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Molecular Pathogenesis

Protein Kinase LegK2 Is a Type IV Secretion System Effector Involved in Endoplasmic Reticulum Recruitment and Intracellular Replication of Legionella pneumophila

Eva Hervet, Xavier Charpentier, Anne Vianney, Jean-Claude Lazzaroni, Christophe Gilbert, Danièle Atlan, Patricia Doublet
J. B. Bliska, Editor
Eva Hervet
1Université de Lyon, Université Lyon 1, CNRS UMR 5240 “Microbiologie, Adaptation et Pathogénie,” 69622 Villeurbanne, France
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Xavier Charpentier
2Department of Microbiology, Columbia University Medical Center, New York, New York 10032
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Anne Vianney
1Université de Lyon, Université Lyon 1, CNRS UMR 5240 “Microbiologie, Adaptation et Pathogénie,” 69622 Villeurbanne, France
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Jean-Claude Lazzaroni
1Université de Lyon, Université Lyon 1, CNRS UMR 5240 “Microbiologie, Adaptation et Pathogénie,” 69622 Villeurbanne, France
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Christophe Gilbert
1Université de Lyon, Université Lyon 1, CNRS UMR 5240 “Microbiologie, Adaptation et Pathogénie,” 69622 Villeurbanne, France
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Danièle Atlan
1Université de Lyon, Université Lyon 1, CNRS UMR 5240 “Microbiologie, Adaptation et Pathogénie,” 69622 Villeurbanne, France
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Patricia Doublet
1Université de Lyon, Université Lyon 1, CNRS UMR 5240 “Microbiologie, Adaptation et Pathogénie,” 69622 Villeurbanne, France
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  • For correspondence: patricia.doublet@univ-lyon1.fr
J. B. Bliska
Roles: Editor
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DOI: 10.1128/IAI.00805-10
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  • Fig. 1.
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    Fig. 1.

    Putative protein kinases encoded by the L. pneumophila genome. (A) Multiple sequence alignment of the protein kinase domains of LegK1 to LegK5. The highly conserved amino acid residues of the Hanks subdomains I, II, VIB, VII, and VIII are indicated in boldface. (B) Schematic representations of the legK5 genomic region. The GC content of each gene of the region is indicated in the upper scheme. The genes conserved in the five sg1 L. pneumophila strains Lens, Paris, Philadelphia, Corby, and Alcoy are represented in gray, while the genes specific to L. pneumophila Lens are represented in black. Encoded proteins are named below.

  • Fig. 2.
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    Fig. 2.

    Biochemical activities of recombinant LegK proteins. (A) SDS-PAGE analysis of purified GST-LegK1, GST-LegK2, GST-LegK3, 6His-LegK4, and 6His-LegK5 after staining with Coomassie blue. Molecular mass standards are indicated on the left. (B) Effects of cations on autokinase activities of GST-LegK2, GST-LegK3, 6His-LegK4, and 6His-LegK5. Purified LegK proteins were subjected to in vitro autophosphorylation assays in the presence of [γ-32P]ATP and Mg2+ or Mn2+. Phosphoproteins were separated by SDS-PAGE and then revealed by autoradiography. (C) Protein kinase activities of LegK proteins. The eukaryotic substrate myelin basic protein (MBP) was incubated with each LegK recombinant protein in the presence of [γ-32P]ATP. Phosphoproteins were visualized by autoradiography after SDS-PAGE separation.

  • Fig. 3.
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    Fig. 3.

    Dot/Icm-dependent translocation of LegK4 into J774 cells. (A) Western blot analysis of TEM fusion expression detected with an α-TEM antibody. (B) J774 cells were infected with L. pneumophila wild-type or dotA mutant strains harboring TEM-FabI, TEM-VipA, TEM-LegK4, and TEM-LegK5 expression plasmids at an MOI of 50. Infected cells were loaded with CCF4/AM, and translocation was determined by a comparison of cleaved to uncleaved CCF4 that gives blue and green fluorescence, respectively. Images were obtained by using epifluorescence microscopy on individual assay wells.

  • Fig. 4.
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    Fig. 4.

    Role of LegK proteins in virulence toward A. castellanii. (A) Cytotoxicity of L. pneumophila. Cells were infected at an MOI of 10. The viability of amoeba cells present in infected monolayers at 48 h postinfection was quantified by using the Alamar blue dye. These data are representative of two independent experiments done in triplicate. (B) Release of bacteria from amoebae infected with L. pneumophila. After 24 h of infection, the number of extracellular bacteria was evaluated by the standard plate count assay. The results are expressed in CFU ml−1 and are representative of two independent experiments performed in triplicate. Error bars represent the standard deviations. (C) Intracellular growth of L. pneumophila in amoebae. Amoebae were infected at an MOI of 10 by L. pneumophila cells expressing the mCherry gene on a plasmid. Bacteria multiplication was automatically monitored by measuring the fluorescence of mCherry at an excitation of 587 nm and an emission of 610 nm every 2 h for 66 h. Fluorescence data were subjected to background subtractions (uninfected cells).

  • Fig. 5.
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    Fig. 5.

    Complementation of the legK2 mutant virulence defect by other legK genes. (A) Cytotoxicity of L. pneumophila measured as described in Fig. 4A. (B) Release of bacteria from amoebae evaluated as described in Fig. 4B. (C) Intracellular growth of L. pneumophila in amoebae monitored as reported in Fig. 4C.

  • Fig. 6.
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    Fig. 6.

    LegK2 is required for ER recruitment on LCV. (A) Uptake and survival ability of legK2 mutant strain. A. castellanii cells were infected at an MOI of 10 with wild-type, dotA mutant, and legK2 mutant strains. After different periods of contact with L. pneumophila, monolayers were treated for 1 h with gentamicin to kill adherent bacteria and disrupted with 0.04% Triton X-100. Viable intracellular bacteria were diluted and plated onto BCYE agar plates for colony enumeration. The results are expressed as a relative value (%) compared to a control invasion experiment with the wild-type strain. These data are representative of three independent experiments performed in triplicate; error bars represent the standard deviations. (B) Bacterial uptake assay. A. castellanii cells were infected with fluorescein-labeled Legionella at an MOI of 20, in the presence of cytochalasin D when indicated (+ CytoD). After 30 min of incubation, the medium was replaced by trypan blue solution to quench the fluorescence of noninternalized bacteria. The fluorescence of internalized bacteria was measured using an excitation of 485 nm and an emission of 530 nm. Fluorescence data were corrected for differences in labeling efficiency between the tested strains. Labeling efficiencies between strains varied by ca. 10%. (C) Recruitment of calnexin-GFP. Fifty Legionella containing vacuoles were scored from each sample by confocal laser scanning micrographs of calnexin-GFP-labeled D. discoideum AX3 infected at an MOI of 100 with mCherry-labeled L. pneumophila. Calnexin-positive vacuoles were numbered for amoeba cells infected by the wild-type L. pneumophila Lens strain, its derivative dotA and legK2 mutant strains, and the complemented legK2(plegK2) and legK2(plegK2K112M) mutant strains. The data are representative of three independent experiments; each error bar represents the standard deviation. (D) Localization of ectopically produced LegK2(K112M)-c-Myc in D. discoideum cells during infection. Confocal laser scanning micrographs of DH1 cells expressing legK2(K112M)-c-myc, either uninfected or infected with mCherry-labeled L. pneumophila legK2 mutant and dotA mutant strains or XL1-Blue E. coli. LegK2(K112M)-c-Myc was detected by an α-c-Myc antibody. The experiments were reproduced twice with similar results.

Tables

  • Figures
  • Table 1.

    Strains and plasmids used in this study

    Strain or plasmidRelevant propertiesSource or reference
    Strains
        E. coli
            XL1-BlueendA1 gyrA96(Nalr) thi-1 recA1 relA1 lac glnV44 F′[::Tn10 proAB+ lacIq Δ(lacZ)M15] hsdR17(rK+ mK−)Stratagene
            BL21(DE3)(pREP4-groESL)F− ompT gal dcm lon hsdSB(rB− mB−) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])2
        L. pneumophila
        CIP 108286Virulent L. pneumophila serogroup 1, strain Lens8
            ΔdotA mutantLens lpl2613::Km23
            ΔlegK1 mutantLens lpl1545::KmThis study
            ΔlegK2 mutantLens lpl2066::KmThis study
            ΔlegK3 mutantLens lpl2481::KmThis study
            ΔlegK4 mutantLens lpl0262::KmThis study
            ΔlegK5 mutantLens lpl2476::KmThis study
        A. castellaniiEnvironmental isolateP. Pernin, Faculty of Pharmacy, Université Lyon 1
        D. discoideum
            DBS0302388Wild-type DH1 strain12
            DBS0236184[act15]:cnxA:GFP55
            Dd03[act15]:legK2(K112M):c-mycThis study
            J774A.1ATCC TIB-67
    Plasmids
        pGEX-6P-3E. coli expression vector69
        pGEX-legK1lpl1545 inserted in BamHI-SalI of pGEX-6P-3 for GST-LegK1 overproductionThis study
        pGEX-legK2lpl2066 inserted in BamHI-SalI of pGEX-6P-3 for GST-LegK2 overproductionThis study
        pGEX-legK3lpl2481 inserted in BamHI-SalI of pGEX-6P-3 for GST-LegK3 overproductionThis study
        pQE30E. coli expression vectorQiagen
        pQE30-legK4lpl0262 inserted in BamHI-SalI of pQE30 for 6His-LegK4 overproductionThis study
        pQE30-legK5lpl2476 inserted in PstI-SacI of pQE30 for 6His-LegK5 overproductionThis study
        pKD13Used for amplification of Km14
        pAV695sacB, OriT OriV, cml (pCDP05 with a deletion of a 4.3-kb fragment)Modified from reference 60
        p695-legK1::KmpAV695 derivative carrying lpl1545 interrupted by KmThis study
        p695-legK2::KmpAV695 derivative carrying lpl2066 interrupted by KmThis study
        p695-legK3::KmpAV695 derivative carrying lpl2481 interrupted by KmThis study
        p695-legK4::KmpAV695 derivative carrying lpl0262 interrupted by KmThis study
        p695-legK5::KmpAV695 derivative carrying lpl2476 interrupted by KmThis study
        pXDC50Legionella expression vector carrying mCherryX. Charpentier
        plegK1pXDC50 derivative Legionella expression vector carrying mCherry; expression of legK1 under the control of legK2 promoter (400 bp)This study
        plegK2pXDC50 derivative Legionella expression vector carrying mCherry; expression of legK2 under its promoter (400 bp)This study
        plegK2(K112M)pXDC50 derivative Legionella expression vector carrying mCherry; expression of legK2(K112M) under its promoter (400 bp)This study
        plegK3pXDC50 derivative Legionella expression vector carrying mCherry; expression of legK3 under the control of legK2 promoter (400 bp)This study
        plegK4pXDC50 derivative Legionella expression vector carrying mCherry; expression of legK4 under the control of legK2 promoter (400 bp)This study
        plegK5pXDC50 derivative Legionella expression vector carrying mCherry; expression of legK5 under the control of legK2 promoter (400 bp)This study
        p3041pXDC50 derivative Legionella expression vector carrying mCherry and gentamicin resistance geneThis study
        pEP46D. discoideum expression vector carrying legK2(K112M)-c-myc under the control of the actin promoterThis study
        pEP73Legionella expression vector carrying gfp-legK2; production of the GFP-LegK2 hybrid proteinThis study
  • Table 2.

    Primers used in this study

    No.NameSequence (5′–3′)aDescription
    1N-lpl1545-BamHIATAGGATCCCCTCGTACAATGTTTTTTTCCGST-LegK1 overproduction
    2C-lpl1545-SalIATAGTCGACTTACTCAGCCACTAACCATAAGGGST-LegK1 overproduction
    3N-lpl2066-BamHIATAGGATCCGTTTATTACATAAATTTGAAGGAACGST-LegK2 overproduction
    4C-lpl2066-SalIATAGTCGACTTAGCTTGGGCCTCGCATCGST-LegK2 overproduction
    5N-lpl2481-BamHIATAGGATCCTTTGATAGAAATATAAAAGAAATAATCGST-LegK3 overproduction
    6C-lpl2481-SalIATAGTCGACTTATAATTCAAAGCCTGAATGST-LegK3 overproduction
    7N-lpl0262-BamHIATAGGATCCAAATTGCTTCGGTTTCATGAATT6His-LegK4 overproduction
    8C-lpl0262-SalIATAGTCGACTTAATATGGCAAAATGATGACGT6His-LegK4 overproduction
    9N-lpl2476-SacIATAGAGCTCGGGATTATCATGGCTACAGT6His-LegK5 overproduction
    10C-lpl2476-PstIATACTGCAGTTATTTTATGAAATCGGCCTTTA6His-LegK5 overproduction
    11Kan-FRTSCACGTCGACAGCGATTGTGTAGGCTGGAGCKm amplification
    12Kan-FRTRGGGGATCCGTCGACCTGCKm amplification
    13P1-dotA-NotIAAAAGCGGCCGCGCTCTCGCTGAAAGTGGCTClpl2613 deletion
    14P2-dotA-SalICGCGTCGACTGCTTGCAAGCTCTTGGTTGlpl2613 deletion
    15P3-dotA-SalICGCGTCGACGCCATTTCCTACATCCAATCGlpl2613 deletion
    16P4-dotA-NotIAAAAGCGGCCGCCCGGTTTAGAGCTTGGTCCAlpl2613 deletion
    17P1-legK1-NotIAAAAGCGGCCGCCGTTGATGCCGCTAATCTCClpl1545 deletion
    18P2-legK1-SalICGCGTCGACCGGTTTAACCGCTATATGCCClpl1545 deletion
    19P3-legK1-SalICGCGTCGACAAGGCTATCAAGCAGTTTTCCClpl1545 deletion
    20P4-legK1-NotIAAAAGCGGCCGCTTTGAGAAAATAATCCCAGGCGlpl1545 deletion
    21P1-legK2-NotIAAAAGCGGCCGCAATTTGAAGGAACAACCCTTACCTClpl2066 deletion
    22P2-legK2-SalICGCGTCGACAAGTTTTTCCAGGACACATCCCTlpl2066 deletion
    23P3-legK2-SalICGCGTCGACTTCGAATTTACAGGCTTACAAGGATClpl2066 deletion
    24P4-legK2-NotIAAAAGCGGCCGCGCCTCGCATCAATGAAGGTGlpl2066 deletion
    25P1-legK3-NotIAAAAGCGGCCGATTTACTTCCGGGCACTGGlpl2481 deletion
    26P2-legK3-SalICGCGTCGACGGCATTCGTTTCATCGTCAGlpl2481 deletion
    27P3-legK3-SalICGCGTCGACAGCAACTTGCGTCCATTACGlpl2481 deletion
    28P4-legK3-NotIAAAAGCGGCCAGCTTCGCTTGCATGCAAAlpl2481 deletion
    29P1-legK4-NotIAAAAGCGGCCGCAAGCCAATCATCGTTCCCAClpl0262 deletion
    30P2-legK4-SalICGCGTCGACGTTCTGGTGCTAAATAGCTTGCGlpl0262 deletion
    31P3-legK4-SalICGCGTCGACCTGCCACATCAAGTCCCCTClpl0262 deletion
    32P4-legK4-NotIAAAAGCGGCCGCTGGCAAAATGATGACGTTGClpl0262 deletion
    33P1-legK5-NotIAAAAGCGGCCGCCATGGCTACAGTAGATTCCGlpl2476 deletion
    34P2-legK5-SalICGCGTCGACGAACGTTAGCTTCACGCTCTlpl2476 deletion
    35P3-legK5-SalICGCGTCGACCACTGAAAATCGCGGATATCGlpl2476 deletion
    36P4-legK5-NotIAAAAGCGGCCGCGTTCCATGTCAATTTTAGGGClpl2476 deletion
    375-promolpl2066-SacIATAGAGCTCCAGGGTAACTGAATAAGCCClpl2066 complementation
    383-promolpl2066-KpnICGGGGTACCTCTCCTACAAATCAATTGCClpl2066 complementation
    395′SphI-promolpl2066ATAGCATGCCAGGGTAACTGAATAAGCCClpl2066 complementation
    40lpl2066(K112M)-sensCCCAAAGAGAATATACTGATGGTTTTATATCAAAATTTTAGTAATGTCGlpl2066 mutagenesis
    41lpl2066(K112M)-revCGACATTACTAAAATTTTGATATAAAACCATCAGTATATTCTCTTTGGGlpl2066 mutagenesis
    • ↵a Restriction enzyme sites are indicated in boldface.

  • Table 3.

    L. pneumophila Lens putative protein kinases

    Table 3.
    • a Domains inferred from electronic annotations: TM, transmembrane helix; STPK, serine/threonine protein kinase domain; YPK, tyrosine protein kinase domain; Ca-depPK, calmodulin-dependent protein kinase domain; EF-HAND-1, specific protein domain; AA, amino acid position of the first PK domain.

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Protein Kinase LegK2 Is a Type IV Secretion System Effector Involved in Endoplasmic Reticulum Recruitment and Intracellular Replication of Legionella pneumophila
Eva Hervet, Xavier Charpentier, Anne Vianney, Jean-Claude Lazzaroni, Christophe Gilbert, Danièle Atlan, Patricia Doublet
Infection and Immunity Apr 2011, 79 (5) 1936-1950; DOI: 10.1128/IAI.00805-10

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Protein Kinase LegK2 Is a Type IV Secretion System Effector Involved in Endoplasmic Reticulum Recruitment and Intracellular Replication of Legionella pneumophila
Eva Hervet, Xavier Charpentier, Anne Vianney, Jean-Claude Lazzaroni, Christophe Gilbert, Danièle Atlan, Patricia Doublet
Infection and Immunity Apr 2011, 79 (5) 1936-1950; DOI: 10.1128/IAI.00805-10
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