The cell death response to enteropathogenic Escherichia coli infection

Microreview

The cell death response to enteropathogenicEscherichia coli infection

Tania Wong Fok Lung, Jaclyn S. Pearson,Ralf Schuelein and Elizabeth L. Hartland*Department of Microbiology and Immunology, Universityof Melbourne at the Peter Doherty Institute for Infectionand Immunity, Melbourne 3000, Australia.

Summary

Given the critical roles of inflammation and pro-grammed cell death in fighting infection, it is notsurprising that many bacterial pathogens haveevolved strategies to inactivate these defences.The causative agent of infant diarrhoea, enter-opathogenic Escherichia coli (EPEC), is anextracellular, intestinal pathogen that blocks bothinflammation and programmed cell death. EPECattaches to enterocytes, remains in the gut lumenand utilizes a type III secretion system (T3SS) toinject multiple virulence effector proteins directlyinto the infected cell, many of which subvert hostantimicrobial processes through the disruption ofsignalling pathways. Recently, T3SS effector pro-teins from EPEC have been identified that inhibitdeath receptor-induced apoptosis. Here we reviewthe mechanisms used by EPEC T3SS effectors tomanipulate apoptosis and promote host cell sur-vival and discuss the role of these activities duringinfection.

Introduction

The gastrointestinal epithelium utilizes a plethora ofinnate immune defence mechanisms to fight entericpathogens. The host intestinal epithelium is com-posed of enterocytes, mucus-secreting goblet cells,enteroendocrine cells and Paneth cells and lies above thelamina propria where lymphocytes and other immunecells reside (Fig. 1A). All these cell types play a vital rolein sensing bacterial pathogens through recognition ofpathogen-associated molecular patterns (PAMPs) by a

range of surface-located and intracellular pattern recog-nition receptors including Toll-like receptors (TLRs), NOD-like receptors, RIG-I like helicases and C-type lectinreceptors (Creagh and O’Neill, 2006). Receptor stimula-tion leads to a cascade of signalling events that ultimatelyresults in the production of inflammatory cytokines, infil-tration of inflammatory immune cells and/or death of theinfected cell.

Although EPEC is an extracellular pathogen thatremains attached to the apical surface of enterocytes(Fig. 1B), EPEC can interfere with intracellular signallingpathways by injecting type III secretion system (T3SS)effectors into the enterocyte, many of which subvert hostcell signalling pathways (Giogha et al., 2014). Forexample, EPEC utilizes NleE, NleC and NleD to inhibitinflammation by blocking distinct steps in the nuclearfactor-κB (NF-κB) and mitogen-activated protein kinase(MAPK) signalling pathways that lead to the production ofpro-inflammatory cytokines (Marchès et al., 2005; Nadleret al., 2010; Newton et al., 2010; Yen et al., 2010; Baruchet al., 2011; Muhlen et al., 2011; Pearson et al., 2011;Zhang et al., 2012). EPEC not only blocks inflammationbut recent work has shown that EPEC T3SS effectors arealso potent inhibitors of cell death. Hence, host cell sur-vival and the prevention of apoptosis have emerged ascritical virulence mechanisms of EPEC and other attach-ing and effacing (A/E) pathogens.

Death receptor-mediated apoptosis

Apoptosis can be stimulated by either intracellularligands, known as the intrinsic or mitochondrial pathway,or by extracellular ligands, known as the extrinsic or deathreceptor pathway. Both pathways converge at the point ofactivation or cleavage of the executioner caspase,caspase-3, resulting in the late morphological changesof apoptosis (Igney and Krammer, 2002). The extrinsicpathway of apoptosis involves a family of transmembranereceptors termed death receptors that are members ofthe tumour necrosis factor receptor (TNFR) superfamily.Members of this family have conserved cysteine-richregions in the extracellular ligand-binding domain anda cytoplasmic domain of about 80 amino acids calledthe death domain (DD; Ashkenazi and Dixit, 1998). A

Received 19 August, 2014; revised 24 September, 2014; accepted26 September, 2014. *For correspondence. E-mail [email protected]; Tel.: +613 8344 8041; Fax +613 9347 1540.

Cellular Microbiology (2014) 16(12), 1736–1745 doi:10.1111/cmi.12371First published online 30 October 2014

© 2014 John Wiley & Sons Ltd

cellular microbiology

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functional member of the TNFR superfamily is typicallya trimeric complex that is stabilized by intracysteinedisulfide bonds formed between the cysteine-rich regionsof individual subunit members (Banner et al., 1993).However, TNFRs can also exist in a soluble form (Grussand Dower, 1995). The DD of death receptors is essentialfor relaying the death signal from the cell surface to the

intracellular signalling pathway via a range of adaptormolecules that ultimately results in activation of theinitiator caspase, caspase-8. The best-characterizeddeath receptors include Fas/CD95/APO-1, TNFR1/p55/CD120a, TNF-related apoptosis-inducing ligand receptor1 (TRAIL-R1)/DR4 and TRAIL-R2/DR5/Apo2/KILLER andtheir respective ligands are the FasL, TNF and TRAIL/

DED

DED

DD

DD DD

Fas

Pro-caspase-8

FADD

FADD

GlcNAc

NleB1

Fas

FasL FasL

Lamina propria

Apoptosis

Nucleus

Actin

Activatedcaspase-8

NleB1

Mucus

Gobletcell Enteroendocrine

cell

Panethcell

Enterocyte

Commensal bacteria

sIgA

Lamina propria

p18 p43/41

caspase-3

B

41

? ?

EPECDnleB

T3SS

Enterocyte

Neutrophil

Gut lumenA C

Fig. 1. EPEC attachment and inhibition of Fas signalling in enterocytes during EPEC infection.A. Diagram of the cellular architecture of the intestinal epithelium. Enterocytes constitute the majority of the intestinal epithelium, which alsocontains goblet cells, enteroendocrine cells and Paneth cells. The epithelium lies above the lamina propria and is covered with a layer ofmucus in the gut lumen. Red dashed circle indicates that A/E pathogens target the tips of intestinal villi for attachment.B. Transmission electron micrograph of C. rodentium (asterisk) attachment during infection showing A/E lesion formation.C. Schematic diagram of NleB1-mediated inhibition of FasL-induced cell death during EPEC infection. Wild-type EPEC inhibits caspase-8activation and apoptotic cell death using NleB1,which GlcNAcylates FADD (blue squares) and prevents formation of the DISC. EPEC lackingNleB1 (ΔnleB1) cannot inhibit FasL-driven apoptotic cell death. The specific source of FasL during EPEC infection has not yet been identified.

EPEC and cell death 1737

© 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1736–1745

Apo2L (for TRAIL-R1 and TRAIL-R2; Chicheporticheet al., 1997; Ashkenazi and Dixit, 1998; Peter andKrammer, 1998; Suliman et al., 2001; Rubio-Moscardoet al., 2005). These trimeric ligands belong to the TNFfamily of cytokines and are mainly expressed astransmembrane proteins although they can also bereleased as soluble cytokines by proteolytic cleavage.Recent studies have demonstrated that only themembrane-bound form of FasL possesses apoptosis-inducing capacity (O’Reilly et al., 2009).

FasL and Fas signalling

Fas is expressed ubiquitously and although it is particu-larly abundant in lymphoid tissues and on activatedmature T lymphocytes, functional Fas is also expressedon the basolateral membrane of intestinal epithelial cells(Moller et al., 1994; Strasser et al., 2009). In contrast tothe ubiquitous expression of Fas, FasL is expressed on aselective cellular pool including activated cytotoxic T-cells,natural killer cells (NK) and NK T-cells (NKT). Upon stimu-lation of Fas by FasL, there is an aggregation of pre-assembled Fas trimers at the plasma membrane, whichleads to internalization of Fas (Lee et al., 2006). Fas thenrecruits the adaptor protein, Fas-associated protein withDD (FADD), via homotypic interactions between DDsfound in the cytoplasmic region of Fas and the C-terminusof FADD. FADD recruits procaspase-8 via a secondhomotypic interaction, this time involving the death effec-tor domain (DED) of FADD and procaspase-8. The inter-actions between Fas, FADD and procaspase-8 form thedeath-inducing signalling complex (DISC) that inducesproximity-induced activation (cleavage) of caspase-8(Kischkel et al., 1995). Activated caspase-8 then cleavesand activates the executioner caspase-3 leading toapoptotic cell death. In certain cell types, including epithe-lial cells, FasL stimulation induces apoptotic cell deaththrough caspase-8 mediated cleavage of a pro-apoptoticBcl-2 homology 3 (BH3)-only protein called Bid (Li et al.,1998; Jost et al., 2009). Bid is a member of the Bcl-2family of proteins, which is involved in the mitochondrialcell death pathway and so its activation by caspase-8 isan example of a crosstalk between the intrinsic and extrin-sic pathways of apoptosis.

The TRAIL and TNF pathways

The death receptors, TRAIL-R1 and TRAIL-R2, haveextensive tissue distribution, whereas the ligand, TRAIL,is expressed mainly on NK cells, NKT cells andmacrophages. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis by binding toeither TRAIL-R1 or TRAIL-R2, although TRAIL-R2 is moresignificant in inducing apoptosis. Receptor binding of

TRAIL leads to recruitment of FADD via homotypic DDinteractions, which in turn recruits procaspase-8 to theDISC via DED interactions leading to activation of acaspase cascade in a similar manner to FasL-induced celldeath (Kuang et al., 2000).

Similar to TRAIL, two different TNF receptors exist,TNFR1 and TNFR2. Although both receptors sharesequence similarity in their extracellular ligand-bindingregion, their cytoplasmic region sequences are different,thereby accounting for their different signalling activities(Tartaglia et al., 1991; Vandenabeele et al., 1995). BothTNFR1 and TNFR2 can bind TNF. However, only TNFR1is involved in death receptor signalling as TNFR2 lacks anintracellular DD. TNFR1 is expressed ubiquitously and isrequired for TNF-induced apoptosis of pathogen-infectedcells (Pfeffer et al., 1993; Zhao et al., 2000). Upon stimu-lation of TNFR1 by TNF, TNFR1 translocates to lipid rafts(Legler et al., 2003), whereupon receptor-interactingprotein kinase 1 (RIPK1), TNFR type1-associated DDprotein (TRADD), TNF receptor-associated factor 2(TRAF2) and cellular inhibitor of apoptosis protein 1 and 2(c-IAP1/2) are recruited to form the membrane-boundreceptor complex I (Micheau and Tschopp, 2003). Thereceptor complex is then internalized by endocytosisduring which RIPK1, TRAF2 and TRADD dissociate fromTNFR1 enabling the DD of TRADD to bind to the DD ofFADD. This in turn recruits procaspase-8 via homotypicDED interactions to the newly formed cytosolic complex II(Micheau and Tschopp, 2003; Schneider-Brachert et al.,2004). Activated caspase-8 then cleaves the executionercaspases leading to cell death by apoptosis. If caspase-8 is depleted or inhibited, complex II cannot induceapoptotic cell death and instead TNFR1 stimulation leadsto a recently described form of cell death known as pro-grammed necrosis or necroptosis, which relies on RIPK3and mixed lineage kinase domain-like (MLKL) (Murphyet al., 2013; Newton et al., 2014). Most recently, RIPK1was shown to play a dual regulatory role in gut homo-eostasis by inhibiting both apoptosis and necroptosis inintestinal epithelial cells through its scaffolding function(Dannappel et al., 2014; Takahashi et al., 2014).

Even though TNFR1 stimulation can lead to cell death,the primary function of TNFR1 is in inflammation. In thispathway, RIPK1 in membrane-bound receptor complex Iis polyubiquitinated at lysine-377 by the E3 ubiquitinligases, cIAP-1/2 (Ea et al., 2006; Li et al., 2006).Polyubiquitinated RIPK1 recruits TAK1 and the associ-ated TAK1-binding proteins 1/2/3 (TAB1/2/3; Ea et al.,2006). TAK1 then activates the IKK complex leading tophosphorylation of the NF-κB inhibitory protein, IκB,which is then ubiquitinated and degraded enablingnuclear translocation of NF-κB and transcriptionalactivation of NF-κB-dependent genes including pro-inflammatory and anti-apoptotic genes (Wang et al.,

1738 T. Wong Fok Lung et al.


1998). In fact, inhibiting the NF-κB pathway sensitizescells to TNF-induced apoptosis (Van Antwerp et al.,1996).

The intrinsic or Bcl-2 pathway of apoptosis

The intrinsic pathway of apoptosis is stimulated by arange of non-surface receptor-mediated stimuli such asDNA damage, endoplasmic reticulum (ER) stress or viralinfection, which ultimately induce the release of signallingfactors from the mitochondria (Strasser et al., 1995;Czabotar et al., 2014). A number of intrinsic stimuli acti-vate the BH3-only proteins, which promote cell death byantagonizing the inhibitory effects of the anti-apoptoticBcl-2 proteins. BH3-only proteins also activate theproapoptotic Bcl-2 family members such as Bax and Bakin the outer mitochondrial membrane resulting in therelease of cytochrome c into the cytoplasm (Czabotaret al., 2014). Cytochrome c stimulates the formation of amultimeric protein complex called the apoptosome thatincludes Apaf-1 and procaspase-9 (Czabotar et al., 2014).Activation of the initiator caspase, caspase-9, then leadsto cleavage of the executioner caspases resulting inapoptotic cell death.

EPEC attachment to the intestinal mucosa

While the gastrointestinal epithelium is well equipped withimmune defence mechanisms, such as inflammation andapoptosis, pathogens have also evolved strategies toovercome these responses. A critical step in EPECpathogenesis is colonization of the gastrointestinal tract.EPEC adheres to the intestinal mucosa in a distinctivemanner via A/E lesion formation (Moon et al., 1983). A/Elesions are typified by intimate bacterial adherence toenterocytes, the formation of actin-rich pedestals adjacentto the sites of bacterial attachment, as well as localizeddestruction of brush border microvilli (Fig. 1B). The abilityto form A/E lesions is encoded by genes on a chromo-somal pathogenicity island (PAI) termed the locus ofenterocyte effacement (LEE; McDaniel et al., 1995; Wonget al., 2011). The LEE PAI encodes a T3SS and severalLEE-encoded T3SS effectors and is highly conservedamong all A/E pathogens including enterohaemorrhagicEscherichia coli (EHEC) and the murine pathogenCitrobacter rodentium (Wong et al., 2011). Since humanEPEC strains do not colonize mice efficiently and do notcause disease or form A/E lesions in mice, C. rodentiumserves as a convenient small animal model to studyEPEC pathogenesis in vivo (Mundy et al., 2005; 2006).The T3SS translocates a number of effector proteins intoenterocytes, some of which play a direct role in A/E lesionformation including the translocated intimin receptor, Tir(Wong et al., 2011).

EPEC infection and host cell viability

A number of studies have shown that EPEC surface prop-erties and some translocated effectors can activateapoptotic signalling in vitro; purified outer membraneEPEC proteins induce TNF expression and activatecaspase-3-induced apoptosis (Malladi et al., 2004;Shankar et al., 2009) and EPEC strains expressingbundle-forming type IV pili (Bfp) induce cell death in cul-tured epithelial cells more readily than strains that lackBfp, suggesting that attachment plays a role in triggeringcell death (Abul-Milh et al., 2001). The modes of cell deathtriggered by EPEC infection have not been fully eluci-dated and inconsistencies exist in the analysis of caspaseactivation (Crane et al., 1999; Flynn and Buret, 2008).

Some individual effector proteins have been directlyimplicated in inducing cell death. T3SS effectors such asEspF and Map compromise mitochondrial and tight junc-tion integrity leading to increased apoptotic cell death(Kenny and Jepson, 2000; McNamara et al., 2001; Deanand Kenny, 2004; Nougayrede and Donnenberg, 2004;Dean et al., 2006; Ma et al., 2006; Nougayrede et al.,2007). EspF is a LEE-encoded effector with a range ofattributed functions including microvillus effacement, dis-ruption of the epithelial barrier, depletion of host cell DNAmismatch repair proteins and the induction of apoptosis(Holmes et al., 2010; Maddocks et al., 2013). EspF trig-gers intrinsic apoptosis by disrupting mitochondrial mem-brane potential, resulting in the release of cytochromec into the host cell cytosol and caspase-9 activation(Nougayrede and Donnenberg, 2004). EspF also bindsthe host protein Abcf2 within mitochondria and in thepresence of EspF, the level of Abcf2 in cells decreaseswhile caspase-9 cleavage increases (Nougayrede et al.,2007). Furthermore, cells treated with siRNA to knock-down abcf2 showed increased caspase-3 activation whenstimulated with staurosporine, suggesting that Abcf2 maybe involved in apoptosis (Nougayrede et al., 2007). Giventhe localization of Map to mitochondria, this T3SS effectorhas also been implicated in reduced host cell survival,although the precise role of Map in cell death remains tobe determined (Kenny and Jepson, 2000; Ma et al.,2006).

Another EPEC effector, Cif, affects host cell cycle pro-gression inducing a delayed form of apoptosis (Marcheset al., 2003; Taieb et al., 2006; Samba-Louaka et al.,2008). More recently, Cif was found to target NEDD8,a ubiquitin-like protein involved in cell cycle control(Morikawa et al., 2010). Cif harbours a papain-likecysteine protease domain that deamidates NEDD8 andthereby compromises the activity of neddylated Cullin-RING ubiquitin ligases leading to defective ubiquitin-dependent degradation of multiple host cell proteinsand increased cytotoxicity (Cui et al., 2010). EspH also



induces a cytotoxic effect and induces caspase-3 cleav-age, which is related to disassembly of focal adhesions(Dong et al., 2010; Wong et al., 2012). EspH binds theDH-PH domain in multiple RhoGEFs, which prevents acti-vation of Rho and therefore Rho-dependent events suchas phagocytosis as well as disruption of the host actincytoskeleton structure (Dong et al., 2010).

Anti-apoptotic effectors of EPEC

Despite the fact that the effectors described above havebeen associated with cytotoxic effects and/or activation ofexecutioner caspases, in general, cells infected withEPEC do not display characteristic late apoptotic featuressuch as membrane blebbing, nuclear condensation andcell shrinkage. In addition, in vivo studies report thatapoptosis is not observed in tissues from humans infectedwith EPEC (Rothbaum et al., 1982; 1983; Fagundes-Netoet al., 1996) or in tissues from C57BL/6 mice infected withC. rodentium (Vallance et al., 2003). Hence, apoptosis isnot a characteristic feature of infection with A/E patho-gens. In fact, compared with Salmonella, Shigella andYersinia, three genera of invasive enteric bacteria, whichare known to induce host cell death, EPEC is experimen-tally much weaker in its ability to cause cell death(Monack et al., 1996; 1997; Crane et al., 1999).

The first EPEC T3SS effectors associated with the inhi-bition of cell death were NleH1 and NleH2. nleH1/H2double mutants of EPEC are highly cytotoxic for cells,which can be complemented by the reintroduction ofeither nleH1 or nleH2 (Hemrajani et al., 2010). Furtherinvestigation of NleH1 showed that the effector inhibitscaspase-3 activation/cleavage even when cells aretreated with a wide range of proapoptotic compoundsincluding staurosporine, tunicamycin and brefeldin A.Caspase-3 inhibition was independent of predicted NleHkinase activity (Hemrajani et al., 2010). The exact mecha-nism by which NleH1 blocks apoptosis has not been fullyelucidated although NleH1 interacts with an anti-apoptoticprotein called Bax inhibitor-1 (BI-1; Hemrajani et al.,2010). BI-1 is an ER protein that when overexpressed inmammalian cells suppresses apoptosis induced by avariety of stimuli including deprivation of growth factor,treatment with the anti-cancer agent, etoposide as well asstaurosporine and the proapototic protein Bax (Robinsonet al., 2011). NleH1 also blocks cell death inducedby the TcdB toxin from Clostridium difficile. TcdB is aglucosyltransferase that inactivates Rho family proteinscausing disruption of the cell cytoskeleton and cell death(Matarrese et al., 2007). The modification of Rho by TcdBis detected in cells by a newly described pyrin-containinginflammasome (Xu et al., 2014) that activates caspase-1and induces cell death. More work is needed to elucidatethe mechanism by which NleH blocks cell death, espe-

cially given the ability of NleH1 to inhibit cell death fromsuch a diverse range of intrinsic stimuli. Interestingly, bothNleH1 and NleH2 are also associated with inhibition of IκBphosphorylation and NF-κB activation in response to TNFstimulation (Royan et al., 2010). The mechanism by whichNleH inhibits NF-κB activation is also not fully understoodas one study implicated the kinase activity of NleH (Royanet al., 2010), while a kinase-independent mechanisminvolving the ribosomal protein S3 was proposed byothers (Wan et al., 2007; Gao et al., 2009).

The NleD effector is a zinc metalloprotease that cleavesthe c-Jun N-terminal kinase (JNK) and MAPK, p38(Baruch et al., 2011). In response to stimulation of TLRs,the interleukin 1 receptor or TNFR, several mitogen-activated protein 3 kinases are activated that can activateJNK, which in turn phosphorylates c-Jun, a component ofthe AP-1 transcription factor that regulates cell prolifera-tion, apoptosis and inflammation (Shaulian and Karin,2001; Eferl and Wagner, 2003). JNK activation involvesphosphorylation on threonine-183 and tyrosine-185located in the activation loop (Kallunki et al., 1994). NleDcleaves JNK in the activation loop, which blocks JNK-driven apoptosis (Baruch et al., 2011). NleD also cleavesp38 in the conserved activation loop and the combinedinactivation of JNK and p38 by NleD is linked to suppres-sion of the inflammatory response via inhibition of IL8expression (Baruch et al., 2011).

Inhibition of the extrinsic cell death response

EPEC and EHEC express two homologues of the T3SSeffector NleB, NleB1 and NleB2. Initial studies in EPECidentified a role for NleB1 in the inhibition of (NF-κBactivation upon TNF but not IL-1β stimulation Newtonet al., 2010). Despite this, however, NleB1 did not inhibitIL-8 production during EPEC infection (Pearson et al.,2013). Instead, recent studies have demonstratedthat NleB1 inhibits the extrinsic apoptotic pathway bymodifying DD-containing proteins including FADD,RIPK1 and TRADD (Li et al., 2013; Pearson et al.,2013). The fact that RIPK1 and TRADD form part of theTNFR1 receptor complex is most likely the reason thatNleB1 inhibits NF-κB activation under certain experi-mental conditions, namely, ectopic expression or over-expression in the absence of nleE (Newton et al., 2010;Li et al., 2013).

NleB1 is a glycosyltransferase (Gao et al., 2013) thatbinds to and modifies the DD of FADD by adding a singleGlcNAc moiety at arginine 117 (Arg117), thereby inhibitingTNF or FasL-induced DISC formation and subsequentcaspase-8 activation and apoptotic cell death (Li et al.,2013; Pearson et al., 2013). Arg117 in FADD is essentialfor Fas-FADD as well as TRADD–FADD DD interactions(Imtiyaz et al., 2005; Wang et al., 2010) and the critical



role of this conserved residue in apoptosis is evident inthe embryonic lethality of mice carrying an R117Q muta-tion (Imtiyaz et al., 2009). NleB1 also GlcNAcylatesTRADD at the equivalent arginine residue (Arg235), pre-venting recruitment of TRADD, TRAF2 and ubiquitinatedRIPK1 to TNFR1 (Li et al., 2013). Furthermore, NleB1inhibits TRAIL-induced DISC formation and apopto-sis (Li et al., 2013). NleB2 is also a predictedglycosyltransferase but does not bind FADD and has noimpact on the inhibition of cell death during EPEC infec-tion (Li et al., 2013; Pearson et al., 2013).

The role of NleB1 in vivo has been investigated usingnleB deletion mutants of C. rodentium, which carriesonly one copy of nleB. nleB mutants are attenuated forcolonization (Kelly et al., 2006) and mice infected withC. rodentium lacking nleB show increased numbers ofcells positive for cleaved caspase-8 in the colon (Pearsonet al., 2013). The importance of the FasL-Fas signallingpathway during C. rodentium infection was demonstratedfurther by infecting Fas-deficient (Faslpr/lpr) and FasL-deficient (Fasgld/gld) mice with C. rodentium (Pearson et al.,2013). Both strains developed severe, watery diarrhoeaand showed increased colitis in response to C. rodentiumcompared with wild-type mice. Although the source ofFasL during infection is unclear, this model suggests thatFasL-positive immune cells recognize and delete infectedcells by inducing apoptosis, thereby controlling pathogenload. In the presence of NleB, this cell death response isthwarted and the bacteria maintain their attachment to aviable enterocyte (Fig. 1C).

While NleB1 is specific for the extrinsic apoptoticpathway, NleF has been implicated in the inhibition ofapoptosis stimulated by both the intrinsic and extrinsicpathways (Blasche et al., 2013). Because of the ability ofNleF to bind and inhibit caspase-9 activation, the mainfocus of work on NleF has been the inhibition of intrinsicapoptosis. However, NleF also prevents TRAIL-inducedapoptosis in HeLa cells by binding to and inhibitingcaspase-8 (Blasche et al., 2013). NleF-mediated inhibi-tion results from insertion of the carboxy terminal end ofNleF into the active site of the caspase (Blasche et al.,2013). However, the role of NleF in apoptosis inhibitionduring infection is still unclear and may be secondary toeffectors such as NleB1, as no significant differenceswere observed in the activation of downstream execu-tioner caspases-3 and 7 in HeLa cells infected withwild-type EPEC or a ΔnleF EPEC derivative (Blascheet al., 2013). NleF also binds to caspase-4, which wasrecently described as an innate sensor of intracellularlipopolysaccharide (LPS; Shi et al., 2014). The CARDdomain of caspase-4 binds directly to LPS leading tostimulation of pyroptosis. Hence, during infection, the roleof NleF may be to counteract inflammasome formation ininfected enterocytes.

Protecting epithelial integrity

Two further T3SS effector proteins influence cell survivalthrough maintaining cellular junctions. EspZ (SepZ) is aLEE-encoded effector protein that promotes maintenanceof the intestinal epithelium by stabilizing focal adhesionsduring infection (Deng et al., 2004; Shames et al., 2010;Berger et al., 2012). Epithelial cells infected with an EPECΔespZ mutant exhibit increased cytotoxicity compared tocells infected with wild-type EPEC. EspZ-mediated pro-tection from cytotoxicity has been attributed to its ability tobind CD98 (Shames et al., 2010). CD98 enhances signal-ling from the β1-integrin receptor, including pro-survivalsignals from the Akt kinase and focal adhesion kinase(FAK; Feral et al., 2005). Interestingly, the T3SS effector,OspE, from the invasive enteric pathogen Shigellaflexneri, also stabilizes focal adhesion sites of host intes-tinal cells, thereby reinforcing enterocyte adherence to thebasement membrane and preventing the exfoliation ofinfected cells into the gut lumen (Kim et al., 2009). Themechanism of action of OspE is via binding to integrin-linked kinase (ILK), which contributes to the inhibition offocal adhesion disassembly rather than affecting focaladhesion assembly (Kim et al., 2009). Indeed, thephosphorylation of both FAK and paxillin, indicating dis-assembly, was significantly reduced in cells expressingOspE and ILK (Kim et al., 2009). The residue impor-tant for OspE binding to ILK was identified astryptophan-68 (Trp68). Trp68 is highly conserved amongOspE homologues from EHEC, EPEC and C. rodentium(termed EspO), which also bind to ILK (Kim et al., 2009).Hence EspO effectors in A/E pathogens may act inconcert with EspZ and effectors that regulate the actincytoskeleton to block sloughing of infected cells into thegut lumen (Morita-Ishihara et al., 2013).

Conclusions

The exposure of intestinal cells to EPEC-derived PAMPs,intimate attachment of the bacteria as well as the activityof effector proteins such as Map, EspF, Cif and EspH allstimulate intrinsic apoptosis pathways and stressresponses in the host cell that are countered by effectorssuch as NleH, NleD, NleF, EspZ and EspO. The action ofthese cell death-suppressing effectors promotes survivalof the infected cell thereby prolonging EPEC adherence tothe intestinal mucosa. In addition, EPEC has evolvedspecific mechanisms to inhibit death receptor-mediatedinflammation and apoptosis in response to immunesystem surveillance and host death ligand-mediatedelimination of infected cells. A diagrammatic summary ofthese effectors and comparison with other pathogens ispresented in Giogha et al. (2014). Importantly, the activityof NleB has revealed a previously unappreciated role for



Fas signalling in fighting gut infection and exemplifies howEPEC has adapted to avoid the intestinal innate immuneresponse.

There is still much to understand about the activities ofEPEC T3SS effector proteins, in particular where hostbinding targets and biological effects have not been con-firmed in the context of infection. In addition, the discoveryof new enzymatic activities for several EPEC effectorproteins warrants re-examination of other effectors thatmay also be novel enzymes. Lastly, especially in the fieldof cell signalling, studies relying on in vitro models andectopic expression of effectors should be interpretedwith caution if not confirmed by infection, ideally usingC. rodentium as an in vivo model for EPEC infection.

Acknowledgements

We gratefully acknowledge Ms Vicki Bennett-Wood for the elec-tron micrograph of C. rodentium. This work was supported byfunding awarded to ELH from the Australian National Health andMedical Research Council (APP1044061). TWFL was supportedby a University of Melbourne International Research Scholarship(MIRS).

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The cell death response to enteropathogenic Escherichia coli infection

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