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The Shiga toxi fail, a group of structurall ad fuc-tioall related exotoxis, icludes Shiga toxi froShigella dysenteriae serotpe 1 ad the Shiga toxisthat are produced benterohaemorrhagic Escherichia coli(EHEC) strais. The existece of differet, iterchage-able ters to describe ver siilar toxis has historicalreasos. The Japaese icrobiologist Kioshi Shiga asthe first to characterize the bacterial origi of dsetercaused bS. dysenteriae, i 1897. I 1977, Kooalchukdiscovered a group ofE. coli isolates that produced afactor that as able to kill Vero cells i culture1. The fac-tor as tered verotoxi, ad the bacteria ere tered

verotoxi-producig E. coli (VTEC). OBrie ad col-leagues recogized i the earl 1980s that soe E. coliisolates produced a toxi that as related to Shiga toxiad aed these orgaiss Shiga-like toxi-producigE. coli (STEC)2. I 1983, it as recogized that STECstrais are associated ith haemolytic uraemic syndrome(HUS)3. Researchers evetuall realized that the erestudig idetical or highl related toxis.

Shiga toxi is the prototpe of the Shiga toxi failad earl idetical to the E. coli-produced Shiga toxi 1(Stx1), differig b a sigle aio acid i the catalticA subuit of the toxi. STEC ca produce Stx1 variats(Stx1 ad Stx1c), Stx2 variats (Stx2, Stx2c, Stx2d, Stx2e,Stx2f) or variats of both i a rage of cobiatios(TABLE 1). Hoever, severe disease has bee epideio-logicall liked to the presece of Stx2 (REF. 4). AlthoughStx1 ad Stx2 share a coo receptor ad possessthe sae itracellular echais of actio, the areiuologicall distict ad ol 56% idetical at theaio acid sequece level5. Stx2 variats are 8499%hoologous to Stx2.

The Shiga toxis i S. dysenteriae ad STEC areecoded b diverse labdoid bacteriophages6. Thesephages are highl obile geetic eleets that plaa iportat part i horizotal gee trasfer ad,hece, i geoe diversificatio7. Located i the lategee regio dostrea of the late prooters adupstrea of the lsis cassette, stx gees are highlexpressed he the ltic ccle of the phage is activated.Phages regulate the productio of Shiga toxis throughthe activit of phage gee prooters, through theaplificatio of gee cop uber ad through toxirelease8,9. Coditios uique to the periplas ofGra-egative bacteria are required for toxi subuitsto efficietl fold ad asseble. Oce assebled, toxisecretio sees to be achieved b phage-ediatedbacterial lsis10.

Little is ko about the ifluece of itestial factorso the productio of Shiga toxis. Hua eutrophilactivatio products such as hdroge peroxide iducetoxi release b EHEC11. The stx1 gees are also regulated

b iro, ith toxi sthesis beig repressed at high irococetratios12, thus liitig the site of toxi productioto the distal sall itestie ad colo.

Hua ifectio bS. dysenteriae or EHEC strais,hich results iitiall i diarrhoea, has bee associ-ated ith the igestio of cotaiated iced beef,

vegetables, ilk, juice or ater. Frequetl, the dis-ease progresses ito dseter ad haeorrhagic coli-tis, hich a further develop ito life-threateigssteic extra-itestial coplicatios, such as acutereal failure ad cetral ervous sste coplica-tios that soeties result i death13. Haeorrhagiccolitis is priaril a disease of oug childre ad

Institut Curie Centre de

Recherche and CNRS

UMR144, Traffic, Signalling

and Delivery Laboratory,

26 rue dUlm, 75248 Paris

Cedex 05, France.

Correspondence to L.J.

e-mail:ludger.johannes@

curie.fr

doi:10.1038/nrmicro2279

Published online

21 December 2009

EnterohaemorrhagicEscherichia coli

(EHEC). Escherichia colistrains

that cause haemorrhagic colitis

and haemolytic uraemic

syndrome. EHEC constitutes

a subset of serotypes called

Shiga toxin-producing E. coli

(STEC), in which these toxins

are virulence factors.

Haemolytic uraemicsyndrome(HUS). A life-threatening

complication characterized by

microangiopathic haemolytic

anaemia, thrombocytopenia

and acute renal failure.

Shiga toxins from cell biology tobiomedical applicationsLudger Johannes and Winfried Rmer

Abstract | Shiga toxin-producingEscherichia coli is an emergent pathogen that can induce

haemolytic uraemic syndrome. The toxin has received considerable attention not only from

microbiologists but also in the field of cell biology, where it has become a powerful tool to

study intracellular trafficking. In this Review, we summarize the Shiga toxin family members

and their structures, receptors, trafficking pathways and cellular targets. We discuss howShiga toxin affects cells not only by inhibiting protein biosynthesis but also through the

induction of signalling cascades that lead to apoptosis. Finally, we discuss how Shiga toxins

might be exploited in cancer therapy and immunotherapy.

R E V I E W S

nATURE REVIEwS |Microbiology VOLUmE 8 | FEBRUARy 2010 |105

20 Macmillan Publishers Limited. All rights reserved10
mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]


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elderl people14. The ost coo strai ofE. colithat causes haeorrhagic colitis is E. coli serotpeO157:H7. This toxigeic bacteriu aturall ihabitsthe gastroitestial tract of doestic aials ithouta ill effect for the host. Up to 15% of patiets ithhaeoltic colitis caused bE. coli serotpe O157:H7go o to develop HUS15. STEC strais that produceStx2 are ore likel to cause HUS tha those thatol produce Stx1 (REF. 16). moreover, strais pro-ducig Stx2 aloe ere foud to cause ore severeeurological sptos i gotobiotic piglets thastrais producig ol Stx1 or both Stx1 ad Stx2,hereas Stx1-producig strais iduce ol diarrhoeaad o ssteic coplicatios17. Affected orgas ihuas ad experietal aials sho sigs of pro-foud vascular daage. The ost severel affectedpatiets require blood trasfusios ad dialsis ther-ap. Haeodialsis ad peritoeal dialsis have beeused i the past, depedig o the age ad eight ofpatiets, ad peritoeal dialsis is the treatetof choice for chroic real failure18. Several iter-

vetio strategies are curretl beig developedto prevet orga daage b Shiga toxis, the ostproiet of hich are suarized i BOX 1.

Toxin structure and cellular receptors

Shiga toxi fail ebers have a AB5

olecu-

lar cofiguratio, as revealed b X-ra crstallog-raph19,20: A ezaticall active ooeric Asubuit, StxA (hich has a olecular ass of 32 kDa)is o-covaletl associated ith a petaer of ide-tical B fragets (each B fraget has a olecularass of 7.7 kDa) that for the B subuit, StxB, hichis resposible for bidig to cell surface receptors(FIG. 1a). StxB fors a doughut-shaped structure itha cetral pore ito hich the carboxl terius of StxAiserts19. StxA ad the StxB fragets are secreted itothe bacterial periplas21, here the asseble o-covaletl ito the holotoxi, as as iitiall describedfor heat-labile eterotoxis fro E. coli 22.

StxA possesses a highl specific RnA N-glcosidaseactivit that cleaves a adeie base at positio4,324 o the -sarci loop located o doai VI of 28Sribosoal RnA (rRnA) of eukarotic ribosoes23,24,thereb ihibitig elogatio factor-depedet aio-acl tRnA bidig ad subsequet chai elogatio25.Bacterial ribosoes are also a substrate for StxA, adexposure to Stx1 results i decreased proliferatioof susceptible bacteria26. Although Shiga toxis areextreel potet ribosoe-odifig ezes, itshould be oted that their actio is ot liited to theihibitio of protei sthesis. As discussed belo,the have several cellular effects, icludig the iduc-tio of ctokie expressio b acrophages, hichi tur a icrease the susceptibilit of certai cellsto toxis.

StxB bids to the eutral glcosphigolipid globo-triaoslceraide (Gb3; also ko as CD77 or the Pkblood group atige), hich is preset o the surfaceof cells2729, leadig to subsequet iteralizatio ofthe toxi. Stx2e uses globotetraoslceraide (Gb4),hich cotais a additioal terial [13]-likedN-acetlgalactosaie residue. I the absece of StxA,StxB still adopts a petaeric structure that is fuctioallequivalet to the holotoxi i receptor bidig30.

The first structures of Shiga holotoxi19 ad of theB subuit of Stx1 aloe20 ere deteried b X-ra

crstallograph i the absece of receptor olecules.molecular odellig studies31,32 ad fluoresceceeerg trasfer easureets33 idicated the preseceof to receptor-bidig sites per B fraget ooer.I 1998, the crstal structure as deteried for theStx1 B subuit i coplex ith a trisaccharide receptoraalogue of Gb3. This stud revealed the existece ofthree trisaccharide-bidig sites per B fraget oo-er34(FIG. 1b,d). All 15 of the Gb3-bidig sites i theB subuit hoopetaer are facig i the sae directio,distal to the A subuit bidig site, thereb idetifigthe ebrae iteractio surface. The Gb3-bidigsites do ot iteract. At least oe of the bidig sites

Table 1 |The family of Shiga toxins

oasm Tx Squ smat t Satx

caatsts Sms cuapts

A suut b suut*

Shigelladysenteriae

Shigatoxin

N/A N/A N/A N/A Gb3

STEC Stx1 97% 98% N/A SLTI and VT1 Gb3Stx1c 97% 98% N/A SLTIc and VT1c Gb3

Stx2 53% 64% Associated with severedisease in humans

SLTII and VT2 Gb3

Stx2c 53% 61% N/A SLTIIc and VT2c Gb3

Stx2d 54% 61% N/A SLTIId and VT2d Gb3

Stx2e 53% 61% Associated with thepiglet edema disease

SLTIIe and VT2e Gb3 andGb4

Stx2f 54% 60% N/A SLTIIf and VT2f Gb3

Gb3, globotriaosylceramide; N/A, not applicable; SLT, Shiga-like toxin; STEC, Shigella toxin-producing Escherichia coli; Stx1, Shigatoxin 1; VT: Verotoxin. *This is the sequence similarity for mature B fragments, without signal sequences.

R E V I E W S

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(site 1) is coposed of residues fro adjacet oo-ers ad therefore ol exists i assebled petaericStxB. mutatioal aalsis has sho that site 1 ad site 2ediate high affiit receptor bidig ad are the ostrelevat for cell ctotoxicit, hereas site 3 ediates therecogitio of additioal lo-affiit Gb3 epitopes35,36.Stable isotope-assisted high-resolutio nmR ethodsere used to deterie the solutio structure of the co-

plex betee the Stx1 B subuit ad a 13C-eriched Gb3aalogue37. I cotrast to the crstal structure, ol oeof the three possible bidig sites per ooer, site 2,as occupied b the trisaccharide i solutio. The ulti-ple Gb3 bidig sites o the hoopetaeric B subuitexplai ho the protei achieves a rearkabl highaffiit for cells (ith bidig costats i the orderof 109 m1)38, despite the uch loer affiit of Gb3olecules for idividual bidig sites (ith bidigcostats of aroud 103 m1)39.

Aalsis of the crstal structure of Stx2 predictedthe presece of the correspodig trisaccharide bid-ig sites o its B subuit but also deostrated that the

coforatio at site 2 differs distictivel fro that ofthe Shiga toxi ad Stx1 B subuits19. Stx1 ad Stx2cideed prefer Gb3 species that differ i the ature oftheir fatt acid chais40, ad the tightest toxi bidigis observed for defied ixtures of these receptor spe-cies41. Iterestigl, Stx1 has a 10-fold higher affiitfor Gb3 i cells tha Stx2 (REF. 42), but Stx2 has aapproxiatel 400-fold loer LD

50i ice tha Stx1

(REF. 43). This apparet cotradictio a be explaiedat least i part b differeces i their biodistributio.

Gb3 fatt acid heterogeeit41, hdroxlatio44,chai legth ad degree of usaturatio40 a iflu-ece the lateral obilit of the lipid i the plasaebrae ad affect the coforatio of the trisac-charide head group that is preseted o the cell sur-face. Ideed, all these factors ad the ebraeeviroet, icludig cholesterol levels, eresho to be crucial for the recogitio process40,41,4448.Iterestigl, patiets ho developed HUS after STECifectio ere foud to have a higher hdroxlatedfatt acid cotet i their red blood cell Gb3 tha

Box 1 | Intervention strategies

Clinical observations indicate that conventional anti-microbial therapies for the treatment of infections by Shiga

toxin-producing bacteria have inherent limitations and may even be counterproductive146148, as killing the bacteria may

accelerate toxin release. Hence, a need exists for the development of new therapeutic modalities. Possible therapeutic

options include the use of agents that block Shiga toxin binding at the cell surface or its intracellular transport, thereby

protecting people from toxin-mediated pathology and development of haemolytic uraemic syndrome (HUS).

Stt Sa tx s

To mimic the cell surface binding of Shiga toxin B subunit (StxB) to several globotriaosylceramide (Gb3) molecules at atime, oligovalent water-soluble globotriose ligands, termed STARFISH, were designed on the basis of the crystal structure

of StxB in complex with a Gb3 analogue and were found to have a subnanomolar inhibitory activity 149. However, the

protective activity of STARFISH against Shiga toxins was suboptimal in vivo150. Recent improvements based on

supramolecular templating to create high-avidity inhibitors for multimeric receptors are now being tested151. In another

study, the trisaccharide moiety of Gb3 has been covalently coupled to an inert silicon-based matrix, Chromosorb-P. The

resulting compound was termed Synsorb-Pk152. Co-incubation of human renal adenocarcinoma cells with Shiga toxin 1

(Stx1) and Stx2 from Escherichia coli and Synsorb-Pk for 1 hour resulted in 50% protection of cells, but clinical

trials did not show the expected protective effect in children with diarrhoea-associated HUS 153. SUPER TWIG, a

carbosilane dendrimer carrying variously oriented Gb3 trisaccharides154, and linear polymers of acrylamide with

clustered Gb3 trisaccharides (called Gb3 polymers)155 are promising therapeutic agents for use against Shiga

toxin-producing E. coli infection in humans. These Stx neutralizers function in the circulation and in the gut,

respectively, and exhibit Stx-binding capacities that are 100,000 times as high as those of Synsorb-Pk. Cytotoxic

activities of Stx1 and Stx2 were markedly inhibited, and mice were protected from a challenge with a fatal dose

ofE. coli O157:H7, even when the neutralizers were administered after infection had been established.

Pt ata

A recombinant bacterium based on a non-pathogenic E. coli strain was designed that displays a Stx receptor mimic

on its surface. This probiotic bacterium has an apparent affinity for StxB that is around 10,000 times as high as that

of Synsorb-Pk, and Shiga toxins were indeed very efficiently adsorbed and neutralized. Oral administration of the

bacterium completely protected mice from lethal toxin doses156,157.

Ma ats

The use of monoclonal antibodies specific for both Stx1 and Stx2 may be the most attractive treatment modality at this

stage158,159. These antibodies prolonged the survival of toxin-challenged mice and have also been shown to prevent

toxin-associated disease in a piglet model, even when antibodies were given 612 hours after toxin application.

Sa tx A suut ts

Small-molecule inhibitors of the catalytic sites of ricin and Stx were designed, based on guanine-like compounds such as

pteroic acid160.

Taspt ts

Screens were designed to identify small molecule compounds that inhibit toxin transport along the retrograde route.Examples of some of the identified molecules are Exo1 (REF. 161), Exo2 (REFS 162,163), compounds 75 and 134 (REF.

164), and Golgicide A165. In all these cases, application of the inhibitor affected Golgi morphology, and the specificity

of the compounds and their protective effect in the host organism remain to be established.

LD50The median lethal dose of a

toxic substance; this is the

dose that is required to kill

half of the members of a tested

population.

R E V I E W S




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A1

A2

StxB StxB

a b c d

A1 fragment

StxB

A2 fragment

Site 1

Site 2

Site 3

Furin cleavage site

Cys242

Cys261

RetromerA heteropentameric complex

that associates with the

cytosolic face of endosomes

and mediates retrograde

transport of cargo molecules

from endosomes to the

trans-Golgi network.

patiets ho did ot develop HUS49, strogl sug-gestig that Gb3 olecular species differeces are alsocrucial for toxi fuctio i huas.

Shiga toxin trafficking

Extesive studies have bee perfored o the itracel-lular traffickig of Shiga toxi ad have bee revieedi detailed elsehere5053. Suppleetar iforatioS1 (table) provides a overvie of the cellular factorsthat cotribute to the traffickig of Shiga toxis. weliit our discussio belo to selected fidigs co-cerig edoctosis ad retrograde trasport that havehad a coceptual ipact beod the toxi field. Ideed,StxB has bee developed ito a cell biolog tool54,55 thatallos the quatitative stud of traffickig pathas ofgeeral iportace (revieed i REF. 52).

Endocytosis. Folloig the bidig to Gb3 receptorolecules at the plasa ebrae, Shiga toxi ad theother ebers of the fail are iteralized ito targetcells b edoctosis (FIG. 2). Shiga toxi has bee foudi clathri-coated pits56. whe clathri-depedetedoctosis is ihibited, hoever, Shiga toxi is stilltake up efficietl5759, shoig that the clathri path-a is ot required for the iitial steps of toxi etrito the cell. Ideed, Shiga toxi ca iduce edoctic

plasa ebrae ivagiatios ithout the help of thectosolic achier60. I this case, ebrae bed-ig results fro the toxi-drive clusterig of glco-sphigolipid receptor olecules (FIG. 2), represetig ae echais of curvature geeratio i biologicalebraes. The toxi-iduced ivagiatios are theprocessed b cellular achier ivolvig dai,acti ad plasa ebrae cholesterol.

Retrograde transport. Folloig its etr ito thecell, Shiga toxis localize to earl ad recclig edo-soes. I a pioeerig stud, Sadvig ad va Deursfoud that Shiga toxi could evetuall be detected i

the ebraes of the edoplasic reticulu (ER)61,strogl suggestig that the toxi as traffickig ia retrograde aer through the secretor patha(FIG. 2). It as sho subsequetl that Shiga toxi cabpass the late edoctic patha to be trasferreddirectl fro earl ad recclig edosoes to thetrans-Golgi etork (TGn)62 ad, fro there, o tothe ER. This traffickig sees to be differet fro therecclig loop that had bee idetified previousl foraose-6-phosphate receptors, hich are cargo-shuttlig proteis that ere sho to ccle betee lateedosoes ad the TGn63. It as the foud that theretrograde traffickig route that liks the earl edo-ctic patha to the TGn is also used b several cellularproteis ith diverse fuctios ragig fro cell sig-allig ad glucose trasport to orphoge traffickigad tissue reodellig (revieed i REF. 52).

The echaiss b hich Shiga toxis escapefro the earl edoctic patha have bee ite-sivel studied, ad clathri57,59 ad retromer6466 are ofcrucial iportace. Clathri is a aodoai orga-izig protei ith established fuctios i the ge-eratio of ebrae curvature. Clathri is recruitedto edosoes b a host of proteis, icludig thephosphatidliositol-4-phosphate-bidig proteiEpsi-related protei (EpsiR), for hich a role i ret-

rograde trasport has bee sho59. Retroer is co-posed of a curvature recogitio subuit of to sortigexis (a cobiatio fro SnX1, SnX2, SnX5 adSnX6) ad a cargo recogitio subuit ade of three

vacuolar protei sortig-associated proteis, VPS26,VPS29 ad VPS35 (revieed i REF. 67). Exactl hoclathri ad retroer drive the foratio of retrogradetrasport iterediates o earl edosoes reaisto be established. Recet evidece suggests that bothproteis a fuctio i a sequetial aer65(FIG. 2).Accordig to this odel, clathri is required for thegeeratio of iitial ebrae curvature o earledosoes. Cargo uses clathri adaptors ad the cargo

Figure 1 | Sa tx stutus. a | A cartoon of Shiga holotoxin, consisting of one A subunit (StxA), which is cleaved

into fragments A1 and A2, and five B fragments that constitute the homopentameric B subunit (StxB). | A ribbon

diagram of Shiga toxin, highlighting globotriaosylceramide (Gb3)-binding sites on StxB. Gb3 is shown in a ball-

and-stick representation. | An enlargement of StxA at the site of furin cleavage (Arg25-Met252), and showing the

disulphide bond (between Cys242 and Cys261) that links the A1 and A2 framents. | A ribbon diagram of an

StxB subunit from the membrane-oriented surface, highlighting the three Gb3-binding sites. Gb3 is shown as a

ball-and-stick representation. Note the central pore that is lined by -helices.

R E V I E W S


http://www.nature.com/nrmicro/journal/v8/n2/suppinfo/nrmicro2279.htmlhttp://www.nature.com/nrmicro/journal/v8/n2/suppinfo/nrmicro2279.htmlhttp://www.uniprot.org/uniprot/Q14677http://www.uniprot.org/uniprot/Q13596http://www.uniprot.org/uniprot/O60749http://www.uniprot.org/uniprot/Q9Y5X3http://www.uniprot.org/uniprot/Q9UNH7http://www.uniprot.org/uniprot/Q9UBQ0http://www.uniprot.org/uniprot/Q96QK1http://www.uniprot.org/uniprot/Q96QK1http://www.uniprot.org/uniprot/Q9UBQ0http://www.uniprot.org/uniprot/Q9UNH7http://www.uniprot.org/uniprot/Q9Y5X3http://www.uniprot.org/uniprot/O60749http://www.uniprot.org/uniprot/Q13596http://www.uniprot.org/uniprot/Q14677http://www.nature.com/nrmicro/journal/v8/n2/suppinfo/nrmicro2279.htmlhttp://www.nature.com/nrmicro/journal/v8/n2/suppinfo/nrmicro2279.html


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Golgi

Plasma membrane

Retrogradetraffic to ER

Cytosol

Retro-translocation

Retrogradetraffficto TGN

Recyclingtubule

ER

Nucleus

Earlyendosome

Clathrinflat lattice

Retromer

Shiga toxin

Coat protein complex IA large coat protein complex

that initiates the vesicle

budding process on the

cis-Golgi and retrograde

transport to the rough

endoplasmic reticulum.

ChaperoneA protein that assists in the

folding or unfolding of target

proteins.

ER-associated degradation(ERAD). A cellular pathway that

targets misfolded proteins in

the endoplasmic reticulum for

ubiquitylation and subsequent

degradation by the

proteasome.

recogitio subuit of retroer to localize to the sites oftrasport iterediate foratio. nascet retrogradetubules are the processed through scissio ito retro-grade trasport iterediates, probabl also ivolvigretroer i a curretl ill-defied process. A detaileddiscussio of this odel ca be foud i REF. 52.

Ho do Shiga toxis, hich bid to the Gb3 glcolipidi the exoplasic ebrae leaflet, couicate iththe ctosolic sortig achier, ad h does their traf-fickig deped o clathri i the earl edosoes but ot

at the plasa ebrae? The first poit a be related tothe fidig that Shiga toxis iduce ebrae reorder-ig60 ad associates ith ebrae eviroets thatfor deterget-isoluble coplexes after cell lsis68,69.This fractioatio behaviour has bee correlated ithefficiet retrograde sortig i HeLa cells, ad the abseceof deterget-isoluble doais has bee correlated ithiefficiet retrograde sortig i hua oocte-derivedacrophages ad dedritic cells that are totall toxiresistat68. Toxi-iduced chages i lipid repartitio ithe exoplasic leaflet a drive siilar aocopart-etalizatio at the level of the ctoplasic leaflet adfavour the recruitet of sortig achier.

The requireet of clathri o edosoes but otat the plasa ebrae a be to sides of the saecoi: although the toxi iduces curvature at the plasaebrae through daic cluster foratio60, oceclustered the toxi a localize preferetiall to thecurved eviroet of retrograde tubules of earl edo-soes, leadig to geoetr-drive sortig. Shiga toxisare trasported fro the TGn i a coat protein complex I(COPI)-idepedet aer through the Golgi appara-tus to the ER70,71. The exact echaiss that uderlie thistraffickig step still reai largel uexplored.

Retro-translocation. Shiga toxis do ot iduce pore for-atio. Istead, the rel o host cell achier to tras-locate the cataltic A subuit across cellular ebraesad to gai access to the ctosol. The echaiss bhich this occurs are startig to be uderstood. Durigthe earl etr process (probabl at the stage of earledosoes), a protease-sesitive loop (residues 242261)located i the C-terial regio of StxA is cleaved bthe ebrae-associated edoprotease furi at site

Arg251-met252 (FIG. 1), dividig StxA ito the catalticA1 fraget (cosistig of residues 1251; 27.5 kDa) ada StxB-associated A2 fraget (cosistig of residues252293; 4.5 kDa)72. The A1 fraget reais likedith the StxA2StxB coplex b virtue of a disulphidebod betee Cs242 i StxA1 ad Cs261 i StxA2(REFS 7276). This disulphide bod is ultiatel reducedi the ER lue, liberatig the ezatic A1 fraget,hich is subsequetl traslocated to the ctosol.

Shiga toxis have bee co-iuoprecipitated ithER chaperones ad trasloco copoets, suggest-ig that the toxis use the cellular ER-associated proteindegradation (ERAD) patha. Ideed, Shiga toxis erefoud to iteract ith HEDJ (also ko as ERdj3 adDnAJB11), BiP ad 94 kDa glucose-regulated protei(GRP94) i pre-associated large ulti-chaperoe co-plexes before their trasport across the ER ebrae7780.Furtherore, iuoprecipitatio studies deostratedthat substatial aouts of HEDJ-associated Shiga toxiiteract ith the itegral ebrae Sec61 traslococore uit79. It as suggested that HEDJ ad its iteractigparters recruit isfolded proteis to the Sec61 traslo-co ad that Shiga toxis exploit this achier for theiro trasport across the ER ebrae79(FIG. 3).

mebrae traslocatio sees to be the rate-liitigstep for the itoxicatio of cells b Shiga toxis. mostof the cell-associated StxA is cleaved but reais boud

to StxB, as the disulphide bod betee the A1 ad A2fragets fails to be reduced. Ol a sall fractio (4%)of StxA sees to be traslocated to the ctosol81, cosist-ig of cleaved, reduced ad StxB-separated A1 fragets.Iteractio of the A1 fraget ith the ERAD achierdoes ot see to ivolve a specific export sequece butrather relies o a degeerated structural eleet, suchas a hdrophobic sequece i its C-terial regio 78.It reais to be deteried hether such a sequecedirectl iteracts ith the lipid bilaer to iduce co-foratioal chages that are the recogized b theERAD achier, as has bee suggested for otherprotei toxis82.

Figure 2 | Ta Sa txs. An overview of intracellular trafficking of

Shiga toxins. Toxin binding to the plasma membrane induces local spontaneous

curvature, membrane-mediated clustering and the toxin-driven formation of

endocytic invaginations. The toxin then undergoes retrograde sorting in early

endosomes, in which retrograde tubules are formed in a clathrin-dependent manner,

and Shiga toxins preferentially localize to this tubular environment. Retrograde tubules

are processed by scission in a retromer-dependent manner. Shiga toxins bypass the late

endocytic pathway and are transferred directly from the early endosome to the trans-

Golgi network (TGN)62 and, from there, on to the endoplasmic reticulum (ER). Finally,

Shiga toxins use the ER-associated degradation (ERAD) machinery to facilitate retro-

translocation into the host cell cytosol. By contrast, the transferrin receptor protein (TfR)

(Y-shaped) becomes enriched in recycling tubules in the early endosome and recyclesback to the plasma membrane.

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ProteasomeEscape fromdegradation

FoldedA1 fragment

StxBStxA2Shigaholotoxin

Proteasomaldegradation

Ubiquitylation

and release

Transportthroughthe pore

Sec61 Lipid bilayer

ER lumen

Cytosol

Multi-chaperonecomplex

Unfoldingandtargeting

Misfoldedprotein

UnfoldedA1 fragment

Ub

p97

HEDJ

HEDJ

Sec61

BiP

GRP94

p97

a Misfolded proteins b Shiga toxin

Interleukin-8A chemokine of the C-X-C

subfamily that is known to

recruit polymorphonuclear

cells into areas of infection.

Mitogen-activated proteinkinase(MAPK). One of a family of

threonine-directed kinases

with important roles in the

regulation of diverse cellular

functions, including cytokine

release. MAPKs are

categorized into three main

pathways and include the

extracellular signal-regulated

kinases (ERKs), the JUN

amino-terminal kinases (Jnks;

also called the stress-activated

protein (SAP) kinases) and

MAP kinase p38.

Ulike isfolded edogeous proteis, the A1 frag-et of Shiga toxis ust avoid the proteasoe utilit reaches its substrate i the ctosol. It is ver probablethat the A1 fraget is protected fro degradatio ithe ctosol b its lack of lsie residues, together ithits abilit to refold rapidl83. Lactacsti, a proteasoeihibitor, icreases the aout of ctosolic A1 fragetb 30% ad ehaces ctotoxicit b 50%, suggestigthat a fractio of the traslocated A1 fraget ca bedegraded b the proteasoe achier81, as describedfor rici ad cholera toxi84,85.

Intoxication by Shiga toxinsThe kide ad gastroitestial tract are the ost co-ol affected orgas i patiets ith HUS, but cliicalaifestatios ca also be see i soe patiets at thelevel of the cetral ervous sste ad other orgas. Thereader is referred to revies focused o disease-relatedissues for further iforatio o this topic13,86. I short,Shiga toxis ca directl ihibit protei biosthesis.I this a, coesal bacteria i the lue of thegut ca be targeted, thereb reducig their proliferatioad providig a copetitive advatage for Shiga toxi-producig bacteria. Other targets are host cells. I thecase of the itestial ucosa, iitial daage is caused

b bacterial products other tha toxis, as hua guteteroctes express ver lo levels of Gb3 (REFS 27,87).Hoever, Shiga toxis daage the icrocirculatio,causig vasculitis, hich exacerbates ucosal da-age. Ifarctio of the ucosa leads to bleedig ito theboel ad blood diarrhoea. This a favour bacterialsurvival, as essetial utriets, icludig iro ad othergroth prooters, are provided. Although it as logthought that the fuctio of Shiga toxis i HUSpatho-geesis as liited to their role as protei biosthesisihibitors, it is o clear that these toxis also triggera sigallig cascades that ifluece their o traf-fickig as ell as other cellular fuctios such as ctokiesecretio ad the iductio of cell death b apoptosis.notabl, the toxi-iduced release of pro-iflaatorctokies fro ooctes or acrophages afavour disease progressio b upregulatig Gb3 expres-sio o edothelial cells. Aother effect of Shigatoxis is the ctokie-stiulated activatio of pol-orphouclear leukoctes, hich release reactiveoxge etabolites that cause edothelial cell ijur.

Interleukin-8 (IL-8) sees to pla a crucial part i thiscotext. The coplex odulatio of the iue ss-te b Shiga toxis a dape specific iuit ad,through local iflaatio, ehace ctotoxicit.

Ribotoxic stress. The ost proiet echais bhich a sigallig respose is iduced b Shiga tox-is stes fro the odificatio of ribosoes ad istered the ribotoxic stress respose88. The 3 ed of28S ribosoal RnA (rRnA) fuctios i aioacltRnA bidig, peptidltrasferase activit ad ribosoaltraslatio. whe these activities are disrupted, oigto the rRnA odificatio iduced b Shiga toxis,for exaple, JUn n-terial kiase (Jk) proteisad mitogen-activated protein kinase (mAPK) p38 areactivated ad extracellular sigal-regulated kiase 1(ERK1; also ko as mAPK3) ad ERK2 (alsoko as mAPK1) sigallig is altered8992. Ribotoxicstress is specific i that ot all protei sthesis ihibi-tors iduce this patha. Ho daage i the 3 ed of28S rRnA triggers the dostrea sigallig kiasesis still ot uderstood. Oe possibilit is that eloga-tio factors or other related ribosoe-bidig factorsiteract ith the mAPK sigallig cascade.

Triggering signalling pathways. O bidig to their recep-tors i the plasa ebrae, Shiga toxis iduce the

recruitet ad activatio of Gb3-cotaiig, glcolipid-eriched ebrae fractios of several trosie kiases,such as splee trosie kiase (SyK) i HeLa cells93, yES iACHn real cells94 ad Lyn i Burkitts lphoa Raoscells95. Oce activated, kiases acquire icreased detergetsolubilit94,95. Activatio of SyK leads to rapid trosie phos-phorlatio of several proteis, icludig clathri heavchais, ad has a effect o toxi uptake93. Iside cells,the toxis also activate kiases such as protein kinase C(PKC)96 ad p38 (REF. 97). I the case of PKC, a directeffect o the retrograde trasport of the toxi has beeobserved96, ad the role of p38 i ribotoxic stress isetioed above.

Figure 3 | The pasm tuumassat pt aat patwa.

a | A simplified view of misfolded protein translocation across the endoplasmic reticulum

(ER) membrane. Misfolded proteins are recognized by ER-localized luminal chaperone

complexes and targeted to a protein-conducting translocation channel termed the

translocon, which includes Sec61. Polypeptides are then tagged by ubiquitin (Ub) at

the cytosolic face of the ER membrane, and the polyubiquitylated polypeptides are

degraded by the proteasome after release from the ER membrane into the cytosol. The

cytosolic chaperone p97 (also known as VCP), which is an AAA (ATPase associated with

various cellular activities) ATPase, together with its cofactors, may be a driving force of

retro-translocation. | Translocation of Shiga toxins. Following cleavage by furin, the

Shiga toxin A subunit is released from the B subunit following disulphide bond reduction.

The A subunit associates with host ER chaperones (HEDJ (also known as ERdj3 and

DNAJB11), BiP and 94 kDa glucose-regulated protein (GRP94)) and is targeted to the

translocon channel for retro-translocation into the cytosol.

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Protein kinase CdA member of the protein

kinase C family, which contains

at least 12 isoforms of serine/

threonine kinases that are

involved in signal transduction,

regulation of gene expressionand myeloid differentiation.

GM-CSF(Granulocyte-macrophage

colony-stimulating factor). A

protein that is often secreted

by macrophages, T cells,

endothelial cells and

fibroblasts.

Tumour necrosis factorA cytokine involved in systemic

inflammation. Its primary role

is the regulation of immune

cells.

The echaiss b hich the Shiga toxis stiu-late ctosolic kiases reai to be cofired, althougha protei ith a apparet olecular ass of 27.2 kDathat co-iuoprecipitates ith Gb3-specific oo-cloal atibodies is a possible cadidate for likigGb3 ith yES i ACHn cells98. Alterativel, Shigatoxi-iduced Gb3 clusterig i the exoplasicleaflet a iduce lipid repartitio i the ctosolic leaflet,leadig to aggregatio-ediated autophosphorlatioof SRC fail protei trosie kiases94,95.

The bidig of Shiga toxis to the plasa e-brae iduces reodellig of the acti ad icrotu-bule ctoskeleto99,100. This process is accopaiedb the redistributio of a uber of proteis, iclud-ig ezri, CD44, vieti, ctokeratis, paxilli adfocal adhesio kiases. Both StxB bidig ad Gb3-specific atibod bidig iduces phosphorlatioof ezri, a protei that liks the acti ctoskeletoto the plasa ebrae. Shiga toxi-iduced ezriphosphorlatio is ihibited b loerig the levels ofplasa ebrae cholesterol or b ihibitig SRC,

phosphoiositide-3-kiase or Rho-associated proteikiase 1 (ROCK1)100.

Cytokine synthesis and release. ma edothelial adepithelial cells i the kide ad the cetral erv-ous sste are exquisitel sesitive to ctotoxicitiduced b Shiga toxis, hereas others, such asperipheral blood ooctes, have bee reported tobe resistat to itoxicatio, despite their expressioof Gb3 (REFS 101,102). Toxi-resistat cells respod totoxi bidig ad iteralizatio b sthesizig adreleasig pro-iflaator ctokies. I tur, thesectokies stiulate Gb3 biosthesis ad expressioo uerous edothelial cells ad sesitize target cellsto the ctotoxic actio of the toxis103,104 i a processthat ofte ivolves sigallig through the p38 path-a105,106. Therefore, the host iate iue resposea exacerbate the vascular daage that is iitiated bShiga toxis (see above).

Shiga toxis regulate ctokie expressio throughultiple echaiss. O release fro the bacteriu,Shiga toxis iteract ith the itestial epitheliu,here it iduces the ribotoxic stress respose to causep38-ediated secretio of the pro-iflaatorctokie IL-8 (REF. 97). I Vero cells as ell as i ites-tial epithelial HCT-8 cells, the ZAK isofors ZAKad ZAK ere idetified as mAP3Ks (mAPK kiase

kiases). ZAK trasduces the sigal fro itoxicatedribosoes to the host mAPKs p38 ad Jk proteis,hich i tur iitiate a cascade of evets that ulti-atel proote upregulatio of IL-8 (REF. 91). Shigatoxis have also bee sho to iduce sthesis ofIL-8 i itestial Caco-2 ad T84 cells107. After cross-ig the itestial epitheliu ad eterig the vascu-lature, Shiga toxis stiulate circulatig ooctes(hich do ot udergo apoptosis) to secrete ctokies,icludig GM-CSF ad tumour necrosis factor (TnF),i a p38-depedet aer 89,101,108,109. Patiets ithHUS frequetl have elevated levels of TnF i theirurie89.

The stiulatio of differetiated, acrophage-like cells of the hua ooctic cell lie THP1 bShiga toxis activates yES, hich the upregulates theexpressio ad activit levels oftissue factor, a keeleet of the coagulatioiflaatiothrobosiscircuit, through phosphoiositol-3-kiase-ediatedactivatio of the ihibitor of nF-B kiase subuit-proteasoeuclear factor-BREL ad mAPK/ERKkiase (mEK)ERK2earl groth respose protei 1pathas110. These reactios a also cotribute totoxi-iduced edothelial cell ijur.

Apoptosis. Apoptosis is a cell death patha that is char-acterized b several uclear chages, such as chroaticodesatio ad DnA fragetatio. The seque-tial activatio of a cascade of csteie-depedet,aspartate-specific proteases, called caspases, is a kecopoet of the prograed cell death achier.Although Shiga toxis a ot iduce apoptosis iall cell tpes, there is aple evidece to suggest thatapoptosis is crucial for the developet of vascular

lesios ad tissue daage folloig traslocatio ofthe toxis ito the bloodstrea. The data published todate suggest that Shiga toxis sigal apoptosis throughdifferet echaiss i differet cell tpes (FIG. 4).

Stx1-iduced apoptosis i THP1 cells requiresretrograde trasport through the Golgi apparatusto the ER ad the activatio of the executioer cas-pase, caspase 3(REF. 111). Icubatio of THP1 cellsith Stx1 activates the ER stress respose, a sigal-lig echais triggered b the accuulatio ofufolded or isfolded proteis i the ER, hichi tur triggers the iductio of prograed celldeath. Stx1 treatet icreases activatio of the ERebrae-associated stress sesors IRE1, PRKR-likeER kiase (PERK; also ko as EIF2K3) ad cclicAmP-depedet trascriptio factor 6 (ATF6) adicreases the expressio of the trascriptioal regula-tor C/EBP-hoologous protei (CHOP; also ko asDDIT3) ad the death receptor 5 (DR5; also koas TnFRSF10B) at RnA ad protei levels (FIG. 4).The level of the survival factor B cell lphoa 2(BCL-2) decreases folloig Stx1 itoxicatio, hereassecretio of TnF-related apoptosis-iducig ligad(TRAIL; also ko as TnFSF10) icreases.

ER stress, elicited b Stx1, leads to Ca2+ release froER stores ad the activatio of the Ca2+-depedetcsteie protease calpai, hich cotributes to the earl

activatio (through cleavage) ofcaspase 8(REF. 112)(FIG. 4). Cleavage of caspase 8 a directl triggerthe activatio of caspase 3 or a iitiate apoptosisthrough the itochodrial patha b cleavage ofthe iactive, 22 kDa for of BH3-iteractig doai(BID) to the active, 15 kDa for called trucated BID(tBID). Oe stud foud that prograed cell deathrequired the deliver of the fuctioal holotoxi (otStxB aloe or holotoxi olecules coprised of StxAith poit utatios that draaticall reduce thetoxis ezatic activit) to the ER, suggestig thatthis orgaelle ideed serves as the iitial locatio forapoptotic sigallig i THP1 cells113.

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TRAIL

Nucleus

Proteasome

FLIP

Golgi

Earlyendosome

Stx1 and Stx2

ER

ER stress,IRE1, PERKand ATF6

Calpain Ca2+

DR5

CHOP BCL-2Cytochrome C

BAK and BAXchannels

?

tBID

BID

XIAP

APAF-1

Caspase 8

Mitochondria

Plasma membrane

Procaspase 8

Caspase 9

Procaspase 9

Procaspase 3

Procaspase 6

Caspase 6

Caspase 3Lamin A

Gb3 Stx

Mitochondria

Intrinsic ormitochondrialpathway

Retrograde transportand enzymatic activityof Stx1 and Stx2

Extrinsic pathway

Apoptosis

Siilar apoptotic sigallig pathas are triggeredb Shiga toxis i differet cell lies, ael oo-ctic THP1 cells, HeLa cells ad hua brai icro-

vascular edothelial cells (HBmEC)112115. I HeLacells, Stx1-iduced apoptosis occurs through a path-a requirig caspase 8, caspase 6 ad caspase 3 butot caspase 9(REF. 114). I these cells, Stx1 as foudto icrease the expressio of the caspase 9 ihibitorX-liked ihibitor of apoptosis protei (XIAP). IHBmEC, Stx2 iduces DnA fragetatio, ad cleav-age activatio of caspase 3, caspase 6, caspase 8 adcaspase 9 is ediated b CHOP upregulatio ad thecoplete degradatio of the ati-apoptotic proteiFLICE-like ihibitor protei (FLIP; also ko asCFLAR), hich i tur ehaces the activatio ofcaspase 8 (REF. 115).

Apoptosis iduced b Stx1 ca be associated ithehaced expressio of the pro-apoptotic proteiBAX116, ad overexpressio of BCL-2 protects cells

agaist Stx1-iduced cell death117. Shiga toxis alsoihibit the expressio of the ati-apoptotic BCL-2fail eber mCL1 (REF. 118).

Gb3-specific oocloal atibod ca iduceapoptosis-like cell death i Burkitts lphoa cells.Strikigl, hoever, Gb3-specific atibod ad Stx1trigger differet apoptotic sigallig pathas. Stx1-iduced apoptosis ivolves caspase activatio aditochodrial depolarizatio, hereas oxidativestress ediates Gb3-specific atibod-iduced celldeath119. I alost all other cells Gb3-specific ati-bodies do ot trigger cell death ad StxA is requiredto iduce apoptosis111,120.

Figure 4 | Apptss patwas tat a u Sa txs. Shiga toxins induce apoptosis through differentmechanisms, resulting in activation of the apoptosis initiator caspase, caspase 8, and the executioner caspase,

caspase 3. See main text for details. This figure summarizes findings from different cell types. In most cases, the

enzymatic activity of Shiga holotoxins is required for the induction of apoptosis. APAF-1, apoptotic protease-activating

factor 1; ATF6, cyclic AMP-dependent transcription factor 6; BAK, BCL-2-homologous agonist/killer; BCL-2, B cell

lymphoma 2; BID, BH3-interacting domain; CHOP, C/EBP-homologous protein (also known as DDIT3); DR5, death

receptor 5 (also known as TNFRSF10B); ER, endoplasmic reticulum; FLIP, FLICE-like inhibitory protein (also known as

CFLAR); Gb3, globotriaosylceramide; PERK, PRKR-like ER kinase (also known as EIF2K3); tBID, truncated BID; TRAIL,TNF-related apoptosis-inducing ligand; XIAP, X-linked inhibitor of apoptosis protein.

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Topoisomerase IA group of proteins belongingto the topoisomerase family,

which contains enzymes that

catalyse ATP-independent

breakage of one of the two

strands of DNA, passage

of the unbroken strand through

the break and rejoining of the

broken strand. Topoisomerase I

enzymes reduce the

topological stress in the DNA

structure by relaxing the

superhelical turns and knotted

rings in the DNA helix.

BenzodiazepineA psychoactive drug, the core

chemical structure of which

is the fusion of a benzene

ring and a diazepine ring.

Benzodiazepines have varying

sedative, hypnotic, anxiolytic,

anticonvulsant, muscle relaxant

and amnesic properties, which

make them useful in treating

anxiety, insomnia, agitation,

seizures, muscle spasms and

alcohol withdrawal and as a

pre-medication for medical or

dental procedures.

Major histocompatibility

complex class I(MHC class I). A moleculefound on every nucleated cell

of the body. Its function is to

display fragments of proteins

from within the cell to T cells,

so that healthy cells will be left

alone and cells with foreign

proteins will be attacked by the

immune system. Because MHC

class I molecules present

peptides that are derived from

cytosolic proteins, the pathway

of MHC class I presentation is

often called the cytosolic or

endogenous pathway.

Biomedical applications

The B subuit of Shiga toxis are aturall evolveddeliver tools that trasport the cataltic A subuitfro its site of productio i the itestie to periph-eral tissue locatios. As a deliver tool, the B subuitexhibits olecular characteristics that have beeacquired durig co-evolutio ith its hosts, icludigstabilit at extree pH ad i the presece of proteases,the capacit to cross tissue barriers ad to distributei the orgais, ad resistace to extra- ad itracellulariactivatio (revieed i REF. 51). The B subuit recep-tor, Gb3, has a restricted tissue expressio patter ad ispreset o to cell tpes that are of particular iterest ibioedical research ito ovel strategies for the cliicalaageet of cacer ad ifectious diseases: cacercells theselves ad dedritic cells, hich are atige-presetig cells ith ke fuctios i the iductio ofpriar iue resposes.

Tumour targeting. The expressio ad etabolis ofcell surface glcolipids is odified durig ocogeic

trasforatio, ad altered glcoslatio patters affecttuour ivasio ad etastasis121. Gb3 is overexpressedb various cacer cell lies ad hua cacers, iclud-ig colorectal87,122, breast123,124 ad ovaria125 carcioasad Burkitts lphoas126. These fidigs suggest thatthe Gb3-bidig specificit of StxB could be exploitedto target hua tuours.

I pioeerig studies, it as foud that itratuoralijectio of the Stx1 holotoxi ihibits tuour grothi ouse xeograft odels127130. Stx1 also eliiatescologeic tuour cells i ex vivo purgig applicatios131.Hoever, the use of holotoxi as a therapeutic aget ihuas a be liited b the fact that the toxic effectof the A subuit is ot tuour specific. The B subuithas therefore bee coupled to ctotoxic copouds thathave preferetial effects o cacer cells. Furtherore,cotrast agets for o-ivasive tuour iagig havealso bee liked to the B subuit.

Usig these liked cotrast agets, it as sho thatthe B subuit targets Gb3-expressig spotaeous adeo-carcioas of the gut folloig oral uptake or itraveousijectio i a trasgeic ouse odel132. The tuourscould be detected b o-ivasive iagig usig fibredcofocal fluorescece edoscop ad positro eissiotoograph. It as also sho that, oig to traffickigthrough the retrograde route, the B subuit stabl associ-ates ith tuour cells, i hich it ca be detected for sev-

eral das after ijectio. The cocept of usig the B subuitas a deliver tool as recetl exteded to hua color-ectal carcioa. Priar cultures of tuoral eteroctesfro surgical saples are targeted b the B subuit87, adit is also efficietl take up b xeografts of priarhua tuours i ice133. Iterestigl, edothelial cellsof tuour eovascularizatio accuulated the B subuitin vivo, hich a allo for targeted therapeutic iterve-tio eve i cases i hich the tuours cells theselvesdo ot express Gb3 (REFS 128,133).

A uber of ctotoxic copouds have bee coupledto the B subuit, icludig: the topoisomerase I ihibitorSn38, hich is the active etabolite of captotheci 11

(a copoud that is used for the treatet of colorectalcarcioas)134; the bezodiazepie RO5-4864, hichis a ligad for itochodrial peripheral benzodiazepinereceptors135; ad photosesitizig agets, such as theporphri-based copouds TPP(p-O--GluOH)

3

(REF. 136) ad Chlori e6 (Ce6)137. It as sho that aB subuitTPP(p-O--GluOH)

3cojugate as ore

efficiet at photodaic cell killig tha the porph-ri copoud aloe, oig to B subuit-iduced retro-grade deliver ito the Golgi apparatus136. The efficacof each of these ovel forulatios is curretl uderscruti i aial experiets.

Immunotherapy. The uique traffickig patha of Shigatoxis ad the selective expressio of Gb3 o huadedritic cells ake the B subuit a attractive o-livead o-toxic sthetic vector for the developet oftherapeutic vaccies to treat hua cacers ad ifec-tious diseases. whe geeticall fused or cheicall cou-pled to the B subuit, various exogeous cacer ad viralatiges ere delivered to the ctosolic copartet

of dedritic cells, leadig to proteasoe-depedetatige processig, TAP-depedet peptide loadigoto eosthesized major histocompatibility complex class I(mHC class I) olecules i the lue of the ER ad sub-sequet mHC class I olecule-restricted atige prese-tatio at the cell surface138141. Dedritic cells are curretlcosidered to be the ost potet iducers of CD8+ cto-toxic T lphocte (CTL) resposes to viral ifectiosad tuours. Vacciatio of ice ith B subuit-basedcojugates ideed iduces strog CTL resposes ith T

H1

polarizatio. These CTLs are log lastig142 ad also effi-ciet, as the protect ice agaist challeges b viruses ortuours i prophlactic ad therapeutic settigs142,143.

Conclusions

we have discussed the a as i hich Shiga toxisiteract ith aalia hosts. Although it is eas to seeho daage of the itestial ucosa ould beefit thetoxi-producig bacteria b providig iproved grothcoditios, it is less eas to explai h daage at periph-eral sites such as the kide or cetral ervous ssteould be beeficial. It is possible that these orgas areot the priar targets. Furtherore, harbourig a bac-teriophage that ecodes Shiga toxis a protect a hostsicrobiota fro bactivorous predators like the ciliatedprotozoa Tetrahymena thermophila144,145. I this respect,huas ight be coicidetl caught i the crossfire of

a icrobial predatorpre relatioship. Hoever, eitherShiga toxi-bidig lipids or close hoologues of thegee that ecodes the aalia Gb3 sthase havebee idetified i T. thermophila144. Alterative receptorscould be preset, or toxis ight be captured through theoral apparatus.

Shiga toxis have becoe poerful tools for theivestigatio of cellular processes, otabl i the co-text of itracellular traffickig. Oe of the ost ipor-tat challeges i this cotext is to coe to a fuctioaluderstadig of ho a protei that associates ith theexoplasic ebrae leaflet ca couicate effi-cietl ith the ctosolic achier. Addressig this

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challege ill require ovel techologies for live-tiead high-resolutio iagig of the daics of Gb3lipids, as ell as iput fro the field of ebraebiophsics to forulate cocepts that are expected toipact our uderstadig of ebrae echaicalprocesses beod the field of Shiga toxi research.

Shiga toxis have started to attract attetio as deliv-er tools, exploitig to this ed a of the character-istics that the toxis have aturall acquired throughiteractio ith their hosts. Although i the field of

iuotherap proof of cocept could be obtaied ithe ouse odel, the tuour deliver approach is stilli a earl phase of developet. Crucial issues such asthe iuogeicit of the vector i log-ter, high-dose treatets, its toxicit to peripheral targets suchas the kide ad its efficac as a high-capacit deliv-er tool eed to be explored ssteaticall. This lieof research holds the proise of edical beefit froolecules that, as holotoxis, are still a ajor threat forhua health toda.

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AcknowledgementsWe thank L. Roberts, J.M. Lord, J. Wiels and C. Lamaze for

helpful discussion, and J. Mntrey for help with figure 1.Work in the laboratory of the authors was supported byCEFIPRA (The Indo-French Centre for the Promotion of

Advanced Research; programme n3803), the HumanFrontier Science Program (grant RGP26/2007), the ImmuCanprogramme of the Ple de Comptitivit MdicenParis Rgion, the Direction Gnrale de lArmement pro-gramme of Paracell, the Cancerimmunotherapy project in theEuropean Unions Seventh Framework Programme and theProgramme indicactif et coopratif (PIC) on Tumour Deliveryat the Institut Curie.

Competing interests statementThe authors declare no competing financial interests.

DATABASESUniProtKB:http://www.uniprot.org

BAX|BCL-2 |BiP |caspase 6|caspase 3 | caspase 8 |caspase 9|

CHOP|DR5 |EpsinR|ERK1 | ERK2 |ezrin | FLIP| furin|

GRP94 |HEDJ | IL-8 |IRE1| PERK |SNX1 | SNX2 | SNX5 | SNX6 |tissue factor|TRAIL |VPS29|VPS35 | XIAP

FURTHER INFORMATIONLudger Johannes homepage:http://www.curie.fr/

recherche/themes/detail_equipe.cfm/lang/_gb/id_

equipe/101.ht

2010_johannes_natrevmicrobiol

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