LETTERS

Listeria monocytogenes impairs SUMOylation forefficient infectionDavid Ribet1,2,3, Melanie Hamon1,2,3, Edith Gouin1,2,3, Marie-Anne Nahori1,2,3, Francis Impens4,5,Helene Neyret-Kahn6,7, Kris Gevaert4,5, Joel Vandekerckhove4,5, Anne Dejean6,7 & Pascale Cossart1,2,3

During infection, pathogenic bacteria manipulate the host cell invarious ways to allow their own replication, propagation and escapefrom host immune responses. Post-translational modifications areunique mechanisms that allow cells to rapidly, locally and speci-fically modify activity or interactions of key proteins. Some of thesemodifications, including phosphorylation and ubiquitylation1,2,can be induced by pathogens. However, the effects of pathogenicbacteria on SUMOylation, an essential post-translational modifica-tion in eukaryotic cells3, remain largely unknown. Here we showthat infection with Listeria monocytogenes leads to a decrease in thelevels of cellular SUMO-conjugated proteins. This event is trig-gered by the bacterial virulence factor listeriolysin O (LLO), whichinduces a proteasome-independent degradation of Ubc9, an essen-tial enzyme of the SUMOylation machinery, and a proteasome-dependent degradation of some SUMOylated proteins. The effectof LLO on Ubc9 is dependent on the pore-forming capacity of thetoxin and is shared by other bacterial pore-forming toxins likeperfringolysin O (PFO) and pneumolysin (PLY). Ubc9 degradationwas also observed in vivo in infected mice. Furthermore, we showthat SUMO overexpression impairs bacterial infection. Together,our results reveal that Listeria, and probably other pathogens,dampen the host response by decreasing the SUMOylation levelof proteins critical for infection.

L. monocytogenes is a facultative intracellular pathogen responsiblefor human listeriosis, a severe food-borne disease, and has emerged as amodel for the study of host–pathogen interactions4. This bacterium isable to cross the intestinal, maternofetal and blood–brain barriers, toresist macrophage killing and to enter into normally non-phagocyticcells and replicate therein. During infection, L. monocytogenes exploitsan amazingly high number of mammalian cell functions for its ownbenefit. In particular, Listeria interferes with several signalling path-ways and is able to regulate host protein activities by modifyingtheir ubiquitylation or phosphorylation5–7. However, the impact ofL. monocytogenes on SUMOylation, an essential post-translationalmodification, remains completely unknown.

SUMOylation is a reversible modification in which SUMO, anubiquitin-like polypeptide of approximately 10 kDa, is covalentlylinked to target proteins. This conjugation results from the formationof an isopeptide bond between the carboxy-terminal Gly residue of amature SUMO moiety and a Lys side chain of the substrate protein3.The human genome encodes three functional SUMO isoforms thatcan be linked to distinct and overlapping sets of proteins8. Covalentlinkage of SUMO to its substrate requires a set of different enzymes, inan analogous fashion to ubiquitylation. In humans, the SUMOylationmachinery is composed of an E1 SUMO enzyme (the SAE1/SAE2heterodimer), an E2 SUMO enzyme (Ubc9) and E3 SUMO enzymes

that enhance SUMO conjugation of specific targets. In contrast to theubiquitylation machinery in which several dozen E2 enzymes arefound9, the E2 SUMO enzyme is unique in mammals and is requiredfor viability10. The SUMOylation level of cellular proteins is tightlyregulated by SUMO-specific proteases that catalyse the deconjugationof SUMO from its substrates11. SUMOylation, like ubiquitylation, isessential for different cellular functions. Several hundreds of SUMO

1Institut Pasteur, Unite des Interactions Bacteries-Cellules, Departement de Biologie Cellulaire et Infection, F-75015 Paris, France. 2INSERM, U604, F-75015 Paris, France. 3INRA,USC2020, F-75015 Paris, France. 4Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium. 5Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.6Institut Pasteur, Unite Organisation Nucleaire et Oncogenese, Departement de Biologie Cellulaire et Infection, F-75015 Paris, France. 7INSERM, U579, F-75015 Paris, France.

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Figure 1 | Decrease in SUMO-conjugated proteins upon infection withListeria. a, Comparison of SUMO-conjugated protein patterns fromuninfected HeLa cells (2) or cells infected for 1, 3 or 5 h with L.monocytogenes (L.m.) or L. innocua (L.i.). Whole-cell lysates were analysedby immunoblotting experiments using antibodies specific for SUMO1(upper panel) or SUMO2/3 (lower panel) isoforms. b, Analysis of SUMO1-conjugated protein patterns from uninfected HeLa cells (2) or cells infectedfor 3 h with wild-type (WT), DinlB or Dhly L. monocytogenes. c, Analysis ofSUMO1- and SUMO2/3-conjugated protein patterns from HeLa cellsincubated with 3 nM LLO for 20 min.

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targets have now been identified, involved in transcription regulation,maintenance of genome integrity, intracellular transport, stress res-ponses, protein stability and many other biological processes (forreview, see refs 3, 12).

Interestingly, some viruses interfere with the SUMOylation of hostproteins13. We thus tested the hypothesis that pathogenic bacteria,like viruses, also alter host protein SUMOylation for the orchestra-tion of the onset, establishment and/or persistence of the infectiousprocess, and addressed this issue in the case of infection with L.monocytogenes.

To investigate whether L. monocytogenes was able to modifySUMOylation of host cell proteins upon infection, we comparedthe global pattern of proteins conjugated to SUMO1 or SUMO2/3in uninfected cells with that of cells infected by L. monocytogenes orincubated with Listeria innocua, a non-pathogenic Listeria species.HeLa cells infected with L. monocytogenes displayed, after 3 h ofinfection, a decrease in both SUMO1- and SUMO2/3-conjugatedproteins of high molecular mass, compared with uninfected cells(Fig. 1a). This decrease was not observed with L. innocua and is thusspecific to the pathogenic L. monocytogenes species. This globalreduction in protein SUMOylation was confirmed by proteomicanalysis of SUMO-conjugated proteins isolated from cells infectedor not by L. monocytogenes, using the stable isotope labelling of aminoacids in cell culture technique (SILAC)14 (Supplementary Fig. 1 andSupplementary Tables 1 and 2). A decrease in SUMOylated proteinsupon infection with Listeria is of particular interest as it clearly differsfrom the reported increase in SUMOylation observed for cells sub-jected to various environmental stresses15,16.

A Listeria DinlB mutant impaired in entry into HeLa cells stillinduced a decrease in SUMO-conjugated proteins (Fig. 1b), indi-cating that this decrease can be triggered by extracellular bacteria,and involves a surface or secreted protein. We thus tested a ListeriaDhly mutant, defective for LLO, a secreted pore-forming toxin with apotent signalling activity, involved in the escape of Listeria from theinternalization vacuole17. Strikingly, this mutant had no effect on hostSUMOylated proteins (Fig. 1b), which strongly suggested that LLOplays a role in the decrease in SUMOylation observed with wild-typeListeria. We next analysed the global SUMOylation pattern of cellsincubated with purified LLO alone, at a non-cytotoxic concentration7.LLO was sufficient to trigger a massive decrease in SUMOylated pro-teins in the host cell, both for SUMO1 and SUMO2/3 isoforms(Fig. 1c). This effect was not restricted to HeLa cells and was observedin Jeg3 cells (Supplementary Fig. 2).

As many different SUMO targets were affected, we hypothesizedthat LLO could have an effect on the SUMOylation machinery. In

vitro SUMOylation of RanGAP1, a well-characterized SUMO sub-strate18, was not affected by adding increasing amounts of LLO in thereaction, revealing that LLO has no direct inhibitory effect on E1 orE2 SUMO enzymes (Fig. 2a). We then quantified the levels of E1 orE2 SUMO enzymes in HeLa cells incubated with purified LLO.Strikingly, LLO induced a dramatic decrease in the level of Ubc9,in a dose-dependent manner, whereas the levels of SAE1 and SAE2remained unaffected (Fig. 2b, d). The messenger RNA levels of Ubc9were not affected upon LLO incubation (Supplementary Fig. 3).Moreover, LLO strongly reduced Ubc9 half-life (from more than8 h to less than 10 min), and inhibition of cellular protein synthesisby cycloheximide did not prevent the LLO-triggered decrease inUbc9 levels (Fig. 2b, c and Supplementary Fig. 3). Together, theseresults indicate that LLO does not affect Ubc9 translation but rathertriggers its degradation. To our knowledge, LLO is the first bacterialfactor inducing the degradation of the E2 SUMO enzyme. Thedecrease in Ubc9 level triggered by LLO was also observed duringbacterial infection of HeLa and Jeg3 cells but was not observed in cellsinfected with a L. monocytogenes Dhly mutant (Fig. 2e, f and Sup-plementary Fig. 2).

To determine whether LLO pore formation is required to triggerUbc9 degradation, we first preincubated LLO with A4-8, a monoclonalantibody preventing the binding of the toxin to cellular membranes19.This preincubation impaired the LLO-dependent decrease in Ubc9levels in HeLa cell extracts, indicating that binding of the toxin tomembranes is required for Ubc9 targeting (Fig. 3a). We then monitored

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Figure 2 | L. monocytogenes induces a specific degradation of Ubc9. a, Invitro RanGAP1 SUMOylation assay in the presence of increasing amounts ofLLO. b, Immunoblot analysis of HeLa cells incubated with 3 nM LLO for20 min. c, Kinetics of Ubc9 degradation in HeLa cells incubated with 3 nMLLO. d, Dose-dependent degradation of Ubc9 in HeLa cells incubated withLLO for 20 min. e, Quantification of Ubc9 levels in cell extracts from HeLacells infected with L. monocytogenes (n 5 3; error bars, s.d.). f, Immunoblotanalysis of HeLa cells infected for 3 h with wild-type (WT) or Dhly L.monocytogenes.

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Figure 3 | Characterization of LLO-mediated Ubc9 degradation.a, Immunoblot analysis of HeLa cells incubated with either LLO or LLOpreincubated with A4-8 or control antibody (Ig). b, Immunoblot analysis ofHeLa cells incubated with LLO or LLO variants. c, Immunoblot analysis ofHeLa cells incubated with LLO, PFO or PLY. d, Immunoblot analysis ofHeLa cells either expressing Gam1 (left panel) or incubated with LLO (rightpanel), in the presence of DMSO or MG132. e, Immunoblot analysis of HeLacells pretreated with DMSO or pepstatin-A-methylester (PME) andincubated with LLO. f, Analysis of RanGAP1 and SUMO*RanGAP1 levels inHeLa cells incubated with LLO (n 5 4; error bars, s.d. *P , 0.01).g, Immunoblot analysis of HeLa cells pretreated with DMSO orMG132 1 lactacystin (MG1321Lc) and incubated with LLO. For all LLOtreatments, cells were incubated for 20 min with 3 nM of the toxin.

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the effect of three different LLO variants able to bind cellular mem-branes but affected in their haemolytic activities (SupplementaryTable 3), namely LLOC484A and LLOW492A, which carry substitutionsin the undecapeptide sequence conserved among cholesterol-dependentpore-forming toxins20, and LLOY206A, designed by homology toPFOY181A, a mutant of PFO unable to form pores21. Analysis of HeLacells incubated with these three LLO mutants revealed a correlationbetween the haemolytic activities of the mutants and degradation ofUbc9 (Fig. 3b). Together, these data reveal that both binding of LLOto cellular membranes and pore formation are required to trigger Ubc9degradation.

To determine whether the effect of LLO on Ubc9 is a propertyshared by other pore-forming toxins, we analysed the effect of PFOand PLY, two toxins of the same family as LLO, encoded by two otherbacterial pathogens (Clostridium perfringens and Streptococcuspneumoniae, respectively). Interestingly, like LLO, PFO and PLYtriggered a degradation of Ubc9 (Fig. 3c). Thus, other pathogenicbacteria, including extracellular pathogens, can target theSUMOylation machinery with mechanisms similar to that used byListeria.

To identify the mechanisms underlying Ubc9 degradation, weused different pharmacological inhibitors. Inhibition of proteasomeactivity by MG132 has no effect on the level of Ubc9, which stilldecreased in HeLa cells upon incubation with LLO (Fig. 3d). As acontrol, we verified that HeLa cells expressing Gam1, a viral proteintriggering the proteasomal degradation of SAE1, SAE2 and Ubc922,23,displayed a reduction in the levels of these enzymes compared withcontrol cells, whereas MG132-treated cells did not (Fig. 3d).Together, these data indicate that LLO-induced Ubc9 degradationis proteasome-independent and highlight differences in the mech-anism of action of LLO and Gam1, although both proteins target thehost cell SUMOylation machinery. Previous reports have shown thatLLO can activate different signalling pathways through triggering ofcalcium influx from the extracellular medium and activation of mito-gen-activated protein kinases (MAPKs)17,24,25. We showed that LLO-mediated Ubc9 degradation is independent of the calcium influxtriggered by LLO and independent of p38 or ERK activities (Sup-plementary Fig. 4). In contrast, pepstatin-A-methylester, a cell per-meable aspartyl-protease inhibitor, was able to partly impairUbc9 decrease (Fig. 3e). The effect of pepstatin-A-methylester on

LLO-induced Ubc9 degradation seems specific as it had no effecton histone H3 Ser 10 dephosphorylation, another LLO-inducedmodification7. Together, our results strongly suggest that the LLO-mediated Ubc9 degradation is induced through a non-classical path-way, which involves an aspartyl-protease remaining to be identified.

We then focused on the downstream effects of Ubc9 degradation.We hypothesized that degradation of Ubc9 is a key event that notonly triggers a blockade of SUMOylation but also leads to thedeSUMOylation of SUMO-conjugated proteins owing to the activityof deSUMOylases. Consistently, whereas the total level of RanGAP1protein (that is, SUMO*RanGAP11RanGAP1 in Fig. 3f) remainsmostly unchanged upon LLO treatment in HeLa cells, the quantityof unSUMOylated RanGAP1 significantly increases, in agreementwith a deSUMOylation of this target (Fig. 3f). However, one cannotexclude that the observed decrease in some SUMO-conjugated pro-teins might also be due to degradation, rather than deSUMOylation.Indeed, inhibition of the proteasome partly abrogates the decrease inSUMO-conjugated proteins triggered by LLO (approximately 50%inhibition, Fig. 3g). We thus conclude that LLO triggers both a globaldeSUMOylation event and degradation of some SUMO-conjugatedproteins.

To investigate further the downstream effects of the LLO-mediated decrease in SUMO-conjugated host proteins, we selectedas a read-out the transforming growth factor b (TGF-b) response ofthe cell, a pathway regulated by SUMOylation. Indeed, SUMOylationstabilizes SMAD4, a central intracellular signal transducer for TGF-bsignalling26 and regulates the TGF-b receptor TbR127. We firstobserved that infection with wild-type L. monocytogenes, as well asincubation with LLO, leads to a decrease in SMAD4 level, in agree-ment with the reported role of SUMOylation in SMAD4 stabilization(Fig. 4a, b). This decrease in SMAD4 level was not observed in cellsinfected with a Listeria Dhly mutant (Fig. 4a). Overexpression ofSUMO1 in HeLa cells counteracted the decrease in SMAD4 inducedby LLO, thus confirming that this decrease is linked to the effect of thetoxin on the SUMOylation of host proteins (Fig. 4b). We then quan-tified the capacity of cells infected by Listeria to respond to TGF-b byusing a reporter plasmid encoding luciferase under the control ofSMAD transcriptional response elements. TGF-b response ofinfected 293FT cells was strongly impaired compared with non-infected cells or cells infected with a Listeria Dhly mutant (Fig. 4c).

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Figure 4 | Roles of SUMOylation in Listeria infection. a, Immunoblotanalysis of HeLa cells infected for 5 h with L. monocytogenes. b, Immunoblotanalysis of HeLa cells transfected with a SUMO1 expression vector or acontrol plasmid and treated with 3 nM LLO. c, Luciferase activities, inresponse to hTGF-b1, in 293FT cells transfected with a SMAD reporterplasmid and infected with wild-type (WT) or Dhly L. monocytogenes (n 5 3;error bars, s.d.). d, Immunoblot analysis of HeLa cells transfected with an

expression vector for SUMO1, SUMO2 or the corresponding empty vectoras a control. e, Number of intracellular bacteria recovered from HeLa cellstransfected as in d and infected with L. monocytogenes for 7 h (n 5 3; errorbars, s.d.). f, Quantification of Ubc9 levels in liver homogenates from C57Bl/6J mice infected with L. monocytogenes (n 5 8 animals per group; error bars,s.d.; *P , 0.01).

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Together, these results suggest that loss of SUMO-conjugated proteinsupon L. monocytogenes infection affects the TGF-b response of thehost cell. However, we cannot rule out that other pathways affected byLLO might participate in the observed impairment of TGF-b responsein infected cells. Of note, TGF-b is known to play a role in pathogeninfections28 and, in particular, it has been proposed to have a protec-tive role in host resistance against infection with L. monocytogenes29.Impairment of TGF-b signalling might thus be beneficial for Listeriato replicate and disseminate in its host.

To characterize the putative role of SUMOylation during infection,we increased the level of SUMOylated proteins in HeLa cells by over-expressing SUMO1 or SUMO2 (Fig. 4d). We infected these cells with L.monocytogenes and determined the number of intracellular bacteria 7 hafter infection. This number was significantly reduced in cells over-expressing SUMO1 or SUMO2 compared with control cells (Fig. 4e).Thus, induction of an increase in SUMOylation is detrimental forbacterial invasion and/or replication. Conversely, Ubc9 degradationtriggered by L. monocytogenes is probably important to promote bac-terial infection by lowering SUMOylation of different host proteins.

To examine the relevance of our in vitro data during infection, wetested whether Ubc9 degradation observed in tissue-cultured cellswas also detectable in vivo. Quantification of Ubc9 levels in liversof C57Bl/6J mice infected with L. monocytogenes showed a significantreduction at 48 h and 72 h after infection (Fig. 4f). Thus, Ubc9 degra-dation occurs in vivo in organs infected by L. monocytogenes.

In conclusion, we report here a previously unknown strategy usedby Listeria to promote its infection capacity. By targeting an essentialenzyme of the SUMO pathway, L. monocytogenes has developed anefficient strategy to modify simultaneously activity of many differentproteins of the host cell. That toxins produced by other pathogensalso induced Ubc9 degradation highlights that inhibition ofSUMOylation by pathogens may be a widespread phenomenon.

METHODS SUMMARY

Plasmids, bacterial strains, antibodies, cell culture and transfection conditions

are described in the Methods. For in vitro infections, bacteria were added to

HeLa, Jeg3 or 293FT cells at a multiplicity of infection of 50. After 1 h of infec-

tion, cells were incubated with 10mg ml21 gentamicin to kill extracellular bac-

teria. Quantification of intracellular bacteria was performed by plating bacteria

released from infected cells lysed in PBS-Triton X-100 0.2%. For in vivo infec-

tion, 106 bacteria were injected intravenously to C57Bl/6J mice. Livers of mice

were then collected and lysed as described in the Methods before immunoblot-

ting experiments. LLO, PFO and PLY proteins were added directly in the culture

medium of cells as indicated. Immunoblotting experiments were performed as

described in the Methods. All displayed immunoblots are representative of at

least three independent experiments. Quantification of immunoblots was per-

formed using a G:Box Gel documentation system (Syngene). In vitro

SUMOylation assays were performed using the BioMol SUMOylation kit

according to the manufacturer’s protocol. Luciferase activities were quantified

on a Tristar LB491 luminometer (Berthold Technologies) using the Dual

Luciferase Assay (Promega). Means 6 standard deviations are shown in the

figures and P values were calculated using a two-tailed two-sample equal vari-

ance Student’s t-test.

Full Methods and any associated references are available in the online version ofthe paper at www.nature.com/nature.

Received 20 November 2009; accepted 24 February 2010.

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Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We thank V. Villiers for technical assistance. Work in the P.C.laboratory received financial support from Institut Pasteur, INSERM, INRA andEuropean Research Council (ERC, Advanced Grant 233348). We furtheracknowledge support by research grants from the Fund for Scientific Research –Flanders (Belgium) (project number G.0042.07), the Concerted Research Actions(project BOF07/GOA/012) from Ghent University and the Inter UniversityAttraction Poles (IUAP06). D.R. is supported by a fellowship from the Associationpour la Recherche sur le Cancer, F.I. is a Research Assistant of the ResearchFoundation – Flanders (Fonds Wetenschappelijk Onderzoek – Vlaanderen) and P.C.is an international research scholar of the Howard Hughes Medical Institute.

Author Contributions P.C. planned the project. D.R., K.G., J.V., A.D. and P.C.designed the research, D.R., M.H., E.G., M.-A.N., F.I. and H. N.-K. performed theexperiments. D.R., M.H., K.G., J.V. and P.C. analysed the experiments. D.R. and P.C.wrote the paper and co-authors commented on it.

Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Correspondence and requests for materials should be addressed to P.C.(pascale.cossart@pasteur.fr).

NATURE | Vol 464 | 22 April 2010 LETTERS

1195Macmillan Publishers Limited. All rights reserved©2010

METHODSPlasmids. Cignal SMAD reporter plasmid encodes the firefly luciferase gene under

the control of a minimal cytomegalovirus promoter and tandem repeats of the

SMAD transcriptional response element (SABiosciences Corporation). pSG5-

SUMO1HisHA (BUG2863) and pSG5-SUMO2HisHA (BUG2864) plasmids

encode mature SUMO isoforms with amino-terminal His6- and HA tags. pSG9-

Gam1wt plasmid encodes the adenoviral Gam1 protein (gift of S. Chiocca22).

Plasmids used for purification of His-tagged LLO mutants were derived from

pET29b-LLO-His6 (ref. 30) with punctual mutations inserted by PCR mutagenesis

(pET29b-LLO C484A-His6, BUG2662; pET29b-LLO Y206A-His6, BUG2860;

pET29b-LLO W492A-His6, BUG2664).

Bacterial strains. Listeria strains were grown in brain heart infusion broth

(Difco) or brain heart infusion agar plates. Strains used in this study were

L. innocua (BUG499), L. monocytogenes wild-type strain EGD (BUG600) and

L. monocytogenes isogenic EGD Dhly (BUG2132, Hamon et al., in preparation)

and DinlB (BUG104731) deletion mutants.

Cell culture, transfections and infections. HeLa and Jeg3 cells were cultivated in

MEM-glutamax medium and 293FT cells in DMEM-glutamax medium

(Invitrogen). Both media were supplemented with 10% FCS, MEM non-essential

amino acids and 1 mM sodium pyruvate (Invitrogen). For transfections, HeLa

cells were seeded at 1.25 3 105 cells per 4-cm2 well and transfected the day after

with 1.5mg of DNA, using Lipofectamine LTX reagent (Invitrogen). 293FT cells

were seeded at 1.2 3 105 cells per 2-cm2 well and transfected the day after with

250 ng of DNA, using GeneJuice transfection reagent (Novagen). For bacterial

infection and LLO treatment, HeLa and Jeg3 cells were seeded at 5 3 105 cells per

10-cm2 well the day before treatment. Cells were then serum-starved for 2 h before

infection or addition of LLO.

For infections, exponential-phase bacteria grown in brain heart infusion at

37 uC were washed twice in PBS and added to cells at a multiplicity of infection of

50. After 1 h of infection, cells were washed and incubated with 10 mg ml21

gentamicin (Euromedex) to kill extracellular bacteria. For quantification of

intracellular bacteria, infected cells were lysed 7 h after infection with PBS-

Triton X-100 0.2% (Sigma-Aldrich) and the number of viable bacteria released

from the cells was assessed by plating on brain heart infusion agar plates. Each

experiment was done in duplicate.

For LLO, PFO and PLY treatments, purified proteins were added directly in

the culture medium as indicated in the text. PFO and PLY were used at the same

haemolytic titre as LLO. For inhibition of LLO binding to cellular membranes,

0.1 mg ml21 LLO were pre-incubated for 45 min at 4 uC either with 0.5 mg ml21

A4-8 antibody19 or with 0.5 mg ml21 D21-1-4 control antibody19 before addition

to HeLa cells.

For analysis of Gam1 expressing cells, HeLa cells were transfected with pSG9-

Gam1wt or a control plasmid and treated from the beginning of the transfection

with 10mM MG132 (Calbiochem) or DMSO. Twenty-four hours after transfec-

tion, cells were lysed in Laemmli buffer (0.125 M Tris pH 6.8, 4% SDS, 20%

glycerol, 100 mM DTT) and analysed by immunoblotting experiments. In parallel,

HeLa cells pretreated in the same conditions with MG132 or DMSO were incu-

bated with 3 nM LLO for 20 min before analysis by immunoblotting experiment.

For analysis of SUMO1-conjugated protein degradation, HeLa cells were pre-

treated for 5 h with DMSO or with 20 mM MG132 1 20 mM lactacystin (Sigma-

Aldrich) and then incubated for 20 min with 3 nM LLO.

Inhibition of aspartyl-proteases was done by incubating cells for 1 h with200mM pepstatin-A methylester (Sigma-Aldrich). Cells were then washed twice

before incubation with LLO.

For in vivo infection, 106 bacteria were injected intravenously in C57Bl/6J

mice. Livers of mice were then collected, homogenized and centrifuged for

10 min at 16,000g. One volume of liver supernatant was then added to five

volumes of Laemmli buffer, sonicated and loaded on SDS–polyacrylamide gels

for immunoblotting experiments.

Immunoblotting and LLO purification. Cells infected or incubated with puri-

fied LLO were lysed directly in Laemmli buffer, sonicated and loaded on SDS-

polyacrylamide gels. Primary antibodies used for immunoblot analysis were the

following: mouse anti-SAE1 (ab56957, Abcam), mouse anti-Uba2/SAE2

(611602, BD Biosciences), mouse anti-Ubc9 (610748, BD Biosciences), rabbit

anti-SUMO132, rabbit anti-SUMO2 (51-9100, Zymed Laboratories), mouse

anti-SMAD4 (sc-7966, Santa Cruz Biotechnology), rabbit anti-RanGAP1

(R0155, Sigma-Aldrich), rabbit anti-phospho-Ser10-H3 (05-817, Upstate),

mouse anti-actin (A5441, Sigma-Aldrich) and mouse anti-GST (05-782,

Millipore). A rabbit polyclonal antibody anti-LLO (R176) was obtained by

immunizing rabbits with purified recombinant LLO, followed by affinity puri-fication of the immune serum. Anti-mouse and anti-rabbit HRP-conjugated

secondary antibodies are from AbCys. All displayed immunoblots are representa-

tive of at least three independent experiments. Quantification of immunoblots

was performed using a G:Box Gel documentation system (Syngene). Protein levels

were normalized using actin levels as an internal control. P values were calculated

using a two-tailed two-sample equal variance Student’s t-test. A P value of less

than 0.01 was determined to be statistically significant.

LLO mutant proteins were purified as described30. Purified PFO and PLY were

provided by T. Mitchell.

In vitro SUMOylation assays. In vitro SUMOylation assays were conducted

using a BioMol SUMOylation kit. GST-RanGAP1 SUMO target (250 nM) was

incubated with 70 nM SAE1/SAE2, 350 nM Ubc9 and 700 nM SUMO1 proteins

in reaction buffer. MgATP was added to the reaction mix with various amounts

of purified LLO (ranging from 10 to 1000 nM). After 1 h at 30 uC, reactions were

stopped by adding one volume of Laemmli buffer. SUMOylation products were

analysed by immunoblotting experiments using an anti-GST antibody.

Luciferase assays. For these, 293FT cells were transfected with a DNA mix con-

taining the SMAD reporter plasmid and a plasmid encoding Renilla luciferase(ratio 40:1, SABiosciences Corporation). Twenty-four hours after transfection,

cells were infected with L. monocytogenes (multiplicity of infection of 50). Six hours

after infection, cells were incubated with recombinant human TGF-b1 (Peprotech,

50 ng ml21) for another 18 h. Cells were then harvested and luciferase activities

were quantified on a Tristar LB941 luminometer (Berthold Technologies) using

Dual Luciferase Assay (Promega). Firefly luciferase activities were normalized

using Renilla luciferase activities as an internal control.

30. Glomski, I. J., Gedde, M. M., Tsang, A. W., Swanson, J. A. & Portnoy, D. A. The Listeriamonocytogenes hemolysin has an acidic pH optimum to compartmentalize activityand prevent damage to infected host cells. J. Cell Biol. 156, 1029–1038 (2002).

31. Dramsi, S. et al. Entry of Listeria monocytogenes into hepatocytes requiresexpression of inIB, a surface protein of the internalin multigene family. Mol.Microbiol. 16, 251–261 (1995).

32. Bischof, O. et al. The E3 SUMO ligase PIASy is a regulator of cellular senescenceand apoptosis. Mol. Cell 22, 783–794 (2006).

doi:10.1038/nature08963

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