Control of Listeria Superoxide Dismutaseby Phosphorylation*□S
Received for publication, June 29, 2006, and in revised form, July 27, 2006 Published, JBC Papers in Press, August 11, 2006, DOI 10.1074/jbc.M606249200
Cristel Archambaud1, Marie-Anne Nahori, Javier Pizarro-Cerda, Pascale Cossart2, and Olivier Dussurget3
From the Institut Pasteur, Unite des Interactions Bacteries-Cellules, Inserm, U604, INRA, USC2020, F-75015 Paris, France
Superoxide dismutases (SODs) are enzymes that protect or-ganisms against superoxides and reactive oxygen species (ROS)produced during their active metabolism. ROS are major medi-ators of phagocytesmicrobicidal activity. Here we show that thecytoplasmic Listeria monocytogenesMnSOD is phosphorylatedon serine and threonine residues and less active when bacteriareach the stationary phase. We also provide evidence that themost active nonphosphorylated formofMnSODcanbe secretedvia the SecA2 pathway in culture supernatants and in infectedcells, where it becomes phosphorylated. A�soddeletionmutantis impaired in survival within macrophages and is dramaticallyattenuated in mice. Together, our results demonstrate thatthe capacity to counteract ROS is an essential component ofL. monocytogenes virulence. This is the first example of a bac-terial SOD post-translationally controlled by phosphoryla-tion, suggesting a possible new host innate mechanism tocounteract a virulence factor.
Reactive oxygen species (ROS)4 are continuously producedbymultiple enzymeswithin cells.Whereas a significant amountof ROS is generated in the cytosol of eukaryotic cells, in peroxi-somes and at the plasmamembrane, oxidative phosphorylationby the mitochondrial respiratory chain is the main cellularsource of ROS. Cells beneficially use ROS as antimicrobialagents (1) and regulators of stress signaling pathways, e.g. heat
shock response, NF-�B, and p53 activation, phosphatidylinosi-tol 3-kinase/Akt andmitogen-activated protein kinase cascades(2, 3). However, uncontrolled production of ROS is deleteriousto cells because they can damage nucleic acids, proteins, andlipids (4). Cellular response to oxidative stress is believed to bea major determinant of lifespan (5, 6). Moreover, accumulationof ROS is associated to human pathology including hyperglyce-mic damages (7), carcinogenesis and tumor progression (8),and neurodegenerative disorders, e.g. Alzheimers, Parkinsons(9), and prion diseases (10). To prevent oxidative damage,cells synthesize antioxidant systems. Superoxide dismutase(SOD) catalyzes the conversion of superoxide radical anionsto hydrogen peroxide, using as cofactors manganese in themitochondria or copper and zinc, extracellularly and in thecytosol. Mutations in the human copper-zinc SOD (CuZn-SOD) are associated with a dramatic genetic disease, i.e.familial amyotrophic lateral sclerosis (11), highlighting theimportance and key role of SOD in life. Bacterial SODs arecytoplasmic, periplasmic, or secreted enzymes. They canbind nickel or iron in addition to manganese, copper, andzinc and are involved in basic processes such as growth,senescence, sporulation, and also virulence (12, 13). Theexpression of both eukaryotic and bacterial SODs is tightlycontrolled at the transcriptional and post-transcriptionallevels (14–18). To our knowledge, post-translational regula-tion of bacterial SODs has not been reported.Listeria monocytogenes is a facultative intracellular pathogen
causing a severe food-borne disease in humans and animals(19). Neutrophiles and macrophages are critical cells of hostdefense against L. monocytogenes (20, 21). Once phagocytosed,L. monocytogenes faces the phagosomal oxidative burst (22) andthen escapes from the phagosome because of the secretion oflisteriolysin O (23), phosphatidylinositol-specific phospho-lipase C (24), and other proteins (25). However, how L. mono-cytogenes reacts to this bactericidal aggression has remainedelusive. A single sod gene, which encodes a functional manga-nese-SOD (MnSOD) has been identified (26–28) but it hasremained poorly characterized.Here, we report that L. monocytogenes MnSOD activity is
down-regulated by serine/threonine phosphorylation duringthe stationary phase. We show that the most active, nonphos-phorylated form of MnSOD is secreted via the SecA2 pathwayin bacterial culture and in infected cells where it is phosphoryl-ated. Inactivation of MnSOD by gene deletion resulted inincreased bacterial death within macrophages and dramaticattenuation inmice, demonstrating that the antioxidant poten-tial is a critical factor for L. monocytogenes pathogenesis.
* This work was supported in part from Institut Pasteur (GPH N°9); INRA,INSERM, and the French Ministry of Research (Programme de Microbiolo-gie Fondamentale et Appliquee, Maladies Infectieuses, Environment etBioterrorisme ACI N° MIC 0312). The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.
□S The on-line version of this article (available at http://www.jbc.org) containstwo supplemental experimental procedures, one supplemental Table S1,and four supplemental Figs. S1–S4.
1 Supported by fellowships from the Ministere de la Recherche, the Pasteur-Weizmann Foundation, EuroPathoGenomics (NoE, Contract N° LSHB-CT-2005-512061) and the Howard Hughes Medical Institute.
2 An International Research Scholar of the Howard Hughes Medical Institute.To whom correspondence may be addressed. Tel.: 33-1-40-61-30-32; Fax:33-1-45-68-87-06; E-mail: [email protected].
3 To whom correspondence may be addressed: Unite des Interactions Bacte-ries-Cellules, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France.E-mail: [email protected].
Bacterial Strains and Growth Conditions—All L. monocyto-genes strains were routinely grown in brain heart infusion (BHI)medium (Difco) at 37 °C.When required, chloramphenicol anderythromycin were added at 7 �g/ml and 5 �g/ml, respectively.Escherichia coli strains were grown in Luria-Bertani (LB)medium (Difco) at 37 °C. When required, antibiotics wereincluded at the following concentrations: ampicillin, 100�g/ml, chloramphenicol 7 �g/ml.Antibodies and Western Blot Techniques—Murine poly-
clonal anti-MnSOD serum, produced as described (29), wasused at 1:1000 in 4% Blotto. Rabbit polyclonal antiphospho-serine and antiphosphothreonine antibodies (ZymedLaborato-ries Inc.) were diluted at 1:1000 in blocking buffer (Zymed Lab-oratories Inc.). Rabbit polyclonal anti-Stp R96 (30), anti-ActAR32 (31) purified antibodies and anti-Listeria R11 (32), anti-InlC R117 sera5 were used at 1:1000 in 4% Blotto. After separa-tion on 10% SDS-PAGE, proteins were detected by Westernblotting as previously described (30).Expression and Purification of the L. monocytogenes MnSOD—
The coding region of sod (lmo1439), excluding the startcodon, was amplified by PCR using genomic DNA fromL. monocytogenes EGDe (BUG 1600) and the oligonucleo-tides AC38F and AC39F (supplemental Table S1). The PCRproduct was digested by NdeI and XhoI and cloned into theexpression vector pET-28b (Novagen), creating pET-28b(sod),that was maintained in E. coli XL-1 blue (BUG 2126). E. coliBL21 (DE3) was transformed with pET-28b(sod) and grown at37 °C to A600 nm � 0.8. Overexpression of the N-terminal histi-dine-tagged MnSOD was induced by addition of isopropyl-1-thio-�-D-galactopyranoside (0.5 mM). After 4 h, cultures (200ml) were harvested. The bacterial pellet was resuspended in 20ml of binding buffer (20 mM Tris, pH 7.4, 300 mM NaCl, 1 mMAEBSF, 1 tablet of Complete protease inhibitor mixture, RocheApplied Science). Bacteria were lysed by 5 cycles of sonicationfor 30 s with 15% of amplitude. The recombinant MnSOD wasthen purified on Probond nickel affinity column (Invitrogen)according to the manufacturer’s instructions.Phosphorylation Analysis—Phosphoproteome analysis was
performed as described (30). Briefly, the L. monocytogenesEGDewild-type and�stp cultureswere grownovernight in BHIthen diluted at 1:10 in 200 ml of improved minimal medium(33). Bacteria were harvested when A600 nm � 0.8. Sequentialextraction of bacterial proteins, first dimension separation andelectrophoresis in the second dimension were performed aspreviously described (30). For each strain, the protein extractwas loaded on three gels. One gel was colored with silver stain-ing. The two other gels were analyzed by Western blottingusing either the antiphosphoserine antibodies or the antiphos-phothreonine antibodies. Spots excision from silver-stainedgels and protein identification by mass spectrometry were per-formed by Proteomic Platform (Genopole, Institut Pasteur,Paris, France) (34). For in vitro phosphorylation, L. monocyto-genes purifiedMnSOD (60�g)was incubatedwith 2700 units ofpurified cAMP-dependent protein kinase (PKA) catalytic sub-
unit from bovine heart (Sigma) in PKA buffer (50mMTris-HCl,pH 7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM dithiothreitol, 0.01%Brij 35) overnight at 30 °C in the presence of 1 mM ATP. Twodialysis were performed in 2 liters of Stp buffer (50 mM Tris-HCl, pH 7.5, 1 mM of MnCl2, 0.1 mM Na2EDTA, 5 mM dithio-threitol, 0.01% Brij 35) for 4 h at 4 °C to eliminate residual ATP.Phosphorylated MnSOD (30 �g) was dephosphorylated by Stp(6 �g) for 1 h at 37 °C in Stp buffer. Ten micrograms of phos-phorylated P-�-casein (Sigma) were dephosphorylated using 1�g of alkaline phosphatase (AP, Roche Applied Science) inphosphatase buffer (Roche Applied Science) for 1 h at 37 °C.The phosphorylation level of the resulting �-casein was com-pared with that of the initial P-�-casein. BSA (Pierce) was usedas a nonphosphorylated protein control for Pro-Q diamondstaining. MnSOD, PKA, and Stp-purified proteins, MnSODsamples after PKA dephosphorylation and Stp dephosphoryla-tion, P-�-casein-, �-casein-, and BSA-purified control proteins(2�g)were separated by SDS-PAGE.Gelswere stainedwith thePro-Q and Sypro Ruby protein gel dyes (Molecular Probes) aspreviously described (35).Quantification of L. monocytogenes MnSOD Activity—SOD
activity was measured using the Bioxytech SOD-525 kit (Oxisresearch). Briefly, 4 �g of purified MnSOD were phosphoryla-ted with PKA and dephosphorylated by Stp as described above.Samples and blanks were diluted in 40�l of H2O and incubatedin 900 �l of SOD-525 buffer at 37 °C. Thirty microliters of theR2 reagent were added and incubated at 37 °C for 1 min beforeaddition of 30 �l of the R1 reagent.A525 nm wasmeasured every3 s for 2 min.Preparation of Total Bacterial Extracts and Supernatant
Precipitation—Cultures (10 ml) of the L. monocytogenes EGDewild-type and �stp mutant strains were harvested at differenttime points. Bacterial pellets were recovered after centrifuga-tion at 4000 rpm for 20 min, washed twice in PBS and resus-pended in 100 �l of B-PERII Bacterial Protein Extraction Rea-gent (Pierce) to extract total bacterial proteins. Bacterialsupernatants were precipitated in 16% trichloroacetic acid onice at 4 °C overnight. After centrifugation, the protein pelletswere washed twice with 5 ml of cold acetone, dried, and resus-pended in 350 �l of 1 M Tris (pH 8.8). Protein concentration oftotal bacterial extract and supernatant was determined by theconventional BCA assay (Pierce).MnSOD Immunoprecipitation—Proteins (250 �g) from total
bacterial extracts or from bacterial supernatants were immu-noprecipitated with 2.5 �l of the anti-MnSOD serum using theprotein G immunoprecipitation kit (Sigma) according to themanufacturer’s instructions. Briefly, after overnight incubationof protein samples with anti-MnSOD serum, protein G beadswere transferred in the spin column and incubated for 4 h at4 °C. Beads were washed six times with 1� IP buffer, one timewith 0.1� IP buffer and incubatedwith 60�l of Laemmli buffer.Immunoprecipitates were recovered after centrifugation.Equivalent volumes (30 �l) of immunoprecipitate were sepa-rated by SDS-PAGE and analyzed by Western blotting usinganti-MnSOD serum or antiphosphothreonine antibodies.Immunofluorescence—One milliliter of cultures of the
L. monocytogenes strains growing in BHI at 6% O2 was har-vested atA600 nm� 0.8. Pellets werewashed and resuspended in5 E. Gouin, unpublished data.
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1 ml of PBS. Fifty microliters of suspension were loaded on acoverslip and placed in a 24-well microplate. Bacteria werefixed in 10%paraformaldehyde for 10min. Cells werewashed inPBS and incubated 5 min in 50 mM NH4Cl. After blocking inPBS, 0.5% BSA for 1 h, anti-MnSOD, or anti-Listeria R11 sera,both diluted at 1:100 in 200 �l of PBS, 0.5% BSA, were addedfor 1 h. After washes in PBS, anti-mouse IgG-Alexa-conjugatedor anti-rabbit IgG-FITC-conjugated secondary antibodies(Molecular Probes) diluted at 1:200 in 200 �l of PBS, 0.5% BSA,were used to detect L. monocytogenesMnSODor total bacteria,respectively. Cover slips were mounted with Mowiol and ana-lyzed with an AxioVert microscope (Zeiss) equipped with theMetamorph software (Universal Imaging Corporation).Mutagenesis and Complementation—Upstream and down-
stream 1-kb flanking sequences of the sod gene were ampli-fied by PCR from genomic DNA from L. monocytogenesEGDe using the oligonucleotides SodKO1/SodKO2 andSodKO3/SodKO4 (supplemental Table S1). The upstreamMluI-EcoRI and downstream EcoRI-NcoI fragments werecloned sequentially into the thermosensitive vector pMAD(36). The recombinant pMAD was electroporated intoL. monocytogenes EGDe to generate the sod deletion as pre-viously described (30) except that L. monocytogenes wasgrown at 6% O2. Deletion of sod in the mutant strain (BUG2225) was analyzed by RT-PCR (data not shown) and West-ern blotting (supplemental Fig. S1). For complementation, aDNA fragment containing the sod gene and the 600-bpupstream sequence was amplified by PCR from L. monocy-togenes EGDe genomic DNA with the oligonucleotides Sod-CplmUP and SodCplmDOWN (supplemental Table S1). TheBamHI-XbaI PCR product was cloned into the integrativevector pPL2 digested by BamHI and SpeI (37). The resultingrecombinant plasmid was introduced in E. coli S17-1, whichwas used for conjugation in L. monocytogenes �sod as previ-ously described (37), constructing the L. monocytogenes�sod�sod strain (BUG 2226). MnSOD expression in the�sod�sod complemented strain was analyzed by Westernblotting (supplemental Fig. S1).Sensitivity to ROS and RNS—For the disk assay, L. monocyto-
genes cultures were grown at 6% O2 until A600 nm � 0.6 andplated onto BHI agar plates. Filter disks (6 mm) were placed onthe agar and loaded with paraquat (570 �g, Sigma) and hydro-gen peroxide (210 �g, Sigma). Plates were incubated at 37 °C at6% O2. For the liquid assay, L. monocytogenes cultures weregrown in BHI at 37 °C and 6% O2 until stationary phase. Cul-tures were then diluted in PBS at 1:100 and incubated at 37 °Cwith hypoxanthine (500 �M, Sigma) and xanthine oxidase (0.2units/ml, Sigma) or spermine/NO (2 mM, Sigma). Number ofCFUwas assessed by plating serial dilutions in duplicate onBHIagar plates, whichwere incubated at 37 °C and 6%O2. Student’st test was used for statistical analyses.Infection of Murine Peritoneal Macrophages—Peritoneal
macrophages (PEM) were isolated from 8-week-old femaleBALB/c mice (Charles River) as described (38). 72 h beforethe bacterial survival assay, 1 � 106 PEM per well were acti-vated or not using interferon � (IFN-�, 100 units/ml). PEMwere infected with L. monocytogenes strains (MOI � 10)growing at 6% O2 to an A600 nm � 0.8. PEM were centrifuged
and incubated at 37 °C for 15 min to allow bacterial phago-cytosis. Non-internalized bacteria were eliminated by threewashes in RPMI supplemented with 10% fetal calf serum and10 �g/ml gentamicin was added for 15min, 1 h, and 4 h. PEMwere lysed with 0.2% Triton X-100 for 10 min, and the num-ber of CFU was assessed as described in the previous section.For immunofluorescence labeling, PEM adhering onto glasscoverslips were loaded over 20 min with 7.5 nM of Lyso-tracker Red DND-99 (Molecular Probes) at 37 °C. PEM werethen infected as above for 4 h at 37 °C, washed once with PBSpH 7.5, and fixed with 4% paraformaldehyde for 20 min.After quenching with 50 mM NH4Cl containing 0.05% sapo-nin and 1% BSA for 10 min, nonspecific binding sites wereblocked with 0.05% saponin and 5% horse serum during 45min. Some coverslips were incubated for 30 min with anti-L. monocytogenes serum R11 followed by incubation during30 min with secondary donkey anti-rabbit antibodies cou-pled to Alexa 647 and phalloidin coupled to Alexa 488(Molecular Probes). All coverslips were mounted on Mowioland analyzed with an AxioVert microscope (Zeiss) equippedwith the Metamorph software (Universal Imaging Corpora-tion). Student’s t and ANOVA tests were used for statisticalanalyses.Immunoprecipitation of MnSOD from Infected Macro-
phages—PEM were activated with IFN-� and infected withL. monocytogenes wild-type and �sod mutant strains (MOI �10) as described above. 15 min after adding gentamicin, PEMwere lysed with 0.2% Triton X-100 for 10 min. Bacteria wererecovered from cell lysates as previously described (39), andbacterial proteins were extracted with 100 �l of B-PER II Bac-terial Protein Extraction Reagent. Proteins present in PEMwere recovered in 500�l of Tris, 1 M (pH 8.8) after precipitationof cell lysates with 16% trichloroacetic acid. Bacterial extracts(15�g) and cellular extracts (250�g)were immunoprecipitatedwith 2.5 �l of anti-MnSOD serum using the protein G immu-noprecipitation kit (Sigma) as described above. Equivalent vol-umes (30 �l) of immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting.Animal Studies—L. monocytogenes EGDe cultures were
grown at 6% O2 atmosphere. For quantification of bacterialmultiplication, 8-week-old female BALB/cmice (Charles River)were injected intravenously with �8 � 103 CFU. Liver andspleen were recovered and disrupted in 3 ml PBS at 24 h, 48 hand 72 h after infection. Serial dilutions of organ homogenateswere plated on BHI agar plates and CFU determined. Animalexperiments were performed according to the Institut Pasteurguidelines for laboratory animal husbandry which comply withEuropean regulations.
RESULTS
L. monocytogenes MnSOD Can Be Phosphorylated and Phos-phorylation Down-regulates Its Activity—We previously dem-onstrated that Stp is a serine-threonine phosphatase involved inL. monocytogenes virulence and identified EF-Tu as its first tar-get (30). Here, we identified L. monocytogenes MnSOD as thesecond target of Stp using a phosphoproteomic approach.Analysis of protein extracts of the L. monocytogenes �stpmutant revealed the presence of a protein phosphorylated on
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threonine and serine residues, whichwas not phosphorylated inprotein extracts of the wild-type strain (Fig. 1A). Mass spec-trometry analysis of the corresponding spot identified theMnSOD.To demonstrate a direct dephosphorylation of L. monocyto-
genesMnSOD by Stp, we produced and purified a recombinantMnSOD in E. coli. The recombinant MnSOD could be phos-phorylated using PKA, a cAMP-dependent serine-threonine
kinase (Fig. 1B). PhosphorylatedMnSOD could be fully dephospho-rylated in vitro by Stp (Fig. 1B).We next examined the influence of
MnSOD phosphorylation state on itsactivity. Dephosphorylation of thePKA-phosphorylated MnSOD morethan doubled its activity, revealingthat MnSOD activity can be downregulated by phosphorylation (Fig.1C). Together, these results demon-strate that L. monocytogenesMnSODwhich can be present in a phospho-rylated form in bacteria is dephos-phorylated by Stp, which thusincreases its activity.L. monocytogenes Cytoplasmic
MnSOD Is Phosphorylated uponEntry in Stationary Phase and IsSecreted in a NonphosphorylatedState by the SecA2-dependentMachinery—We investigated MnSODphosphorylation during L. mono-cytogenes growth (Fig. 2A) andperformed immunoprecipitationexperiments on bacterial extractsand culture supernatants. MnSODcould be immunoprecipitatedfrom both bacterial extracts andculture supernatants, in bothexponential and stationary phases(Fig. 2B, panel 1). Whereas thesecreted MnSOD was constantlyfound in its nonphosphorylatedstate, MnSOD from bacterialextracts was nonphosphorylatedin exponential phase and becamephosphorylated upon entry in sta-tionary phase (Fig. 2B, panel 1).Increased MnSOD phosphoryla-tion was concomitant with de-creased Stp production in bacteria(Fig. 2B, panel 2). We analyzed theglobal production of MnSOD dur-ing growth. The MnSOD level,which was high in bacterial ex-tracts in exponential phase, de-creased in stationary phase whilethe amount of MnSOD detected inculture supernatants increased
(Fig. 2B, panel 2). Thus, upon entry in stationary phase, non-phosphorylated MnSOD is increasingly secreted while theremaining cytoplasmic MnSOD is progressivelyphosphorylated.Using the �stp mutant, we next addressed the role of Stp
on MnSOD dephosphorylation and production (Fig. 2B,panel 3 and panel 4). As expected, in the bacterial extracts ofthe �stp mutant, the phosphorylated form of MnSOD was
FIGURE 1. Regulation of L. monocytogenes MnSOD activity by phosphorylation. A, phosphoproteomeanalysis of protein extracts from wild-type EGDe strain (WT) and �stp mutant (30). Phosphorylation of the L.monocytogenes MnSOD (dotted circle) on threonine residues (central panel ) and on serine residues (right panel )was only detectable in the �stp mutant by Western blotting. The spots, corresponding to phosphorylatedMnSOD present in silver-stained two-dimensional gels (left panel ), were analyzed by MALDI-TOF. Molecularmasses are indicated on the left. B, phosphorylation and dephosphorylation of purified MnSOD in vitro. Phos-phoproteins were detected on SDS-PAGE gel stained with Pro-Q Diamond fluorescent dye. The gel was furtherprocessed for total protein staining using Sypro Ruby fluorescent stain. Recombinant MnSOD was phospho-rylated using PKA and dephosphorylated by Stp. Phosphorylated �-casein (P-�-casein), alkaline phosphatase(AP)-dephosphorylated �-casein and nonphosphorylated BSA were used as phosphoprotein controls. Molec-ular masses are indicated on the left. C, MnSOD activity in vitro. SOD activity was quantified by spectrophoto-metric measurement at 525 nm every 3 s for 2 min, using the Bioxytech SOD-525 kit. Recombinant MnSOD (4�g) was phosphorylated by PKA and dephosphorylated by Stp. Mean values are expressed as the measure ofA525 nm � S.D. (n � 3).
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detected earlier, i.e. at mid-logphase, and at a higher level com-pared with the wild-type strain(Fig. 2B, panel 3). Strikingly, Mn-SOD immunoprecipitated fromculture supernatants was still con-stantlydetected in itsnonphospho-rylated form in the �stp mutant.Because phosphorylated listerioly-sin O and phosphorylated EF-Tucontrol proteins could be detectedin supernatants from the wild-typeand �stp mutant respectively(supplemental Fig. S2), these re-sults strongly suggest that secretionof the phosphorylated MnSOD can-not occur. We also observed that thelevel of MnSOD in both bacterial ex-tracts and culture supernatants washigher in the �stpmutant than in thewild-type strain while two controlsproteins,ActA, theactin-basedmotil-ity protein, and the secreted interna-lin InlC, were detected in similaramounts in both strains (Fig. 2B,panel 4). Thus, the presence of Stpcontrols not only MnSOD phospho-rylation but also its production.We then investigated the secre-
tion mechanism of the MnSOD,whose amino acid sequence doesnot contain any signal peptide.Since the accessory secretion pro-tein SecA2hadbeen shown tomedi-ate secretion of proteins, which lacka signal peptide, in Gram-positivebacterial pathogens we analyzedsupernatants from L. monocyto-genes wild-type 10403S, from a�secA2 mutant, and from a complemented strain (40) andfound that the MnSOD was secreted by a SecA2-dependentmachinery (Fig. 2C). Thus, MnSOD belongs, along withL. monocytogenes FbpA (41), to the increasing list of proteinslacking a signal peptide and secreted by the SecA2 pathway inpathogenic bacteria (40).
MnSOD Protects L. monocytogenes against Reactive Oxygenand Nitrogen Species-mediated Toxicity in Vitro—BecauseSOD is a major antioxidant enzyme, we investigated the sensi-tivity to ROS and reactive nitrogen species (RNS) of an isogenic�sodmutant compared with the wild-type strain.We observeda large increase in the doubling time of the �sodmutant grown
FIGURE 2. Analysis of MnSOD phosphorylation, secretion, and production during L. monocytogenes growth. A, growth curve of wild-type EGDestrain (WT) and �stp mutant in BHI at 37 °C. Bacteria were harvested at various time points (arrows). Total bacterial proteins were extracted, and proteinsfrom culture supernatants were precipitated. B, analysis of MnSOD phosphorylation and production in total extracts and culture supernatants from WTand �stp mutant. At the chosen time points, MnSOD was immunoprecipitated from WT (panel 1) and �stp mutant (panel 3) bacteria, using theanti-MnSOD serum. Immunoprecipitates from total bacterial extracts and culture supernatants were separated by SDS-PAGE. MnSOD was detected byWestern blotting with the anti-MnSOD serum. The IgG small chain is indicated (*). Phosphorylated MnSOD was detected by Western blotting using theantiphosphothreonine antibodies. Three additional phosphorylated proteins that could correspond to MnSOD partners, are indicated (**). Positions ofthe molecular mass markers are indicated on the right. At the time points indicated above, WT (panel 2) and �stp mutant (panel 4) bacteria wereharvested. Proteins from total extracts (20 �g) and culture supernatants (70 �g) were separated by SDS-PAGE. Anti-MnSOD serum and anti-Stpantibodies were used to detect MnSOD and Stp by Western blotting, respectively. Immunodetection of ActA and InlC control proteins was performedusing the anti-ActA antibodies and the anti-InlC serum on the same samples. C, SecA2-dependent secretion of MnSOD. Proteins were precipitated fromculture supernatants (80 �g) of the wild-type L. monocytogenes 10403S (WT), the �secA2 mutant and complemented strains (�secA2�secA2) (40).MnSOD was detected by immunoblotting using the anti-MnSOD serum. Immunodetection of the InlC control protein was performed using the anti-InlCantiserum.
FIGURE 3. Role of L. monocytogenes MnSOD in resistance to oxidative stress in vitro. A, effect of soddeletion on aerobic and microaerophilic growth. Growth of the wild-type EGDe strain (WT), the �sodmutant and the complemented strain (�sod�sod) was measured in BHI at 37 °C and 21% O2 (left panel ) or6% O2 (right panel ) atmosphere. Mean values are expressed as the measure of A600 nm � S.D. (n � 3). B,effect of the sod deletion on sensitivity to oxidants. Growth of the WT strain, the �sod mutant and the�sod�sod strain was measured in the presence of the superoxide-generating HX-XO system and/or nitricoxide donor spermine/NO (NO). Mean values are expressed as the number of CFU � S.D. (n � 3), with pvalues of p � 0.05 (*) and p � 0.01 (**) corresponding to the comparison of the CFUs of the WT and �sodmutant strains.
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in normal atmosphere (pO2 � 21%) compared with the wild-type strain and the�sod strain complementedwith the sod genethat had a similar doubling time (Fig. 3A). In microaerophilicatmosphere (pO2 � 6%), the doubling time of the �sodmutantwas similar to that of the wild-type and of the complementedstrains (Fig. 3A).We then tested the sensitivity of the �sodmutant to ROS in
these microaerophilic growth conditions using a disk diffusion
assay. The �sod mutant was hyper-sensitive to paraquat, a reagent thatincreases the intracellular level ofsuperoxides. Indeed, the diameterof growth inhibitory area of the�sod mutant (36.3 mm � 0.8) wasstrongly increased compared withthat of the wild-type and the com-plemented strains whose growthwas not affected (0.0 mm and 0.1mm � 0.1, respectively). Alterna-tively, addition of hydrogen perox-ide on filter disks resulted in a signif-icant albeit less severe differencebetween growth inhibition of the�sod mutant (5.3 mm � 1.3) andthat of the wild-type and the com-plemented strains (2.5 mm � 0.5;1.8 mm � 1.0, respectively). Wenext analyzed growth of the �sodmutant in liquid culture media inthe presence of exogenous ROSand/or RNS. ROSwere generated byxanthine oxidase (XO) from hypox-anthine (HX). We first verified thataddition of HX did not inhibitL. monocytogenes growth (data notshown). Nitric oxide (NO) was pro-duced from spermine/NO. The�sod mutant had a significantlyhigher susceptibility to RNS (Fig.3B, central panel) compared withthat of the wild-type and comple-mented strains. No drastic effect ofthe production of exogenous ROSwas observed in these conditionson the growth of the �sod mutant(Fig. 3B, left panel). However, astrong synergistic effect of ROSand RNS on growth inhibition of
the �sod mutant was observed (Fig. 3B, right panel). Collec-tively, these results show that MnSOD protects L. monocy-togenes from exogenous ROS and RNS.MnSOD Is Found in a Phosphorylated Form in Macro-
phages—Because ROS and RNS are mediators of antibacterialactivity in phagocytes, we next assessed the contribution ofMnSOD to L. monocytogenes intracellular survival in macro-phages. Thewild-type,�sodmutant and complemented strainshad similar multiplication rates in the non listericidal macro-phage-like RAW264.7 cell line and in peritoneal macrophages(data not shown). However, the �sod mutant was killed moreefficiently and significantly faster than the wild-type and com-plemented strains by listericidal macrophages, i.e. peritonealmacrophages activated with IFN-� (Fig. 4A).
To further compare the intracellular behavior of the wild-type, �sod mutant and complemented strains in peritonealmacrophages activated with IFN-�, we indirectly analyzedbacterial escape from phagosomes, by counting after 4 h of
FIGURE 4. Role of L. monocytogenes MnSOD during infection. A, effect of the sod deletion on intracellularsurvival in macrophages. Peptone-elicited peritoneal macrophages (PEM, 106 cells/well) from BALB/c micewere activated with IFN-� and infected at a MOI of 10 with the wild-type strain (WT), the �sod mutant and thecomplemented strain (�sod � sod). The number of CFU was determined at 15 min, 1 h, and 4 h after additionof gentamicin. Mean values are expressed as CFU � S.D., with p values of p � 0.05 (*) and p � 0.01 (**)corresponding to the comparison of WT and �sod mutant CFUs. B, detection of MnSOD during PEM infection.PEM were activated with IFN-� and infected for 15 min with the WT strain and the �sod mutant. Infected PEMwere lyzed and centrifuged to separate L. monocytogenes from PEM extracts (PEM). Proteins were thenextracted from bacteria recovered from infected PEM (BACTERIA) as described under “Experimental Proce-dures.” MnSOD immunoprecipitation was carried out on PEM extracts and bacterial extracts of infected PEMand on PEM extracts of non infected macrophages (NI PEM). Immunoprecipitates were analyzed by immuno-blotting using the anti-MnSOD serum raised against L. monocytogenes MnSOD and with antiphosphothreo-nine antibodies. Molecular masses are indicated on the right. C, effect of the sod deletion on L. monocytogenessurvival in BALB/c mice. Multiplication of the WT strain, the �sod mutant and the �sod � sod strain wasdetermined in the spleen and liver of BALB/c mice infected intravenously with 8 � 103 CFU. For each strain, CFUwere counted in organs of 4 BALB/c mice at 24, 48, and 72 h after infection.
TABLE 1Role of L. monocytogenes MnSOD on phagosomal escape inmacrophages
WT �sod �sod � sod% Infected PEM 99.2 99.2 100% Infected PEM with intracytosolicbacteria labeled with phalloidin
22.3 14.3a 21.7
% Infected PEM with intracytosolicbacteria labeled with lysotracker
56.9 82.5b 63.8
a p � 0.05.b p � 0.0001.
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infection, the number of macrophages containing intracel-lular bacteria surrounded with polymerized actin in thecytosol. As shown in Table 1, all macrophages were infectedwith these bacterial strains. Significantly fewer �sodmutantbacteria were able to polymerize cytosolic actin comparedwith wild-type and complemented strains, suggesting that alower number of �sod bacteria had reached the cytosol. Todetect if actin-negative bacteria were present in a phagoly-sosomal compartment, we loaded macrophages with thelysosome marker LysoTracker Red DND-99. After 4 h ofinfection, the macrophages infected with the �sod mutantcontained a much higher number of LysoTracker Red-posi-tive bacteria than the macrophages infected with the wild-type strain (Table 1). These results strongly suggested thatMnSOD contributes toL.monocytogenes vacuole escape, possiblyby preventing phagosomal bactericidal mechanisms.We then determined the phosphorylation state of L. mono-
cytogenes MnSOD in peritoneal macrophages activated withIFN-�.We immunoprecipitatedMnSOD from protein extractsof bacteria recovered from infectedmacrophages and from cel-lular extracts of infected macrophages (Fig. 4B). As shown inthe control experiment with non infected macrophages, ouranti-MnSOD antibodies cross-reacted with a cellular protein,probably a SOD. However, as expected, MnSOD was detectedin the bacterial extracts from cells infected with the wild-typebacteria and it was not the case when �sod bacteria had beenused to infect cells. Strikingly, we detected a signal correspond-ing to a phosphorylated SOD in infectedmacrophages. Becausethis phosphorylated SOD was only present in macrophagesinfected with the wild-type strain and not with the �sodmutant, it most likely corresponds to L. monocytogenes phos-phorylatedMnSOD (Fig. 4B), suggesting that L. monocytogenesMnSOD can be phosphorylated in macrophages. It is unlikelythat phosphorylatedMnSODwas secreted from the bacteria, asthis has never been observed in vitro.MnSOD Is Required for L. monocytogenes Pathogenesis—All
our results were converging to indicate that MnSOD was criti-cal for virulence of L. monocytogenes. We thus addressed therole of MnSOD in vivo. BALB/c mice were challenged intrave-nously with the wild-type, the �sod mutant and the comple-mented strains. In spleen and liver, which are early sites ofL. monocytogenes replication, growth of the �sod mutant wasstrongly impaired compared with that of the wild-type strain(Fig. 4C). This difference could be detected as soon as 1 dayafter infection in the spleen and 2 days post-infection in theliver (Fig. 4C). At 3 days post-infection, there were 10 timesfewer mutant bacteria in the spleen compared with the wild-type strain and growth of the mutant was controlled by theanimals in the liver. The wild-type and complemented strainsbehaved similarly in animals. Thus,MnSOD is a novel virulencefactor of L. monocytogenes significantly contributing to listeri-osis in mice.
DISCUSSION
In this article, we have characterized a newvirulence factor inListeria and the first exampleof aSODdown-regulatedbyphos-phorylation. In addition, we provide evidence that MnSOD,which is secreted as an active, nonphosphorylated form by the
SecA2-dependent machinery, plays a critical role in intracellu-lar survival in macrophages and is required for full virulence ofL. monocytogenes. The first unexpected result and the startingpoint of this study was that Listeria MnSOD can be phospho-rylated on serine and threonine residues. The second importantresult was that Listeria MnSOD can be secreted and that onlythe nonphosphorylated andmost active form is secreted. Strik-ingly, MnSOD can become phosphorylated inside infectedcells, highlighting a possible cross-talk between the host celland the incoming microbe.That L. monocytogenes MnSOD was present in culture
supernatants as well as in the bacterial cytoplasm was at firstunexpected since its amino acid sequence does not reveal anystandard signal peptide. However, several pathogenic bacteria,including L. monocytogenes and Mycobacterium tuberculosis,have now been shown to secrete proteins lacking a signal pep-tide, through the SecA2 auxilliary secretion system (40–43).Thus MnSOD secretion occurs by the well established mecha-nism, the SecA2-dependent mechanism.ROS are produced during growth in all living cells and
thus the cytoplasmic form of MnSOD protects Listeria fromendogenously produced ROS. During the oxidative burstthat occurs after phagocytosis, ROS are among the mainmediators of the antibacterial activity of phagocytes. Super-oxides produced in the acidic phagolysosome can directlydamage targets on the bacterial surface (44). They can alsopenetrate inside bacteria and react with cytosolic targetssuch as [4Fe-4S] cluster proteins or DNA (45). In addition,superoxides can indirectly be deleterious by reacting withnitric oxide to form peroxynitrite, a toxic oxidant that canfreely enter bacteria. Thus targets of ROS and RNS are pres-ent both inside bacteria and on its surface. Interestingly, ourpreliminary results show that L. monocytogenesMnSOD canbe detected on the bacterial surface (supplemental Fig. S3),as reported for SODs from Mycobacterium leprae (46),Mycobacterium avium (47), and Mycobacterium bovis BCG(48). Therefore Listeria and mycobacterial MnSODs mayprotect bacterial surface components from superoxide dam-age as documented for well-characterized periplasmicCuZnSOD from pathogenic Gram-negative bacteria (13). Asshown by the increase in the bacterial doubling time in vitro,ROS and RNS are indeed toxic for Listeria and the �sodmutant was much more sensitive than the wild type to ROSand RNS, definitively establishing the role of the MnSOD inprotection against these radicals.The generation of superoxides into the phagocytic vacuole
has been reported to induce a potassium influx which increasesthe pH to the optimal value for activation of bactericidal pro-teases (49), which also participate to the phagocytic process.We observed that the �sod mutant was associated with acidiccompartments of infected macrophages, failing to escape fromphagosomes, strongly suggesting that MnSOD allows L. mono-cytogenes to resist phagolysosomal degradation in perfectagreement with the report that localized reactive oxygen andnitrogen intermediates inhibit escape from vacuoles in acti-vated macrophages (22). As expected from its location in theinfected cell, survival of the �sod mutant was impaired in list-ericidal macrophages. Together these results demonstrate that
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MnSOD counteracts a host defense mechanism by destroyingsuperoxide radicals generated by the activatedmacrophages, asreported for SODs produced by other pathogenic bacteria (50–53). In line with the role of MnSOD in intracellular survival inmacrophages, this enzyme was required for full virulence inmice.We also provide here evidence that Listeria MnSOD can be
phosphorylated and that phosphorylation controls its activity.Post-translational modifications of SODs affecting their enzy-matic activity have been reported in eukaryotes. For instance,tyrosine nitration of human MnSOD decreases its activity (54)and glycation of human CuZnSOD, which is associated to dia-betis and aging, inactivates the enzyme (55–57). More recently,Csar et al. (58) reported for the first time, through a proteomicanalysis, transient phosphorylation of a cytoplasmic CuZnSODafter treatment ofmyeloıd cells by the granulocyte colony stim-ulating factor. However, whether phosphorylation could regu-late SOD activity remained elusive. We demonstrate here thatcomplete dephosphorylation of L. monocytogenes MnSOD bythe serine-threonine phosphatase Stp dramatically increasesits activity, possibly through conformational changes affect-
ing the active site. Listeria growingexponentially produced the mostactive, nonphosphorylatedMnSOD, thereby preventing dam-age from ROS produced duringaerobic respiratory metabolism(Fig. 5A) (59). In contrast, whenbacterial division and metabolismslow down upon entry in station-ary phase, MnSOD is found in itsless active phosphorylated state.The increased phosphorylation ofMnSOD observed in stationaryphase is concomitant with adecreased production of the serine-threonine phosphatase Stp, con-firming the regulatory link betweenthe two proteins (Fig. 5A). Alongthese lines, MnSOD phosphoryla-tion, which occurs earlier and at ahigher level during growth of a �stpmutant, correlated with an in-creased, possibly compensatory,production of MnSOD. Interest-ingly, in mammalian cells, a linkbetween phosphorylation of anti-oxidant proteins and growthseems to exist as inactivation ofthe antioxidant human peroxire-doxin I by Cdc2-dependent phos-phorylation, has been hypothe-sized to be important for cell cycleprogression (60). Strikingly, aphosphorylated form of L. mono-cytogenes MnSOD was detected inthe cytoplasm of infected macro-phages. Since secreted MnSOD is
constantly found in its nonphosphorylated form in L. mono-cytogenes, we propose that a cellular kinase down-regulatesMnSOD activity by phosphorylation (Fig. 5B). Secretion ofproteins interfering with phagosomal maturation, e.g. M. bo-vis BCG serine-threonine kinase G (61) and Salmonellapathogenicity island 2 proteins (62), is a strategy used bypathogenic bacteria to promote intracellular survival. Inac-tivation of secreted proteins important for bacterial survivalcould be a strategy used by the host phagocytes to controlintracellular infection. Up to now, and to our knowledge, thisis the first reported case of bacterial protein down-regula-tion through phosphorylation in the host. A previous reporthas nevertheless documented the differential ubiquitina-tion/degradation of Salmonella type III effectors (63).
Are other bacterial SODs phosphorylated and would phos-phorylation also occur during infection with other bacteria?Our preliminary results show unambiguously that the myco-bacterial FeSOD fromM. bovis BCG is phosphorylated on bothserine and threonine residues and can be dephosphorylated invitro (supplemental Fig. S4). Interestingly, it has been reportedthat M. tuberculosis FeSOD, which is essentially identical to
FIGURE 5. Regulation of MnSOD activity by Stp in L. monocytogenes. A, during exponential growth phase,phosphorylation of MnSOD by an unidentified bacterial kinase is counteracted by Stp, generating a pool ofhighly active MnSOD. In stationary phase, decrease in the level of Stp leads to an increase in phosphorylatedMnSOD, while nonphosphorylated MnSOD is actively secreted by the SecA2 system. B, within host macro-phages, phagocytosed L. monocytogenes neutralizes the oxidative burst-generated ROS by secretion ofMnSOD, which in turn is possibly down-regulated by phosphorylation mediated by an unknown cellularkinase.
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M. bovis BCG FeSOD, can be secreted by the SecA2 pathwayand is critical for survival in the host (43, 64). Whether thisFeSODactivity is tightly regulated by phosphorylation inmyco-bacteria has not been determined. Given the present high inter-est inmycobacterial kinases and phosphatases as potential drugtargets, this issue clearly deserves investigation.In conclusion, we have shown that post-translational regula-
tion of ListeriaMnSOD represents a key level of control of thisimportant enzyme. It would be interesting to investigate if asimilar post-translational level of regulation also exists inhumans. Activation and inactivation of SODs or of proteinsregulating SODs may provide tools to fight against diseasesassociated to superoxide-mediated cell injury such as cancer,neurodegenerative, inflammatory, and cardiovascular diseases.
Acknowledgments—We thank Daniel Portnoy for providing L. mono-cytogenes 10403S strains, Fabienne Nicolle and Nathalie Winter forM. bovis BCG culture supernatants and Douglas Young for anti-M. tuberculosis FeSOD antibodies. We are grateful to Claire Poyart,Tony Pugsley, and Jacques D’Alayer for productive discussions. Wethank members of the laboratory for helpful discussion andcomments.
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