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. 2011 May;79(5):1936-50.
doi: 10.1128/IAI.00805-10. Epub 2011 Feb 14.

Protein kinase LegK2 is a type IV secretion system effector involved in endoplasmic reticulum recruitment and intracellular replication of Legionella pneumophila

Eva Hervet  1 , Xavier Charpentier, Anne Vianney, Jean-Claude Lazzaroni, Christophe Gilbert, Danièle Atlan, Patricia Doublet
Affiliations

Protein kinase LegK2 is a type IV secretion system effector involved in endoplasmic reticulum recruitment and intracellular replication of Legionella pneumophila

Eva Hervet et al. Infect Immun. 2011 May.

Erratum in

Abstract

Legionella pneumophila is the etiological agent of Legionnaires' disease. Crucial to the pathogenesis of this intracellular pathogen is its ability to subvert host cell defenses, permitting intracellular replication in specialized vacuoles within host cells. The Dot/Icm type IV secretion system (T4SS), which translocates a large number of bacterial effectors into host cell, is absolutely required for rerouting the Legionella phagosome. Many Legionella effectors display distinctive eukaryotic domains, among which are protein kinase domains. In silico analysis and in vitro phosphorylation assays identified five functional protein kinases, LegK1 to LegK5, encoded by the epidemic L. pneumophila Lens strain. Except for LegK5, the Legionella protein kinases are all T4SS effectors. LegK2 plays a key role in bacterial virulence, as demonstrated by gene inactivation. The legK2 mutant containing vacuoles displays less-efficient recruitment of endoplasmic reticulum markers, which results in delayed intracellular replication. Considering that a kinase-dead substitution mutant of legK2 exhibits the same virulence defects, we highlight here a new molecular mechanism, namely, protein phosphorylation, developed by L. pneumophila to establish a replicative niche and evade host cell defenses.

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Figures

Fig. 1.
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.
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.
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.
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.
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.
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.
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References

    1. Alber T. 2009. Signaling mechanisms of the Mycobacterium tuberculosis receptor Ser/Thr protein kinases. Curr. Opin. Struct. Biol. 19:650–657 - PMC - PubMed
    1. Amrein K., et al. 1995. Purification and characterization of recombinant human p50csk protein-tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL. Proc. Natl. Acad. Sci. U. S. A. 92:1048–1052 - PMC - PubMed
    1. Andrews H. L., Vogel J. P., Isberg R. R. 1998. Identification of linked Legionella pneumophila genes essential for intracellular growth and evasion of the endocytic pathway. Infect. Immun. 66:950–958 - PMC - PubMed
    1. Barz C., Abahji T. N., Trulzsch K., Heesemann J. 2000. The Yersinia Ser/Thr protein kinase YpkA/YopO directly interacts with the small GTPases RhoA and Rac-1. FEBS Lett. 482:139–143 - PubMed
    1. Berger K., Isberg R. 1993. Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol. Microbiol. 7:7–19 - PubMed

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