Control of Listeria Superoxide Dismutase by Phosphorylation
2006; Elsevier BV; Volume: 281; Issue: 42 Linguagem: Inglês
10.1016/s0021-9258(19)84096-6
ISSN1083-351X
AutoresCristel Archambaud, Marie‐Anne Nahori, Javier Pizarro‐Cerdá, Pascale Cossart, Olivier Dussurget,
Tópico(s)Salmonella and Campylobacter epidemiology
ResumoSuperoxide dismutases (SODs) are enzymes that protect organisms against superoxides and reactive oxygen species (ROS) produced during their active metabolism. ROS are major mediators of phagocytes microbicidal activity. Here we show that the cytoplasmic Listeria monocytogenes MnSOD is phosphorylated on serine and threonine residues and less active when bacteria reach the stationary phase. We also provide evidence that the most active nonphosphorylated form of MnSOD can be secreted via the SecA2 pathway in culture supernatants and in infected cells, where it becomes phosphorylated. A Δsod deletion mutant is impaired in survival within macrophages and is dramatically attenuated in mice. Together, our results demonstrate that the capacity to counteract ROS is an essential component of L. monocytogenes virulence. This is the first example of a bacterial SOD post-translationally controlled by phosphorylation, suggesting a possible new host innate mechanism to counteract a virulence factor. Superoxide dismutases (SODs) are enzymes that protect organisms against superoxides and reactive oxygen species (ROS) produced during their active metabolism. ROS are major mediators of phagocytes microbicidal activity. Here we show that the cytoplasmic Listeria monocytogenes MnSOD is phosphorylated on serine and threonine residues and less active when bacteria reach the stationary phase. We also provide evidence that the most active nonphosphorylated form of MnSOD can be secreted via the SecA2 pathway in culture supernatants and in infected cells, where it becomes phosphorylated. A Δsod deletion mutant is impaired in survival within macrophages and is dramatically attenuated in mice. Together, our results demonstrate that the capacity to counteract ROS is an essential component of L. monocytogenes virulence. This is the first example of a bacterial SOD post-translationally controlled by phosphorylation, suggesting a possible new host innate mechanism to counteract a virulence factor. Reactive oxygen species (ROS) 4The abbreviations used are: ROS, reactive oxygen species; SOD, superoxide dismutase; CuZnSOD, copper and zinc superoxide dismutase; MnSOD, manganese superoxide dismutase; PKA, protein kinase A; AP, alkaline phosphatase; BSA, bovine serum albumin; RNS, reactive nitrogen species; XO, xanthine oxidase; HX, hypoxanthine; NO, nitric oxide; PEM, peptone-elicited peritoneal macrophages; IFN-γ, interferon γ; MOI, multiplicity of infection; CFU, colony-forming unit; FeSOD, iron superoxide dismutase; PBS, phosphate-buffered saline; WT, wild type. 4The abbreviations used are: ROS, reactive oxygen species; SOD, superoxide dismutase; CuZnSOD, copper and zinc superoxide dismutase; MnSOD, manganese superoxide dismutase; PKA, protein kinase A; AP, alkaline phosphatase; BSA, bovine serum albumin; RNS, reactive nitrogen species; XO, xanthine oxidase; HX, hypoxanthine; NO, nitric oxide; PEM, peptone-elicited peritoneal macrophages; IFN-γ, interferon γ; MOI, multiplicity of infection; CFU, colony-forming unit; FeSOD, iron superoxide dismutase; PBS, phosphate-buffered saline; WT, wild type. are continuously produced by multiple enzymes within cells. Whereas a significant amount of ROS is generated in the cytosol of eukaryotic cells, in peroxisomes and at the plasma membrane, oxidative phosphorylation by the mitochondrial respiratory chain is the main cellular source of ROS. Cells beneficially use ROS as antimicrobial agents (1Heyworth P.G. Cross A.R. Curnutte J.T. Curr. Opin. Immunol. 2003; 15: 578-584Crossref PubMed Scopus (337) Google Scholar) and regulators of stress signaling pathways, e.g. heat shock response, NF-κB, and p53 activation, phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase cascades (2Finkel T. Holbrook N.J. Nature. 2000; 408: 239-247Crossref PubMed Scopus (7300) Google Scholar, 3Kamata H. Honda S. Maeda S. Chang L. Hirata H. Karin M. Cell. 2005; 120: 649-661Abstract Full Text Full Text PDF PubMed Scopus (1501) Google Scholar). However, uncontrolled production of ROS is deleterious to cells because they can damage nucleic acids, proteins, and lipids (4Imlay J.A. Annu. Rev. 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Bacterial SODs are cytoplasmic, periplasmic, or secreted enzymes. They can bind nickel or iron in addition to manganese, copper, and zinc and are involved in basic processes such as growth, senescence, sporulation, and also virulence (12Inaoka T. Matsumura Y. Tsuchido T. J. Bacteriol. 1999; 181: 1939-1943Crossref PubMed Google Scholar, 13Battistoni A. Biochem. Soc. Trans. 2003; 31: 1326-1329Crossref PubMed Google Scholar). The expression of both eukaryotic and bacterial SODs is tightly controlled at the transcriptional and post-transcriptional levels (14Lynch M. Kuramitsu H. Microbes. Infect. 2000; 2: 1245-1255Crossref PubMed Scopus (88) Google Scholar, 15Knirsch L. Clerch L.B. Biochemistry. 2001; 40: 7890-7895Crossref PubMed Scopus (27) Google Scholar, 16Masse E. Gottesman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4620-4625Crossref PubMed Scopus (876) Google Scholar, 17Zelko I.N. Mariani T.J. Folz R.J. Free Radic. Biol. Med. 2002; 33: 337-349Crossref PubMed Scopus (1599) Google Scholar, 18Ahn B.E. Cha J. Lee E.J. Han A.R. Thompson C.J. Roe J.H. Mol. Microbiol. 2006; 59: 1848-1858Crossref PubMed Scopus (105) Google Scholar). To our knowledge, post-translational regulation of bacterial SODs has not been reported. Listeria monocytogenes is a facultative intracellular pathogen causing a severe food-borne disease in humans and animals (19Hamon M. Bierne H. Cossart P. Nat. Rev. Microbiol. 2006; 4: 423-434Crossref PubMed Scopus (459) Google Scholar). Neutrophiles and macrophages are critical cells of host defense against L. monocytogenes (20Cousens L.P. Wing E.J. Immunol. Rev. 2000; 174: 150-159Crossref PubMed Scopus (79) Google Scholar, 21Pamer E.G. Nat. Rev. Immunol. 2004; 4: 812-823Crossref PubMed Scopus (650) Google Scholar). Once phagocytosed, L. monocytogenes faces the phagosomal oxidative burst (22Myers J.T. Tsang A.W. Swanson J.A. J. Immunol. 2003; 171: 5447-5453Crossref PubMed Scopus (86) Google Scholar) and then escapes from the phagosome because of the secretion of listeriolysin O (23Henry R. Shaughnessy L. Loessner M.J. Alberti-Segui C. Higgins D.E. Swanson J.A. Cell. Microbiol. 2006; 8: 107-119Crossref PubMed Scopus (97) Google Scholar), phosphatidylinositol-specific phospholipase C (24Poussin M.A. Goldfine H. Infect. Immun. 2005; 73: 4410-4413Crossref PubMed Scopus (30) Google Scholar), and other proteins (25Chatterjee S.S. Hossain H. Otten S. Kuenne C. Kuchmina K. Machata S. Domann E. Chakraborty T. Hain T. Infect. Immun. 2006; 74: 1323-1338Crossref PubMed Scopus (289) Google Scholar). However, how L. monocytogenes reacts to this bactericidal aggression has remained elusive. A single sod gene, which encodes a functional manganese-SOD (MnSOD) has been identified (26Brehm K. Haas A. Goebel W. Kreft J. Gene (Amst.). 1992; 118: 121-125Crossref PubMed Scopus (27) Google Scholar, 27Vasconcelos J.A. Deneer H.G. Appl. Environ. Microbiol. 1994; 60: 2360-2366Crossref PubMed Google Scholar, 28Glaser P. Frangeul L. Buchrieser C. Rusniok C. Amend A. Baquero F. Berche P. Bloecker H. Brandt P. Chakraborty T. Charbit A. Chetouani F. Couve E. de Daruvar A. Dehoux P. Domann E. Dominguez-Bernal G. Duchaud E. Durant L. Dussurget O. Entian K.D. Fsihi H. Garcia-del Portillo F. Garrido P. Gautier L. Goebel W. Gomez-Lopez N. Hain T. Hauf J. Jackson D. Jones L.M. Kaerst U. Kreft J. Kuhn M. Kunst F. Kurapkat G. Madueno E. Maitournam A. Vicente J.M. Ng E. Nedjari H. Nordsiek G. Novella S. de Pablos B. Perez-Diaz J.C. Purcell R. Remmel B. Rose M. Schlueter T. Simoes N. Tierrez A. Vazquez-Boland J.A. Voss H. Wehland J. Cossart P. Science. 2001; 294: 849-852PubMed Google Scholar) but it has remained poorly characterized. Here, we report that L. monocytogenes MnSOD activity is down-regulated by serine/threonine phosphorylation during the stationary phase. We show that the most active, nonphosphorylated form of MnSOD is secreted via the SecA2 pathway in bacterial culture and in infected cells where it is phosphorylated. Inactivation of MnSOD by gene deletion resulted in increased bacterial death within macrophages and dramatic attenuation in mice, demonstrating that the antioxidant potential is a critical factor for L. monocytogenes pathogenesis. Bacterial Strains and Growth Conditions—All L. monocytogenes strains were routinely grown in brain heart infusion (BHI) medium (Difco) at 37 °C. When required, chloramphenicol and erythromycin were added at 7 μg/ml and 5 μg/ml, respectively. Escherichia coli strains were grown in Luria-Bertani (LB) medium (Difco) at 37 °C. When required, antibiotics were included at the following concentrations: ampicillin, 100 μg/ml, chloramphenicol 7 μg/ml. Antibodies and Western Blot Techniques—Murine polyclonal anti-MnSOD serum, produced as described (29Cooper H.M. Paterson Y. Curr. Protocol. Immunol. 1995; 2.4: 1-9Google Scholar), was used at 1:1000 in 4% Blotto. Rabbit polyclonal antiphosphoserine and antiphosphothreonine antibodies (Zymed Laboratories Inc.) were diluted at 1:1000 in blocking buffer (Zymed Laboratories Inc.). Rabbit polyclonal anti-Stp R96 (30Archambaud C. Gouin E. Pizarro-Cerda J. Cossart P. Dussurget O. Mol. Microbiol. 2005; 56: 383-396Crossref PubMed Scopus (90) Google Scholar), anti-ActA R32 (31Steffen P. Schafer D.A. David V. Gouin E. Cooper J.A. Cossart P. Cell. Motil. Cytoskeleton. 2000; 45: 58-66Crossref PubMed Scopus (24) Google Scholar) purified antibodies and anti-Listeria R11 (32Gouin E. Dehoux P. Mengaud J. Kocks C. Cossart P. Infect. Immun. 1995; 63: 2729-2737Crossref PubMed Google Scholar), anti-InlC R117 sera 5E. Gouin, unpublished data. were used at 1:1000 in 4% Blotto. After separation on 10% SDS-PAGE, proteins were detected by Western blotting as previously described (30Archambaud C. Gouin E. Pizarro-Cerda J. Cossart P. Dussurget O. Mol. Microbiol. 2005; 56: 383-396Crossref PubMed Scopus (90) Google Scholar). Expression and Purification of the L. monocytogenes MnSOD—The coding region of sod (lmo1439), excluding the start codon, was amplified by PCR using genomic DNA from L. monocytogenes EGDe (BUG 1600) and the oligonucleotides AC38F and AC39F (supplemental Table S1). The PCR product was digested by NdeI and XhoI and cloned into the expression vector pET-28b (Novagen), creating pET-28b (sod), that was maintained in E. coli XL-1 blue (BUG 2126). E. coli BL21 (DE3) was transformed with pET-28b (sod) and grown at 37 °C to A600nm = 0.8. Overexpression of the N-terminal histidine-tagged MnSOD was induced by addition of isopropyl-1-thio-β-d-galactopyranoside (0.5 mm). After 4 h, cultures (200 ml) were harvested. The bacterial pellet was resuspended in 20 ml of binding buffer (20 mm Tris, pH 7.4, 300 mm NaCl, 1 mm AEBSF, 1 tablet of Complete protease inhibitor mixture, Roche Applied Science). Bacteria were lysed by 5 cycles of sonication for 30 s with 15% of amplitude. The recombinant MnSOD was then purified on Probond nickel affinity column (Invitrogen) according to the manufacturer's instructions. Phosphorylation Analysis—Phosphoproteome analysis was performed as described (30Archambaud C. Gouin E. Pizarro-Cerda J. Cossart P. Dussurget O. Mol. Microbiol. 2005; 56: 383-396Crossref PubMed Scopus (90) Google Scholar). Briefly, the L. monocytogenes EGDe wild-type and Δstp cultures were grown overnight in BHI then diluted at 1:10 in 200 ml of improved minimal medium (33Phan-Thanh L. Gormon T. Int. J. Food Microbiol. 1997; 35: 91-95Crossref PubMed Scopus (72) Google Scholar). Bacteria were harvested when A600 nm = 0.8. Sequential extraction of bacterial proteins, first dimension separation and electrophoresis in the second dimension were performed as previously described (30Archambaud C. Gouin E. Pizarro-Cerda J. Cossart P. Dussurget O. Mol. Microbiol. 2005; 56: 383-396Crossref PubMed Scopus (90) Google Scholar). For each strain, the protein extract was loaded on three gels. One gel was colored with silver staining. The two other gels were analyzed by Western blotting using either the antiphosphoserine antibodies or the antiphosphothreonine antibodies. Spots excision from silver-stained gels and protein identification by mass spectrometry were performed by Proteomic Platform (Genopole, Institut Pasteur, Paris, France) (34Saveanu C. Miron S. Borza T. Craescu C.T. Labesse G. Gagyi C. Popescu A. Schaeffer F. Namane A. Laurent-Winter C. Barzu O. Gilles A.M. Protein Sci. 2002; 11: 2551-2560Crossref PubMed Scopus (29) Google Scholar). For in vitro phosphorylation, L. monocytogenes purified MnSOD (60 μg) was incubated with 2700 units of purified cAMP-dependent protein kinase (PKA) catalytic subunit from bovine heart (Sigma) in PKA buffer (50 mm Tris-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. Two dialysis 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 dithiothreitol, 0.01% Brij 35) for 4 h at 4 °C to eliminate residual ATP. Phosphorylated MnSOD (30 μg) was dephosphorylated by Stp (6 μg) for 1 h at 37 °C in Stp buffer. Ten micrograms of phosphorylated P-α-casein (Sigma) were dephosphorylated using 1 μg of alkaline phosphatase (AP, Roche Applied Science) in phosphatase buffer (Roche Applied Science) for 1 h at 37°C. The phosphorylation level of the resulting α-casein was compared with that of the initial P-α-casein. BSA (Pierce) was used as a nonphosphorylated protein control for Pro-Q diamond staining. MnSOD, PKA, and Stp-purified proteins, MnSOD samples after PKA dephosphorylation and Stp dephosphorylation, P-α-casein-, α-casein-, and BSA-purified control proteins (2 μg) were separated by SDS-PAGE. Gels were stained with the Pro-Q and Sypro Ruby protein gel dyes (Molecular Probes) as previously described (35Steinberg T.H. Agnew B.J. Gee K.R. Leung W.Y. Goodman T. Schulenberg B. Hendrickson J. Beechem J.M. Haugland R.P. Patton W.F. Proteomics. 2003; 3: 1128-1144Crossref PubMed Scopus (335) Google Scholar). Quantification of L. monocytogenes MnSOD Activity—SOD activity was measured using the Bioxytech SOD-525 kit (Oxis research). Briefly, 4 μg of purified MnSOD were phosphorylated with PKA and dephosphorylated by Stp as described above. Samples and blanks were diluted in 40 μl of H2O and incubated in 900 μl of SOD-525 buffer at 37 °C. Thirty microliters of the R2 reagent were added and incubated at 37 °C for 1 min before addition of 30 μl of the R1 reagent. A525 nm was measured every 3 s for 2 min. Preparation of Total Bacterial Extracts and Supernatant Precipitation—Cultures (10 ml) of the L. monocytogenes EGDe wild-type and Δstp mutant strains were harvested at different time points. Bacterial pellets were recovered after centrifugation at 4000 rpm for 20 min, washed twice in PBS and resuspended in 100 μl of B-PERII Bacterial Protein Extraction Reagent (Pierce) to extract total bacterial proteins. Bacterial supernatants were precipitated in 16% trichloroacetic acid on ice at 4 °C overnight. After centrifugation, the protein pellets were washed twice with 5 ml of cold acetone, dried, and resuspended in 350 μl of 1 m Tris (pH 8.8). Protein concentration of total bacterial extract and supernatant was determined by the conventional BCA assay (Pierce). MnSOD Immunoprecipitation—Proteins (250 μg) from total bacterial extracts or from bacterial supernatants were immunoprecipitated with 2.5 μl of the anti-MnSOD serum using the protein G immunoprecipitation kit (Sigma) according to the manufacturer's instructions. Briefly, after overnight incubation of protein samples with anti-MnSOD serum, protein G beads were transferred in the spin column and incubated for 4 h at 4 °C. Beads were washed six times with 1× IP buffer, one time with 0.1× IP buffer and incubated with 60 μl of Laemmli buffer. Immunoprecipitates were recovered after centrifugation. Equivalent volumes (30 μl) of immunoprecipitate were separated by SDS-PAGE and analyzed by Western blotting using anti-MnSOD serum or antiphosphothreonine antibodies. Immunofluorescence—One milliliter of cultures of the L. monocytogenes strains growing in BHI at 6% O2 was harvested at A600 nm = 0.8. Pellets were washed and resuspended in 1 ml of PBS. Fifty microliters of suspension were loaded on a coverslip and placed in a 24-well microplate. Bacteria were fixed in 10% paraformaldehyde for 10 min. Cells were washed in PBS and incubated 5 min in 50 mm NH4Cl. After blocking in PBS, 0.5% BSA for 1 h, anti-MnSOD, or anti-Listeria R11 sera, both diluted at 1:100 in 200 μl of PBS, 0.5% BSA, were added for 1 h. After washes in PBS, anti-mouse IgG-Alexa-conjugated or anti-rabbit IgG-FITC-conjugated secondary antibodies (Molecular Probes) diluted at 1:200 in 200 μl of PBS, 0.5% BSA, were used to detect L. monocytogenes MnSOD or total bacteria, respectively. Cover slips were mounted with Mowiol and analyzed with an AxioVert microscope (Zeiss) equipped with the Metamorph software (Universal Imaging Corporation). Mutagenesis and Complementation—Upstream and downstream 1-kb flanking sequences of the sod gene were amplified by PCR from genomic DNA from L. monocytogenes EGDe using the oligonucleotides SodKO1/SodKO2 and SodKO3/SodKO4 (supplemental Table S1). The upstream MluI-EcoRI and downstream EcoRI-NcoI fragments were cloned sequentially into the thermosensitive vector pMAD (36Arnaud M. Chastanet A. Debarbouille M. Appl. Environ. Microbiol. 2004; 70: 6887-6891Crossref PubMed Scopus (704) Google Scholar). The recombinant pMAD was electroporated into L. monocytogenes EGDe to generate the sod deletion as previously described (30Archambaud C. Gouin E. Pizarro-Cerda J. Cossart P. Dussurget O. Mol. Microbiol. 2005; 56: 383-396Crossref PubMed Scopus (90) Google Scholar) except that L. monocytogenes was grown at 6% O2. Deletion of sod in the mutant strain (BUG 2225) was analyzed by RT-PCR (data not shown) and Western blotting (supplemental Fig. S1). For complementation, a DNA fragment containing the sod gene and the 600-bp upstream sequence was amplified by PCR from L. monocytogenes EGDe genomic DNA with the oligonucleotides Sod-CplmUP and SodCplmDOWN (supplemental Table S1). The BamHI-XbaI PCR product was cloned into the integrative vector pPL2 digested by BamHI and SpeI (37Lauer P. Chow M.Y. Loessner M.J. Portnoy D.A. Calendar R. J. Bacteriol. 2002; 184: 4177-4186Crossref PubMed Scopus (358) Google Scholar). The resulting recombinant plasmid was introduced in E. coli S17-1, which was used for conjugation in L. monocytogenes Δsod as previously described (37Lauer P. Chow M.Y. Loessner M.J. Portnoy D.A. Calendar R. J. Bacteriol. 2002; 184: 4177-4186Crossref PubMed Scopus (358) Google Scholar), constructing the L. monocytogenes Δsod+sod strain (BUG 2226). MnSOD expression in the Δsod+sod complemented strain was analyzed by Western blotting (supplemental Fig. S1). Sensitivity to ROS and RNS—For the disk assay, L. monocytogenes cultures were grown at 6% O2 until A600 nm = 0.6 and plated onto BHI agar plates. Filter disks (6 mm) were placed on the agar and loaded with paraquat (570 μg, Sigma) and hydrogen peroxide (210 μg, Sigma). Plates were incubated at 37 °C at 6% O2. For the liquid assay, L. monocytogenes cultures were grown in BHI at 37 °C and 6% O2 until stationary phase. Cultures were then diluted in PBS at 1:100 and incubated at 37 °C with hypoxanthine (500 μm, Sigma) and xanthine oxidase (0.2 units/ml, Sigma) or spermine/NO (2 mm, Sigma). Number of CFU was assessed by plating serial dilutions in duplicate on BHI agar plates, which were incubated at 37 °C and 6% O2. Student's t test was used for statistical analyses. Infection of Murine Peritoneal Macrophages—Peritoneal macrophages (PEM) were isolated from 8-week-old female BALB/c mice (Charles River) as described (38Alford C.E. King Jr., T.E. Campbell P.A. J. Exp. Med. 1991; 174: 459-466Crossref PubMed Scopus (109) Google Scholar). 72 h before the bacterial survival assay, 1 × 106 PEM per well were activated or not using interferon γ (IFN-γ, 100 units/ml). PEM were 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 phagocytosis. Non-internalized bacteria were eliminated by three washes in RPMI supplemented with 10% fetal calf serum and 10 μg/ml gentamicin was added for 15 min, 1 h, and 4 h. PEM were lysed with 0.2% Triton X-100 for 10 min, and the number of CFU was assessed as described in the previous section. For immunofluorescence labeling, PEM adhering onto glass coverslips were loaded over 20 min with 7.5 nm of Lysotracker Red DND-99 (Molecular Probes) at 37 °C. PEM were then infected as above for 4 h at 37 °C, washed once with PBS pH 7.5, and fixed with 4% paraformaldehyde for 20 min. After quenching with 50 mm NH4Cl containing 0.05% saponin and 1% BSA for 10 min, nonspecific binding sites were blocked with 0.05% saponin and 5% horse serum during 45 min. Some coverslips were incubated for 30 min with anti-L. monocytogenes serum R11 followed by incubation during 30 min with secondary donkey anti-rabbit antibodies coupled to Alexa 647 and phalloidin coupled to Alexa 488 (Molecular Probes). All coverslips were mounted on Mowiol and analyzed with an AxioVert microscope (Zeiss) equipped with the Metamorph software (Universal Imaging Corporation). Student's t and ANOVA tests were used for statistical analyses. Immunoprecipitation of MnSOD from Infected Macrophages—PEM were activated with IFN-γ and infected with L. monocytogenes wild-type and Δsod mutant strains (MOI = 10) as described above. 15 min after adding gentamicin, PEM were lysed with 0.2% Triton X-100 for 10 min. Bacteria were recovered from cell lysates as previously described (39Saito S. Shinomiya H. Nakano M. Infect. Immun. 1994; 62: 1551-1556Crossref PubMed Google Scholar), and bacterial proteins were extracted with 100 μl of B-PER II Bacterial Protein Extraction Reagent. Proteins present in PEM were recovered in 500 μl of Tris, 1 m (pH 8.8) after precipitation of cell lysates with 16% trichloroacetic acid. Bacterial extracts (15 μg) and cellular extracts (250 μg) were immunoprecipitated with 2.5 μl of anti-MnSOD serum using the protein G immunoprecipitation kit (Sigma) as described above. Equivalent volumes (30 μl) 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 bacterial multiplication, 8-week-old female BALB/c mice (Charles River) were injected intravenously with ∼8 × 103 CFU. Liver and spleen were recovered and disrupted in 3 ml PBS at 24 h, 48 h and 72 h after infection. Serial dilutions of organ homogenates were plated on BHI agar plates and CFU determined. Animal experiments were performed according to the Institut Pasteur guidelines for laboratory animal husbandry which comply with European regulations. L. monocytogenes MnSOD Can Be Phosphorylated and Phosphorylation Down-regulates Its Activity—We previously demonstrated that Stp is a serine-threonine phosphatase involved in L. monocytogenes virulence and identified EF-Tu as its first target (30Archambaud C. Gouin E. Pizarro-Cerda J. Cossart P. Dussurget O. Mol. Microbiol. 2005; 56: 383-396Crossref PubMed Scopus (90) Google Scholar). Here, we identified L. monocytogenes MnSOD as the second target of Stp using a phosphoproteomic approach. Analysis of protein extracts of the L. monocytogenes Δstp mutant revealed the presence of a protein phosphorylated on threonine and serine residues, which was not phosphorylated in protein extracts of the wild-type strain (Fig. 1A). Mass spectrometry analysis of the corresponding spot identified the MnSOD. To demonstrate a direct dephosphorylation of L. monocytogenes MnSOD by Stp, we produced and purified a recombinant MnSOD in E. coli. The recombinant MnSOD could be phosphorylated using PKA, a cAMP-dependent serine-threonine kinase (Fig. 1B). Phosphorylated MnSOD could be fully dephosphorylated in vitro by Stp (Fig. 1B). We next examined the influence of MnSOD phosphorylation state on its activity. Dephosphorylation of the PKA-phosphorylated MnSOD more than doubled its activity, revealing that MnSOD activity can be down regulated by phosphorylation (Fig. 1C). Together, these results demonstrate that L. monocytogenes MnSOD which can be present in a phosphorylated form in bacteria is dephosphorylated by Stp, which thus increases its activity. L. monocytogenes Cytoplasmic MnSOD Is Phosphorylated upon Entry in Stationary Phase and Is Secreted in a Nonphosphorylated State by the SecA2-dependent Machinery—We investigated MnSOD phosphorylation during L. monocytogenes growth (Fig. 2A) and performed immunoprecipitation experiments on bacterial extracts and culture supernatants. MnSOD could be immunoprecipitated from both bacterial extracts and culture supernatants, in both exponential and stationary phases (Fig. 2B, panel 1). Whereas the secreted MnSOD was constantly found in its nonphosphorylated state, MnSOD from bacterial extracts was nonphosphorylated in exponential phase and became phosphorylated upon entry in stationary phase (Fig. 2B, panel 1). Increased MnSOD phosphorylation was concomitant with decreased Stp production in bacteria (Fig. 2B, panel 2). We analyzed the global production of MnSOD during growth. The MnSOD level, which was high in bacterial extracts in exponential phase, decreased in stationary phase while the amount of MnSOD detected in culture supernatants increased (Fig. 2B, panel 2). Thus, upon entry in stationary phase, nonphosphorylated MnSOD is increasingly secreted while the remaining cytoplasmic MnSOD is progressively phosphorylated. Using the Δstp 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 of the Δstp mutant, the phosphorylated form of MnSOD was detected earlier, i.e. at mid-log phase, and at a higher level compared with the wild-type strain (Fig. 2B, panel 3). Strikingly, Mn-SOD immunoprecipitated from culture supernatants was still constantly detected in its nonphosphorylated form in the Δstp mutant. Because phosphorylated listeriolysin O and phosphorylated EF-Tu control proteins could be detected in supernatants from the wild-type and Δstp mutant respectively (supplemental Fig. S2), these results strongly suggest that secretion of the phosphorylated MnSOD cannot occur. We also observed that the level of MnSOD in both bacterial extracts and culture supernatants was higher in the Δstp mutant than in the wild-type strain while two controls proteins, ActA, the actin-based motility protein, and the secreted internalin InlC, were detected in similar amounts in both strains (Fig. 2B, panel 4). Thus, the presence of Stp controls not only MnSOD phosphorylation but also its production. We then investigated the secretion mechanism of the MnSOD, whose amino acid sequence does not contain any signal peptide. Since the accessory secretion protein SecA2 had been shown to mediate secretion of proteins, which lack a signal peptide, in Gram-positive bacterial pathogens we analyzed supernatants from L. monocytogenes wild-type 10403S, from a ΔsecA2 mutant, and from a complemented strain (40Lenz L.L. Mohammadi S. Geissler A. Portnoy D.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12432-12437Crossref PubMed Scopus (235) Google Scholar) and found that the MnSOD was secreted by a SecA2-dependent machinery (Fig. 2C). Thus, MnSOD belongs, along with L. monocytogenes FbpA (41Dramsi S. Bourdichon F. Cabanes D. Lecuit M. Fsihi H. Cossart P. Mol. Microbiol. 2004; 53: 639-649Crossref PubMed Scopus (121) Google Scholar), to the increasing list of proteins lacking a signal peptide and secreted by the SecA2 pathway in pathogenic bacteria (40Lenz L.L. Mohammadi S. Geissler A. Portnoy D.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12432-12437Crossref PubMed Scopu
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