bacterial E3 ligases would provide information on the fate of the substrate protein. Furthermore, although many bacterial E3 ligases have been identified, only a few of their substrates have been found. This is, in part, because the number of bacterial effectors delivered into host cells during infection is typi-cally extremely low, and it is therefore difficult to detect endogenous bacterial effectors and to monitor their interactions with target host proteins. In addition, some effectors that have E3 ligase activity are highly unstable, owing to their self-ubiquitylation activity within host cells, so they often undergo rapid proteasome-dependent

degradation. To further complicate the issue, interac-tions between bacterial effectors and host factors are thought to occur as transient and dynamic events, which are temporally and spatially controlled by both the pathogen and the host. Advances in bioinformatics and systems biology tools should facilitate the identification of host targets and thereby improve our understanding of bacterial pathogenesis. Furthermore, whole-genome sequencing of bacterial pathogens should prove to be useful for the detection and identification of novel effec-tor proteins that interfere with the host ubiquitin system (for example, E3 ligases, DUBs and ubiquitin-interaction motifs, such as UBDs and F-boxes). To understand the mechanisms of action of such novel or unidentified effectors, we need to exploit systems-wide approaches — this is exemplified by phosphoproteome analysis of S. Typhimurium and S. flexneri infection115–117.

Bacterial pathogens use a broad range of factors that have E3 ligase activity, including NEL-type, HECT-type and RING–U-box-type E3 ligases. The NEL group of effectors, which are represented by S. flexneri IpaH and S. Typhimurium SspH2, are distinct from the typical HECT-type and RING–U-box-type E3 ligases, and they are produced by several Gram-negative bacterial patho-gens. Therefore, the development of novel antibacterial drugs to specifically inhibit bacterial E3 ligase activity has potential. Such antivirulence drugs have predicted benefits compared with traditional antibiotics, as they only target virulence and not bacterial viability; as such, they represent an approach to minimize the emergence of drug-resistant bacteria.

Finally, as mentioned above, our current knowledge of the interplay between pathogens (and their effec-tors) and host ubiquitin systems at the single-cell level and under in vivo conditions is still poor compared with our knowledge of the interactions that occur under in vitro conditions. To circumvent this problem, it is desirable to develop or improve rodent infection models, which should ideally mimic natural bacterial infections, including human diseases. Furthermore, in addition to improving conventional methodologies, we must also take advantage of interdisciplinary and integrated research approaches, such as highly sensi-tive live-imaging systems, in silico analyses, structural biology and bioinformatics to further develop the field. Such efforts have the potential to rapidly increase our understanding of the biological implications of the interplay between bacteria and the host ubiquitin system during infection.

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AcknowledgementsThis work was supported by a Grant-in-Aid for Specially Promoted Research (grant number 23000012 to C.S), a Grant-in-Aid for Young Scientists (A) (grant number 23689027 to M.K), a Grant-in-Aid for Scientific Research on Innovative Areas (grant numbers 25121711 and 25112506 to M.K) and a Grant-in-Aid for Scientific Research (C) (grant number 25460527 to H.A). This work was also supported by research grants from the Yakult Bio-Science Foundation and the Nippon Institute for Biological Science, Japan.

Competing interests statement The authors declare no competing interests.

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