Article Figures & Data
Figures
- 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.
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.
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.
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.
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.
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
- Table 1.
Strains and plasmids used in this study
Strain or plasmid Relevant properties Source or reference Strains E. coli XL1-Blue endA1 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 108286 Virulent L. pneumophila serogroup 1, strain Lens 8 ΔdotA mutant Lens lpl2613::Km 23 ΔlegK1 mutant Lens lpl1545::Km This study ΔlegK2 mutant Lens lpl2066::Km This study ΔlegK3 mutant Lens lpl2481::Km This study ΔlegK4 mutant Lens lpl0262::Km This study ΔlegK5 mutant Lens lpl2476::Km This study A. castellanii Environmental isolate P. Pernin, Faculty of Pharmacy, Université Lyon 1 D. discoideum DBS0302388 Wild-type DH1 strain 12 DBS0236184 [act15]:cnxA:GFP 55 Dd03 [act15]:legK2(K112M):c-myc This study J774A.1 ATCC TIB-67 Plasmids pGEX-6P-3 E. coli expression vector 69 pGEX-legK1 lpl1545 inserted in BamHI-SalI of pGEX-6P-3 for GST-LegK1 overproduction This study pGEX-legK2 lpl2066 inserted in BamHI-SalI of pGEX-6P-3 for GST-LegK2 overproduction This study pGEX-legK3 lpl2481 inserted in BamHI-SalI of pGEX-6P-3 for GST-LegK3 overproduction This study pQE30 E. coli expression vector Qiagen pQE30-legK4 lpl0262 inserted in BamHI-SalI of pQE30 for 6His-LegK4 overproduction This study pQE30-legK5 lpl2476 inserted in PstI-SacI of pQE30 for 6His-LegK5 overproduction This study pKD13 Used for amplification of Km 14 pAV695 sacB, OriT OriV, cml (pCDP05 with a deletion of a 4.3-kb fragment) Modified from reference 60 p695-legK1::Km pAV695 derivative carrying lpl1545 interrupted by Km This study p695-legK2::Km pAV695 derivative carrying lpl2066 interrupted by Km This study p695-legK3::Km pAV695 derivative carrying lpl2481 interrupted by Km This study p695-legK4::Km pAV695 derivative carrying lpl0262 interrupted by Km This study p695-legK5::Km pAV695 derivative carrying lpl2476 interrupted by Km This study pXDC50 Legionella expression vector carrying mCherry X. Charpentier plegK1 pXDC50 derivative Legionella expression vector carrying mCherry; expression of legK1 under the control of legK2 promoter (400 bp) This study plegK2 pXDC50 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 plegK3 pXDC50 derivative Legionella expression vector carrying mCherry; expression of legK3 under the control of legK2 promoter (400 bp) This study plegK4 pXDC50 derivative Legionella expression vector carrying mCherry; expression of legK4 under the control of legK2 promoter (400 bp) This study plegK5 pXDC50 derivative Legionella expression vector carrying mCherry; expression of legK5 under the control of legK2 promoter (400 bp) This study p3041 pXDC50 derivative Legionella expression vector carrying mCherry and gentamicin resistance gene This study pEP46 D. discoideum expression vector carrying legK2(K112M)-c-myc under the control of the actin promoter This study pEP73 Legionella expression vector carrying gfp-legK2; production of the GFP-LegK2 hybrid protein This study - Table 2.
Primers used in this study
No. Name Sequence (5′–3′)a Description 1 N-lpl1545-BamHI ATAGGATCCCCTCGTACAATGTTTTTTTCC GST-LegK1 overproduction 2 C-lpl1545-SalI ATAGTCGACTTACTCAGCCACTAACCATAAGG GST-LegK1 overproduction 3 N-lpl2066-BamHI ATAGGATCCGTTTATTACATAAATTTGAAGGAAC GST-LegK2 overproduction 4 C-lpl2066-SalI ATAGTCGACTTAGCTTGGGCCTCGCATC GST-LegK2 overproduction 5 N-lpl2481-BamHI ATAGGATCCTTTGATAGAAATATAAAAGAAATAATC GST-LegK3 overproduction 6 C-lpl2481-SalI ATAGTCGACTTATAATTCAAAGCCTGAAT GST-LegK3 overproduction 7 N-lpl0262-BamHI ATAGGATCCAAATTGCTTCGGTTTCATGAATT 6His-LegK4 overproduction 8 C-lpl0262-SalI ATAGTCGACTTAATATGGCAAAATGATGACGT 6His-LegK4 overproduction 9 N-lpl2476-SacI ATAGAGCTCGGGATTATCATGGCTACAGT 6His-LegK5 overproduction 10 C-lpl2476-PstI ATACTGCAGTTATTTTATGAAATCGGCCTTTA 6His-LegK5 overproduction 11 Kan-FRTS CACGTCGACAGCGATTGTGTAGGCTGGAGC Km amplification 12 Kan-FRTR GGGGATCCGTCGACCTGC Km amplification 13 P1-dotA-NotI AAAAGCGGCCGCGCTCTCGCTGAAAGTGGCTC lpl2613 deletion 14 P2-dotA-SalI CGCGTCGACTGCTTGCAAGCTCTTGGTTG lpl2613 deletion 15 P3-dotA-SalI CGCGTCGACGCCATTTCCTACATCCAATCG lpl2613 deletion 16 P4-dotA-NotI AAAAGCGGCCGCCCGGTTTAGAGCTTGGTCCA lpl2613 deletion 17 P1-legK1-NotI AAAAGCGGCCGCCGTTGATGCCGCTAATCTCC lpl1545 deletion 18 P2-legK1-SalI CGCGTCGACCGGTTTAACCGCTATATGCCC lpl1545 deletion 19 P3-legK1-SalI CGCGTCGACAAGGCTATCAAGCAGTTTTCCC lpl1545 deletion 20 P4-legK1-NotI AAAAGCGGCCGCTTTGAGAAAATAATCCCAGGCG lpl1545 deletion 21 P1-legK2-NotI AAAAGCGGCCGCAATTTGAAGGAACAACCCTTACCTC lpl2066 deletion 22 P2-legK2-SalI CGCGTCGACAAGTTTTTCCAGGACACATCCCT lpl2066 deletion 23 P3-legK2-SalI CGCGTCGACTTCGAATTTACAGGCTTACAAGGATC lpl2066 deletion 24 P4-legK2-NotI AAAAGCGGCCGCGCCTCGCATCAATGAAGGTG lpl2066 deletion 25 P1-legK3-NotI AAAAGCGGCCGATTTACTTCCGGGCACTGG lpl2481 deletion 26 P2-legK3-SalI CGCGTCGACGGCATTCGTTTCATCGTCAG lpl2481 deletion 27 P3-legK3-SalI CGCGTCGACAGCAACTTGCGTCCATTACG lpl2481 deletion 28 P4-legK3-NotI AAAAGCGGCCAGCTTCGCTTGCATGCAAA lpl2481 deletion 29 P1-legK4-NotI AAAAGCGGCCGCAAGCCAATCATCGTTCCCAC lpl0262 deletion 30 P2-legK4-SalI CGCGTCGACGTTCTGGTGCTAAATAGCTTGCG lpl0262 deletion 31 P3-legK4-SalI CGCGTCGACCTGCCACATCAAGTCCCCTC lpl0262 deletion 32 P4-legK4-NotI AAAAGCGGCCGCTGGCAAAATGATGACGTTGC lpl0262 deletion 33 P1-legK5-NotI AAAAGCGGCCGCCATGGCTACAGTAGATTCCG lpl2476 deletion 34 P2-legK5-SalI CGCGTCGACGAACGTTAGCTTCACGCTCT lpl2476 deletion 35 P3-legK5-SalI CGCGTCGACCACTGAAAATCGCGGATATCG lpl2476 deletion 36 P4-legK5-NotI AAAAGCGGCCGCGTTCCATGTCAATTTTAGGGC lpl2476 deletion 37 5-promolpl2066-SacI ATAGAGCTCCAGGGTAACTGAATAAGCCC lpl2066 complementation 38 3-promolpl2066-KpnI CGGGGTACCTCTCCTACAAATCAATTGCC lpl2066 complementation 39 5′SphI-promolpl2066 ATAGCATGCCAGGGTAACTGAATAAGCCC lpl2066 complementation 40 lpl2066(K112M)-sens CCCAAAGAGAATATACTGATGGTTTTATATCAAAATTTTAGTAATGTCG lpl2066 mutagenesis 41 lpl2066(K112M)-rev CGACATTACTAAAATTTTGATATAAAACCATCAGTATATTCTCTTTGGG lpl2066 mutagenesis ↵a Restriction enzyme sites are indicated in boldface.
- Table 3.
L. pneumophila Lens putative protein kinases
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.
