Post-translational Mechanisms of Host Subversion by Bacterial Effectors

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Review

Post-translational Mechanisms of Host Subversion by Bacterial Effectors Nichollas E. Scott1 and Elizabeth L. Hartland2,3,* Bacterial effector proteins are a specialized class of secreted proteins that are translocated directly into the host cytoplasm by bacterial pathogens. Effector proteins have diverse activities and targets, and many mediate posttranslational modifications of host proteins. Effector proteins offer potential in novel biotechnological and medical applications as enzymes that may modify human proteins. Here, we discuss the mechanisms used by effectors to subvert the human host through blocking, blunting, or subverting immune mechanisms. This capacity allows bacteria to control host cell function to support pathogen survival, replication and dissemination to other hosts. In addition, we highlight that knowledge of effector protein activity may be used to develop chemical inhibitors as a new approach to treat bacterial infections.

Trends Bacterial effector proteins contribute to the success of bacterial infections by interfering with host defense pathways. Bacterial effector proteins are increasingly being recognized as new families of enzymes. Recent developments in mass spectrometry technologies are enabling an easier characterization of effector protein activities. Some highly novel post-translational modifications have been identified in bacterial effectors; these include arginine glycosylation or b elimination of phosphorylated threonine residues.

Bacterial Effector Proteins and Their Modification of Host Cell Biology The translocation of bacterial effector proteins into host cells during infection is a wellestablished virulence mechanism and occurs in a controlled manner, requiring specific molecular triggers. Several bacterial protein secretion machines deliver effector proteins directly into the host cell but the best-characterized systems are known as type III and type IV secretory systems, which are ancestrally related to flagellae and DNA conjugation systems respectively [1,2]. Effector numbers range from tens to hundreds depending on the pathogen. Where bacteria interact with many hosts, effector diversification and expansion is evident, whereas effector rationalization occurs in highly host-adapted organisms [3]. The effector repertoires of pathogens typically target host responses, and sometimes simultaneously target multiple steps within the same pathway leading to functional redundancy between codelivered effectors. Once introduced, effector proteins can be effective at low levels due to their potent biochemical activities such as glycosylation, acetylation, or ADP ribosylation of host proteins. Bacterial effector mechanisms encompass four broad categories of activity (Figure 1 and Table 1, Key Table). First, there are effectors that mediate their activities through physical interactions with host targets. Second, some effectors use functional mimicry (see Glossary) such as host motifs or structural mimicry to subvert host cell processes. This might be related directly to enzymatic function, such as guanine exchange factor (GEF) activity, or be a means of regulating effector activity, such as lipidation motifs. Third, effectors can carry out posttranslational modifications of host proteins. Depending on the modification, these may be permanent or reversible marks, with the same bacteria sometimes supplying the reversible activity. Lastly, some effectors are novel proteases that can lead to changes in the spatial organization or composition of the host proteome. Of course, these broad groupings are not mutually exclusive, as bacterial effectors may have more than one functional domain.

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1

Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia 2 Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton 3168, Australia 3 Department of Molecular and Translational Science, Monash University, Clayton 3168, Australia

*Correspondence: [email protected] (E.L. Hartland).

https://doi.org/10.1016/j.molmed.2017.10.003 © 2017 Elsevier Ltd. All rights reserved.

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Given that a precise understanding of the biochemical mechanism of action of bacterial effector proteins is now possible, the question is whether we can exploit the novel activities of these virulence factors for therapeutic benefit, such as engineering T cell activation in a synthetic biology approach to improve the safety of adoptive immunotherapy [4] or by targeting the activities of effectors themselves for the development of new antimicrobial agents [5]. Many of these principles also apply to classical bacterial toxins, for example, botulinum toxin, which has been successfully developed for therapeutic and cosmetic use [6], and diphtheria toxin, which has been exploited as an experimental anticancer therapy [7], [36_TD$IF]and used to create transgenic mice that conditionally express the human diphtheria toxin receptor so that distinct immune cell populations may be depleted [8].

Blocking or Hijacking Host Cell Function through Direct Effector Interactions A physical interaction between effectors and host proteins is the simplest means by which effectors can exert control within the host cell, since just the target is required, but no other host cofactors or substrates. By inhibiting host protein function through direct binding, effectors can limit the antimicrobial host response by blocking apoptotic and inflammatory signaling [9,10]. Depending on the affinity, such interactions between effectors and host proteins may be targeted for inhibition to attenuate bacterial virulence. Examples of effectors that inhibit apoptosis and pyroptosis by direct binding to host targets are found in the diarrheal pathogens, enteropathogenic Escherichia coli (EPEC) and Shigella. The effector NleF from EPEC acts by binding directly to host caspase-8, -9, and -4 and inserting a short C-terminal domain into the active site of these enzymes [9]. The insertion of this tail sterically blocks access to the caspase catalytic site thereby inhibiting protease activity [9]. Hence, NleF prevents the amplification of proapoptotic signals and limits activation of noncanonical caspase-4 during EPEC infection of human cells [9,11,12]. Similarly, the effector OspC3 from Shigella binds the active caspase-4 subunit, p19, preventing its oligomerization with p10, which is required for proteolytic activity [10]. The inhibition of p19 prolongs epithelial cell viability during Shigella infection of human cells, thereby enhancing bacterial replication, and also limits the release of proinflammatory cytokines [10]. Direct binding of effectors is not restricted to protein–protein interactions, with several effectors now shown to bind host DNA. The archetype of this class of effectors is AnkA from the rickettsial pathogen, Anaplasma phagocytophilum, which infects and replicates in human neutrophils. AnkA binds to AT-rich chromatin sites leading to alterations in the expression of genes associated with antibacterial defense, including CYBB, RAC2, BPI, and MPO in human neutrophils and HL-60 cells [13]. The effect of AnkA on host gene transcription depends on AnkA-associated recruitment and activity of host histone deacetylase (HDAC)1; indeed, pharmacological inhibitor of HDAC1, mocetinostat, abolishes AnkA dependent suppression of gene transcription in human HL-60 cells [14]. Hence, the direct binding of host proteins and DNA by bacterial effectors can be an efficient means to alter host cell function to the advantage of the pathogen.

Fake it ‘Til They Make it: Mimicry of Host Proteins Structural Mimicry Host mimicry is a common strategy utilized by bacterial pathogens and includes the mimicry of host cell function and imitation of host motifs [15]. Host motif mimics are normally, but not always, identifiable by their amino acid sequence similarity to known eukaryotic motifs. Structural mimicry may also occur without amino acid sequence similarity being evident. This

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Glossary Apoptosis: regulated, noninflammatory cell death that requires activation of host cell caspases. Atg8 protein family: small proteins with a ubiquitin-like fold that are directly conjugated to a target membrane during autophagy. The human protein member is LC3, which is commonly used as a marker for autophagosomal membranes in human cells. Autophagic flux: activity of organelle degradation by autophagy, which delivers cellular components to the lysosome. CRISPR-based technologies: CRISPR stands for clustered regularly interspaced short palindromic repeats and is a bacterial defense system that has been repurposed to allow genomic editing in a wide range of organisms including humans. Familial Mediterranean fever: autoinflammatory disease characterized by recurrent episodes of fever and joint pain caused by gain of function mutations in the pyrin inflammasome protein. Focal adhesion complexes: subcellular protein complexes that connect the cell cytoskeleton to extracellular matrix proteins in response to adhesion Functional mimicry: bacterial protein mimics the function or activity of a host protein without necessarily sharing structural or amino acid sequence similarity. Mass spectrometry: an analytical technique that allows the measurement of all masses in a sample through ionization of the sample and sorting of the ions according to their mass-to-charge ratio. Necroptotic signaling: signaling leading to necroptosis; a form of caspase-independent cell death requiring -containing proteins. PDZ-binding motif: short Cterminal motif that binds to a 80–90 structural motif termed the post synaptic density protein Drosophila disc large tumor suppressor zonula occludens-1 protein (PDZ). Pyroptosis: regulated, inflammatory cell death that requires activation of the inflammasome related caspases and release of IL-1 and IL-18.

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is the case for the NleG effector family of E3 ubiquitin ligases from enterohemorrhagic E. coli, which do not resemble eukaryotic E3 enzymes at the amino acid level but share significant structural and functional similarity [16]. This mimicry may also allow effectors to be targeted by host modification systems, such as ubiquitination or SUMOylation [17,18]. In [34_TD$IF]some cases, modification by the host system leads to the regulation of effector activity, ensuring that effector function is restricted to the host cell environment. This is the case for the Salmonella effector, SopB, where monoubiquitination downregulates SopB inositol phosphatase activity at the plasma membrane [17]. However, whether this monoubiquitination is performed by host or bacterial E3 ligases is not clear [17]. Structural mimics may be difficult targets for the development of antimicrobial agents, due to their similarity to host factors. Nevertheless, these effectors may be useful tools to understand host cell biology and cellular responses to infection. Functional Mimicry Numerous functional mimics have been identified among bacterial effectors, including some that are conserved across bacterial genera. Within the effector repertoires of EPEC, Salmonella, and Shigella, a conserved class of effectors containing a WxxxE [34_TD$IF]amino acid motif activate Rholike GTPases by acting as functional mimics of GEFs [19–21]. The GEFs appear to enhance bacterial infection based on evidence showing that WxxxE effector gene deletion mutants are attenuated in mouse infection models and in vitro models of human epithelial cell invasion [22]. In fact, there are many examples of bacterial effectors from diverse mammalian, plant, protozoan, and insect pathogens that function as GEF proteins for host GTPases [345_TD$IF][110].

Q-fever: serious respiratory disease caused by the bacterium Coxiella burnetii, transmitted by farm animals, originally termed query (Q) fever. RHIM-containing proteins: signaling adaptor proteins carrying an RIP homotypic interaction motif (RHIM). RHIM-containing proteins, including TRIF, RIPK1, RIPK3 and ZBP1, are involved in inflammatory and necroptotic signaling. SNARE: host proteins that mediate the tethering of vesicles to a target membrane. Structural mimicry: bacterial protein shares structural or amino acid sequence (motif) similarity with a host protein.

Host Activation of Effector Activity The activation of an effector by the host cell has also been observed for some bacterial effectors. In the case of the acute respiratory pathogen, Legionella pneumophila, the effector VipD binds to host Rab5, a GTPase that regulates endosomal–lysosomal trafficking, and this binding is essential for the activation of VipD phospholipase A1 activity in vitro and in transfected COS-1 cells [23,24]. Upon binding to Rab5, VipD undergoes a conformational change exposing the phospholipase domain [23], leading to the depletion of the essential tethering lipid, phosphaptidyinositol-3 phosphate [24]. This depletion results in the loss of endocytic trafficking of proteins from endosomal membranes, thereby blocking endosomal fusion with Legionellacontaining vacuoles in CHO-FcdRII cells [24]. Similarly, the YopO effector from pathogenic Yersinia bears no serine/threonine kinase activity until translocated to the host cell; in this case, human epithelial HEK293T and HeLa cells [25], whereupon YopO associates with actin, leading to autophosphorylation and the subsequent phosphorylation of host substrates [25]. Indeed, structural analysis has shown that YopO binding to G-actin can stabilize its catalytic loop, allowing kinase function and the modification of actin binding proteins in human cells, including macrophages [26,27], leading to disrupted phagocytosis. In contrast to VipD and YopO, activation of the effector YopJ from Yersinia is not mediated by host protein binding but rather, by the small metabolite inositol hexakisphosphate (IP6), only found within eukaryotic cells [28]. Within mammalian cells, YopJ associates with IP6 to initiate a conformational change that leads to a marked increase in the acetyltransferase activity of YopJ and subsequent acetylation of serine and threonine residues in the activation loops of mitogenactivated protein kinases (MAPK) in the MAPK pathway and IKK a/b kinases in the nuclear factor (NF)-kB pathway [28]. IP6 binding leads to structural stabilization of YopJ that facilitates binding of the effector substrate, acetyl coenzyme A, and subsequent acetyltransferase activity [29]. The requirement for IP6 binding to enable complete activation is observed across YopJ homologs from all human pathogenic Yersinia species and Salmonella, suggesting this process appears to be a conserved and important feature of this effector family [28]. Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

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Physical binding (compeve acvators/ compeve inhibitors/ steric inhibitors)

+

Caspase-8

Mimicry of funcon (allosteric acvators/ allosteric inhibitors/ host substrate)

PTM mediators (transferases/ hydrolase/ chemical conversion) Rab1

NleF

Rho

+

GDP GTP

+ Caspase-8

NleF

SopE

+

Addion of modificaon

+ GTP GDP

Rho

MAPK

Chemical conversion

VipD

SifA

SifA

Rab5

Compeve inhibitor

RavZ

LC3

G

Effector lipidaon

Removal of lipidaon

OspF

Rab5

GTPase acvaon EEA1

+

LC3

P MAPK

SopE EEA1

Pc Rab1

AnkX

Steric inhibion of acvaon

VipD

Proteases (terminal proteases domain specific proteases) /

P

p65

+

NleC

FAK

+

FAK

YopH Removal of modificaon

p65

Cleavage within domain

Figure 1. Broad Classes of Bacterial Effector Mechanisms In Host Cells. We have classified effectors into four categories based on their mode of action. (i) Effectors that exert function through physical interactions with host proteins, such as NleF and VipD; (ii) Effectors that use functional mimicry, such as SopE, which is a guanine nucleotide exchange factor for host GTPases and SifA that is lipidated by host proteins; (iii) Effectors that mediate post-translational protein modifications to block host protein functions such as AnkX, OspF, and YopH. AnkX functions by transferring phosphocholine to Rab1, OspF destroys the site of phosphorylation of MAPKs by b elimination and YopH is a protein tyrosine phosphatase that dephosphorylates peripheral focal complexes. (iv) Effectors that change the spatial organization or composition of the host proteome by proteolysis, such as RavZ, which removes the terminal phosphoethanolamine-linked glycine residue of ATG8 and NleC, which cleaves the Rel homology domain of RelA/p65 preventing NF-kB activation. Abbreviations: ATG8, autophagy-related protein 8; EEA1, early endosome antigen 1; FAK, focal adhesion kinase; MAPK, mitogen activated protein kinase; NF-kB, nuclear factor kB; RHO, Ras homolog gene family; Zigzag line indicates lipids.

Host Motif Mimicry A common form of mimicry used by bacterial effectors is the exploitation of host motifs for subcellular protein trafficking [30]. Some effectors are activated by host caspases such as SipA from Salmonella, which contains a caspase-3 cleavage site [31]. Other effectors are tethered to specific membrane compartments by host-mediated lipidation in a variety of host cells, including mammals and lower eukaryotes such as amoebae. In the case of the Salmonella WxxxE effector, SifA, the last six amino acids, 331[342_TD$IF]CLCCFL336, harbor multiple host lipidation motifs, which when deleted, lead to loss of correct membrane localization and effectorassociated phenotypes [32]. Analysis of these modifications has revealed that SifA is prenylated at C333 and S-acylated at C331, with prenylation of C333 being required to achieve wild-type levels of bacterial replication in mouse RAW 264.7 macrophages and mouse livers [32]. Similarly, the Salmonella effectors SspH2 and SseI are modified with host lipids via palmitoyltransferases at a conserved cysteine within the N terminus of these proteins [33]. Moreover, exploitation of host-mediated effector lipidation has also been seen for many effectors from L. pneumophila [34]. One example is the Soluble NSF attachment protein receptor (SNARE) mimic, LseA, in which mutation of the C-terminal CaaX prenylation motif abolishes farnesylation, thus resulting in loss of LseA Golgi localization in mouse RAW 264.7 macrophages [35].

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Key Table

Table 1. Summary of Selected Bacterial Effector Mechanisms and Host Targets Effector proteins

Pathogen

Mechanism

Host target

Refs

NleF

EPEC

Direct binding

Caspase-8, -9, and -4

[9,11,12]

OspC3

Shigella

Direct binding

Caspase-4 subunit, p19

[10]

AnkA

Anaplasma

Direct binding

AT-rich chromatin sites of host DNA

[13,14]

Map, IpgB2, IpgB1, EspM2, SopEa, SopE2a, SifAb and SifBb

Shigella, EPEC, EHEC, Salmonella

WxxxE host GEF mimics

Rho/Rac GTPases

[19–22,32,36]

LseA

Legionella

Mimicry of host SNARE proteins

Syntaxin 5, 7, and 18, Vti1a, Vti1b, Vamp4, and Vamp8

[35]

Cig57

Coxiella

Host mimicry through the use of a tyrosine-based endocytic sorting motif

FER/CIP4 homology only protein 2 (FCHO2)

[39,40]

SspH22

Salmonella

Modified by host palmitoyltransferases; E3 ubiquitin ligase

SGT1

[33]

YopO

Yersinia

Actin-binding dependent kinase activity

Heterotrimeric G proteins, VASP, other actin binding proteins

[25–27,67–72]

YopJ

Yersinia

Acetylation of serine and threonine residues in signaling proteins

MAPKK6, MAPKK2, IKK-b and TAK1

[28,29,64–66]

VipD

Legionella

Rab5 binding activates phospholipase A1 activity

Phosphaptidyinositol-3 phosphate depletion

[23,24]

NleB1

EPEC

Arg-glycosylation

FADD, TRADD, and RIPK1

[52–54]

Cif

EPEC

Deamidation

Ubiquitin-like modifier NEDD8

[44]

OspI

Shigella

Deamidation

Ubiquitin-conjugating E2 enzyme UBC13

[45,46]

VopC

Vibrio

Deamidation

Rac GTPases

[47]

TecA

Burkholderia

Deamidation

RhoA GTPases

[48,49]

OspF

Shigella

b Elimination

ERK, p38

[50,51]

EspJ

EPEC

Deamidation and ADP ribosylation

Src

[56]

SpvB2[35_TD$IF]

Salmonella

ADP ribosylation

Actin

[57,58]

ExoS

Pseudomonas

ADP ribosylation

Various

[59]

ExoT

Pseudomonas

ADP ribosylation

CT10 regulator of kinases

[59–61]

NleE, OspZ

EPEC, EHEC, Shigella

Cysteine methylation

TAB2/3, [36_TD$IF]zinc finger, RAN-binding domain containing 3 (ZRANB3)

[62,63]

YopH

Yersinia

Tyrosine phosphatase

[67–69,73–77]

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Table 1. (continued) Effector proteins

Pathogen

Mechanism

Host target

Refs

Cas, FAK, paxillin, Fyn-binding protein, and [37_TD$IF]Src kinase associated phosphoprotein 2 (SKAP-HOM) IpaH9.8, IpaH4.5, IpaH2.5 IpaH1.4

Shigella

E3 ubiquitin ligase

Various

[79–83]

SdeA

Legionella

Phosphoribosylation of ubiquitin and deubiquitinase

Rab GTPases and tubular ER protein reticulon 4

[84–86]

SseL2

Salmonella

Deubiquitinase

IkBa and protein aggregations

[89–92]

ChlaDub1

Chlamydia

Deubiquitinase and deneddylase

Various

[88]

ShiCE, RickCE

Shigella, Rickettsia

Deubiquitinase

Various

[87]

AnkX

Legionella

Phosphocholination

Rab1 and Rab35

[93,94]

Lem3

Legionella

Dephosphocholination

Rab1 and Rab35

[93,94]

DrrA/SidM

Legionella

AMPylation

Rab1

[95,96]

SidD

Legionella

De-AMPylation

Rab1

[95,96]

YopT

Yersinia

Cysteine protease

RhoA, Rac, Cdc42

[97,98]

RavZ

Legionella

Cysteine protease

Atg8

[99]

NleC

EPEC, EHEC

Metalloprotease

NF-kB subunits

[100–102]

PipAb[38_TD$IF], GogAb, and GtgAb

Salmonella

Metalloprotease

NF-kB subunits

[104]

EspL

EPEC, EHEC

Cysteine protease

RIPK1, RIPK3, TRIF, ZBP1

[105]

For each effector, the pathogen group, host targets, and mechanism of action are provided, see text for details. Secreted by the Salmonella SPI-1 encoded type III secretion system[340_TD$IF]. b Secreted by the Salmonella SPI-2 encoded type III secretion system[341_TD$IF]. a

The lipid modification of bacterial effectors ensures a level of precision in their localization and site of activity, ensuring that even under conditions of low translocation, effector activity is delivered as efficiently as possible. NleA/EspI, NleH, and Map from EPEC all possess a PDZ-binding motif at the C terminus that facilitates interactions with host PDZ-containing proteins, leading to changes in effector localization [19,36–38]. In the case of the WxxxE effector Map, this PDZ-binding motif facilitates interactions with Ezrin binding protein (EBP)50, which directs the effector to the plasma membrane enabling filopodia formation and actin reorganization in human epithelial HeLa cells and human intestinal Caco-2 and T84 cells [19,36]. The reciprocal situation also occurs, where effectors harbor a eukaryotic binding motif that diverts host proteins to alternative subcellular locations. An example of this is Cig57 from Coxiella burnetii, the causative agent of Q fever, which recruits FER/CIP4 homology only protein 2 (FCHO2) to the Coxiella-containing vacuole in human epithelial HeLa cells, by virtue of its tyrosine-based endocytic sorting motif [39]. By recruiting FCHO2, clathrin-coated vesicles are redirected to Coxiella-containing vacuoles (CCVs). This is believed to enhance intracellular replication of Coxiella given that cig57 mutants 6

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are highly attenuated for intracellular replication and lose clathrin recruitment to CCVs [39,40]. Hence, mimicry of even small eukaryotic protein sorting motifs can have a substantial influence on effector activity and the success of bacterial infection. In addition to direct modification of effectors by host enzymes, many effectors possess binding motifs that can function as adapters to facilitate protein–protein interactions between effectors and host proteins.

Altering Host Cell Function through Post-translational Modification of Host Proteins In recent years, it has become increasingly apparent that a wide range of bacterial effectors mediate their effects through post-translational modification (PTM) of host proteins. These effectors probably offer the best potential for the development of novel anti-infective agents as the bacterial targets are enzymes with distinct biochemical activities, which may be exploited for selective toxicity against bacterial pathogens. A disadvantage of this approach is that inhibitors of these enzymes are not of broad spectrum, as few effector families are shared by all Gram-negative human [347_TD$IF]bacterial pathogens [41]. This has led to a greater focus on the development of drugs that block the secretion systems themselves [42]. However, where the development of bespoke anti-infective agents is warranted, as in the cases of multidrugresistant organisms, or in rare, but highly pathogenic infections, the targeting of effector enzymes emerges as a valid approach. Broadly speaking, effector mediated PTMs can be classified as reversible or irreversible depending on the ability of the PTM to be removed either by host enzymes or other effector proteins. While reversible PTMs provide a means to temporally control host activity, irreversible modifications permanently inactivate or alter host protein function. In addition, such effectors may function by either adding or removing a PTM from a host protein. Irreversible Modifications Irreversible modifications mediated by effectors typically involve the chemical conversion of essential residues to chemically incompatible forms. One means to achieve this is the conversion of glutamine residues to glutamic acid via deamidation, typically mediated through the activities of papain-like deamidase effectors [43]. In the case of the EPEC effector, Cif, conversion of the conserved Q40[346_TD$IF] residue within the ubiquitin-like modifier NEDD8 inhibits the activity of neddylated Cullin-RING ubiquitin E3 ligases (CRLs) and leads to an impairment of ubiquitination and ubiquitin-dependent degradation of CRL substrates [44]. One consequence is accumulation of the small GTPase RhoA, which is thought to contribute to the cytopathic effects observed during EPEC infection of human epithelial HeLa cells [44]. Similarly, OspI from Shigella deamidates Q100 of the ubiquitin-conjugating E2 enzyme UBC13 to abolish tumor necrosis factor (TNF) receptor-associated factor (TRAF)6 polyubiquitylation, thereby inhibiting downstream TRAF6/NF-kB signaling in human epithelial HeLa cells [45,46]. Moreover, ubiquitin-like proteins are not the only targets of papain-like effectors, with the small GTPase Rac also deamidated at Q61 by VopC of Vibrio species [47]. In addition to glutamine deamidases, asparagine deamidase activity has also been noted for the effector TecA from Burkholderia cenocepacia [48]. Translocation of TecA into host cells leads to the inactivation of RhoA at N41, which triggers the pyrin inflammasome to control B. cenocepacia infections [48,49]. Another example of an irreversible PTM mediated by the chemical conversion of amino acid residues is the b-elimination of phosphorylated threonine residues via phosphothreonine lyase activity that converts phosphothreonine to dehydrobutyrine [50]. This modification does not occur naturally within eukaryotic cells and in the case of Shigella; it is mediated by the effector OspF [50]. During Shigella infection, OspF drives the irreversible modification of Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

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phosphothreonine within the pT-X-pY motif of MAPKs in human epithelial HeLa cells [50]. This irreversible block of MAPK phosphorylation results in downstream suppression of histone H3 phosphorylation and the inhibition of IL8, encoding the proinflammatory cytokine, interleukin (IL)-8 in infected human epithelial HeLa cells [50,51]. Irreversible PTMs also occur through the modification of amino acids that are not typically mediated by the host cell. Recently, we and others discovered that the EPEC effector, NleB1, mediates the addition of N-acetylglucosamine (GlcNAc) to arginine within death domain containing proteins, thereby preventing formation of the death-inducing signaling complex (DISC) and blocking extrinsic apoptosis in human epithelial HeLa and HT29 cells [52,53]. ArgGlcNAcylation appears to be resistant to enzymatic removal with known deglycosylating enzymes and hence, remains as a permanent PTM mark within the host cell [52–54]. This means that the infected cell remains protected from apoptosis induced by death ligands such as FasL and TNF [52–54]. Of note, the modification also appears to be highly stable in vitro, which may have useful biotechnological applications in enhancing recombinant protein stability and solubility [54]. ADP ribosylation is a common host PTM that is also mediated irreversibly by bacterial effectors and toxins [55]. In the case of the EPEC effector EspJ, ADP ribosylation of Src kinase results in the inhibition of Src-mediated actin polymerization in mouse J774A.1 macrophages [56]. This inhibition is thought to result from the disruption of an essential salt bridge through the simultaneous amidation and ADP ribosylation of a conserved glutamate residue (E310), yielding glutamine–ADP ribose [56]. Salmonella also disrupts actin networks through ADP-ribosylation of actin by SpvB in infected CHO cells and this occurs via direct modification of actin at arginine177 (R177), as identified by mutagenesis and in vitro ADP-ribosyltransferase assays [57,58]. The ADP-ribosyltransferase activity of SpvB is required for virulence in a mouse infection model as point mutations disrupting the active site of SpvB attenuate the bacteria to a similar degree as a spvB null mutation [57,58]. Similarly, the human pathogen Pseudomonas aeruginosa targets host proteins for arginine ADP ribosylation with two effectors called exoenzyme S (ExoS) and exoenzyme T (ExoT) [59,60]. ExoT predominantly modifies the CT10 regulator of kinases cytoskeletal adaptor proteins (Crk-I and Crk-II), resulting in disrupted phagocytosis [61]. ExoS modifies a large array of proteins including the small GTPase, Ras and moesin, an actinmembrane crosslinking protein, contributing to ExoS-induced cytotoxicity of human epithelial HeLa cells [59,60]. Both ExoT and ExoS are bifunctional effectors that contain N-terminal RhoGTPase activating protein (GAP) activity and C-terminal ADP-ribosyltransferase activity [60]. Hence these toxins can have more than one effect on the host cell cytoskeleton. Other atypical eukaryotic modifications mediated by effectors include cysteine methylation and protein acetylation. NleE from EPEC and OspZ from Shigella methylate cysteine residues, C673/ 692 , in the zinc finger domain of the adaptor proteins, TAB2 and TAB3, thereby blocking their binding to ubiquitinated TRAF proteins and irreversibly blocking NF-kB signaling, leading to a suppression of the proinflammatory response to infection in human epithelial HeLa, HEK293T, and HT-29 cells and mouse bone-marrow-derived dendritic cells [62,63]. YopJ from Yersinia targets the MAPK signaling pathway by acetylating serine and threonine residues [64]. YopJ mediated acetylation occurs within the activation loop of MAPKs, thereby blocking phosphorylation of downstream substrates and halting NF-kB signaling in human epithelial HEK293T and HeLa cells, again leading to a blunted proinflammatory response [65,66]. Hence, bacterial pathogens have evolved multiple means to reduce proinflammatory signaling through the activities of effector proteins, many of which mediate irreversible PTMs.

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Reversible Modifications The phosphorylation of serine, threonine, or tyrosine residues is a reversible and ubiquitous event within eukaryotic cells, and also constitutes a PTM that is commonly augmented by bacterial effectors. For example, within pathogenic Yersinia two effectors alter the phosphorylation status of host proteins, YopO and YopH [67–69]. YopO, also known as the Yersinia protein kinase A (YpkA), phosphorylates heterotrimeric G proteins, Gaq, as well as the vasodilator-stimulated phosphoprotein (VASP) and other actin binding proteins [27,69–71]. Through this phosphorylation mechanism, YopO subverts the cytoskeletal machinery of the cell [72]. The dephosphorylation of various host proteins, including focal adhesion kinase (FAK) and p130Cas from focal adhesion complexes, by the bacterial protein tyrosine phosphatase, YopH, from Yersinia leads to disruption of phagocytosis and signaling in human epithelial HeLa cells, the mouse macrophage lines, J774A.1 and RAW264.7, as well as in primary neutrophils during mouse infection [73–77]. Moreover, strains of Yersinia pseudotuberculosis expressing catalytically inactive YopH are attenuated in mouse models of infection, showing that hosttargeted tyrosine phosphatase activity is an important virulence attribute [67]. Another common reversible host modification exploited to enhance bacterial infection is ubiquitination. Ubiquitination controls a range of cellular processes, and multiple effectors have been identified that usurp host cell protein functions by the addition and removal of ubiquitin [78]. For Shigella, the IpaH effectors represent a large family of bacterial derived E3 ubiquitin ligases responsible for ubiquitinating a range of host proteins [79–83]. Some IpaH effectors (IpaH9.8, IpaH4.5, IpaH1.4, and IpaH2.5) dampen NF-kB signaling by directing proteasomal degradation of NF-kB related proteins in human epithelial HEK293T cells (Figure 2) [80,82,83]. L. pneumophila also produces an abundance of E3 ligase effectors, some of which remarkably do not require host E1 and E2 ligase activity. The SidE family of effectors, such as SdeA, use ADP-ribosylation activity to catalyze the ubiquitination of serine residues in target proteins via an Arg–ribose–phosphodiester–Ser crosslink in human macrophage and epithelial cell lines [84–86]. This illustrates that some biochemical modifications may occur in unconventional ways. Several deubiquitinase (DUB) effectors have also been identified that counteract host and effector mediated ubiquitination [87,88], although there is not always consensus agreement on their effects on the host cell. For example, the Salmonella DUB effector SseL has been reported to limit NF-kB activation by counteracting IkBa ubiquitination and proteasomal degradation in human epithelial HEK293T and HeLa cells and mouse RAW264.7 macrophages (Figure 2) [89]. However, other researchers have failed to find a role for SseL in the inhibition of NF-kB signaling when comparing wild-type and sseL mutant Salmonella strains in NF-kB activation assays in mouse macrophages, or by ectopic coexpression of SseL and an in NF-kB-dependent luciferase reporter assays in HEK293T cells [90]. Instead, the deubiquitinase activity of SseL was suggested to act on host ubiquitinated protein aggregates to reduce autophagic flux in mouse RAW264.7 macrophages, leading to enhanced intracellular replication and reduced accumulation of lipid droplets in mouse epithelial cells and in mouse liver during in vivo infection [91,92]. These discrepancies suggest that the deubiquitinase effectors require more study to understand the range of effects on the host cell during infection. Overall, DUB effectors show a preference for Lys63-linked polyubiquitin chains, which would normally drive endolysosomal destruction of intracellular bacteria [87]. In special cases, effector mediated PTMs, not typically found in eukaryotic systems can be removed by another bacterial effector. For example, the phosphocholination of Rab1 by the L. pneumophila effector AnkX can be removed by the L. pneumophila effector, Lem3 in infected Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

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Cytoplasm K27

ABIN-1

K27 K27

IpaH2.5

K27

NEMO

IKKα

Proteasomal degradaon

M1

M1

HOIL-1L M1 M1

IpaH1.4 HOIP

IKKβ

IpaH9.8

SHARPIN

Proteasomal degradaon

LUBAC

ChlaDub1

IpaH4.5 Proteasomal degradaon

SseL

P

Protein stabilizaon/ blockade of endosomallysosomal trafficking IpaH4.5 Proteasomal degradaon

Ubiqun

Nucleus

IκBα TAK1

NF-κB1 NF-κB2

Immune regulaon, inflammaon genes

RelA/p65 NF-κB1/2

Host proteins (non effector target)

Host proteins (effector target)

Bacterial E3 Ligase

Bacterial deubiquinase

Figure 2. Modulation of NF-kB Signaling by Ubiquitination to Modifying Bacterial Effectors in Host Cells. Multiple effectors target components of NF-kB signaling. During Shigella infection at least four effectors, IpaH9.8, IpaH2.5, IpaH1.4, and IpaH4.5 target NF-kB signaling by modulating ubiquitination and subsequent proteasomal degradation. IpaH9.8 targets NEMO; IpaH4.5 targets RelA/p65 and TAK1, while IpaH2.5 and IpaH1.4 target HOIL-1L of LUBAC. During Salmonella typhimurium and chlamydial infections, SseL and ChlaDub1 defuse NF-kB signaling by preventing deubiquitination and subsequent proteasomal degradation of IkBa. Abbreviations: ABIN-1, A20 binding and inhibitor of NF-kB; HOIL-1[32_TD$IF]L, heme-oxidized IRP2 ubiquitin ligase 1L; HOIP, HOIL-1L-interacting protein; IkBa, inhibitor of kB; IKK, IkB kinase; LUBAC, linear ubiquitin chain assembly complex comprising HOIL-1L, HOIP and SHARPIN; NEMO, NF-kB essential modulator; NF-kB, nuclear factor kB; SHARPIN[3_TD$IF]: SHANK associated RH domain interactor; TAK1, transforming growth factor b-activated kinase.

mouse bone-marrow-derived macrophages [93,94]. Similarly, the L. pneumophila effector DrrA (also known as SidM), AMPylates Rab1, which is then de-AMPylated by SidD in mouse bonemarrow-derived macrophages [95,96]. In these cases, the disruption of Rab1 GTPase activity by DrrA or AnkX and restoration of function by SidD or Lem3 enables temporal control of Rab1 activity during infection, thereby enabling L. pneumophila to exquisitely control the recruitment and activity of Rab1 on Legionella-containing vacuoles (LCVs), as suggested by experiments showing Rab1 cycling on and off the LCV membrane [93–96]. The activities of the effectors that control Rab1 activity during L. pneumophila infection also illustrate that some effector-mediated PTMs can be reversed by other bacterial effectors. Where the host cell is unable to mediate the canceling activity, the bacterial pathogen then has complete control over a particular host process.

Reorganization of Host Proteins through Cleavage and Protein Degradation An effective way to inhibit protein function is through the removal of regions required for the spatial organization of host proteins such as domains modified with lipid moieties. In the case of YopT from the pathogenic Yersinia, the removal of C-terminal prenyl modification sites results in release of the GTPases RhoA/Rac/Cdc42 from the membrane in human epithelial HEK293T cells [97]. This disruption nullifies the chemotactic and phagocytosis functions of primary 10

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human macrophages [98]. Another example of reorganization through protease activity is L. pneumophila effector RavZ, which is an extremely specific cysteine protease required for the disruption of autophagy during infection [99]. Mass spectrometry analysis of synthetic liposomes showed that RavZ cleaves the [31_TD$IF]phosphoethanolamine-linked glycine residue of conjugated LC3, a member of the Atg8 protein family, thereby preventing LC3 sequestration to the autophagosomal membrane and irreversibly blocking the downstream autophagy pathway in infected human epithelial HEK293T cells [99]. Direct inactivation of host proteins through protein cleavage has also been observed for many bacterial effectors. For example, the EPEC effector NleC is a metalloprotease that degrades NF-kB subunits RelA/p65 and p50, resulting in the inhibition of NF-kB signaling in human epithelial HeLa, HT-29, and HEK293T cells [100–102]. NleC binding of the Rel homology domain in p65 requires the recognition of two distinct motifs to enable subsequent cleavage [103]. Recently, a family of metalloprotease effectors from Salmonella, including PipA, GogA, and GtgA, were identified that also cleave p65, suggesting that this may be a shared feature of enteropathogens [104]. EPEC also produces a novel cysteine protease, EspL, that cleaves RHIM-containing proteins in human epithelial HT-29 and HeLa cells to block inflammatory and necroptotic signaling, leading to an inability of the immune system to mount a proinflammatory response, or drive killing of the infected cells by necroptosis [105]. Given that EspL is a member of a large and uncharacterized family of effector cysteine proteases, it is likely that further host targets will be identified for these virulence factors, offering more possibilities to intervene in or exploit effector–host protein interactions.

Outstanding Questions The modification of host proteins by bacterial effectors is an abnormal event that may trigger a cell intrinsic immune response. How these affect the total immune response of the host needs further study. Whether inhibitors targeting effector activity are efficient anti-infective drugs still needs proof of principle studies. The use of effector proteins to produce bioactive molecules for therapeutic use is an exciting possibility, but these may generate antibody responses rendering them ineffective over time. Potential autoimmune responses generated by the modified molecules also need to be considered. Mass spectrometry applications are limited by a lack of reagents recognizing novel modifications, so these need further rapid development.

Concluding Remarks As greater insight into the role of individual bacterial effectors is gained, it is increasingly clear that these proteins utilize a range of mechanisms to subvert the host. Here, we have highlighted notable examples of diverse effector mechanisms, yet this is by no means a complete list. As our understanding of effector activity and biology has increased, so has the recognition of the complex interplay and redundancy in effector function that influences the outcomes of bacterial infections. For example, both NleE and NleC from EPEC potently contribute to inhibition of the proinflammatory response. For some pathogens, this interplay and redundancy is widespread and represents a significant challenge for the characterization of individual effector function, as is the case for L. pneumophila [106] (see [348_TD$IF]Outstanding Questions and Box 1). Looking forward, research on bacterial effector biology will undoubtedly be assisted by technological advances. Two key players are CRISPR-based technologies and mass spectrometry. The streamlining of animal models through the use of CRISPR genome editing

Box 1. Clinician’s Corner Bacterial pathogens share families of virulence-related effector proteins that modify host cell biology and immunity to enable successful infection and transmission of the pathogen. The biochemical activity of effector proteins may be inhibited in a precision approach to alter the course of infection with certain pathogens. In the era of bacteria that are resistant to multiple antibiotics, the development of anti-infective agents targeting effector proteins is a possible avenue of drug development. The novel activities of effector proteins could also be harnessed in biotechnological applications for the production of bioactive molecules or synthetic biology.

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[107] holds significant promise given that our understanding of the in vivo consequences of effector activity still lags significantly behind that of our in vitro observations [30]. By generating precise models that mimic effector inhibition, the results of effector modification within in vivo settings can be explored. Furthermore, the continuous evolution of mass spectrometry renders this technology a vital tool for the study of effector function. By coupling mass spectrometry technologies to specialized affinity reagents, researchers will increasingly be able to probe systems that were out of reach even a few years ago. The power of mass spectrometry has already had significant impact on our understanding of eukaryotic cell biology. For example, it is now possible to monitor tens of thousands of ubiquitination sites within a single experiment [108]. As more tools become available, the ability to probe effector activities will also improve, especially with the generation of tools specifically designed for the detection of effectormediated modifications [109]. Effector proteins may also be used as tools to understand which host responses are critical to bacterial control, as these are the ones generally targeted by pathogens. It is no surprise that many effectors target NF-kB signaling to block a proinflammatory response, given that NF-kB activation is fundamental to the stimulation of proinflammatory responses leading to immune cell recruitment and ultimately, to resolution of infection. However, the inhibition of cell death signaling has been less obvious. Effector proteins have also shed light on the underlying mechanisms of some human diseases. This is best exemplified by the study of familial Mediterranean fever, where gain of function mutations in pyrin mimic the effects of activation of pyrin in response to sensing Rho GTPases modified by bacterial effector proteins and toxins [49]. An important area that has lacked development to date is the targeting of effector activities for the discovery of anti-infective drugs. Since many effector genes are known to be required for optimal host infection, inhibitors that selectively target effector – but not host activities – could aid the treatment of certain infections (Box 1). Moreover, a knowledge of validated host effector targets may point to the repurposing of existing drugs that could be used as adjunct therapies to block pathways required by the pathogen for replication. Lastly, the use of effector proteins in biotechnology and medicine remains largely unexplored beyond proof-ofprinciple demonstrations [4]. Given the range and novelty of many effector activities, from novel glycosylation mechanisms to the inhibition of inflammation, the possibility of exploiting these factors for medical and industrial applications in human diseases seems an attainable goal. Acknowledgments We would like to acknowledge the outstanding work done by all researchers within the field of bacterial effector characterization, and apologize to those whose work we have not highlighted within this review for reasons of space. NES is supported by National Health and Medical Research Council of Australia (NHMRC) Overseas (Biomedical) Fellowship (APP1037373), NHMRC project grant (APP1100164) and the University of Melbourne Early Career Researcher Grant Scheme (Proposal number 603107). ELH is supported by NHMRC project grants[349_TD$IF]APP1098820, APP1098826 and APP1100609.

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