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. 2009 Aug 18;106(33):13725-30.
doi: 10.1073/pnas.0907200106. Epub 2009 Aug 3.

A Legionella type IV effector activates the NF-kappaB pathway by phosphorylating the IkappaB family of inhibitors

Jianning Ge  1 , Hao Xu, Ting Li, Yan Zhou, Zhibin Zhang, Shan Li, Liping Liu, Feng Shao
Affiliations

A Legionella type IV effector activates the NF-kappaB pathway by phosphorylating the IkappaB family of inhibitors

Jianning Ge et al. Proc Natl Acad Sci U S A. .

Abstract

NF-kappaB is critical in innate immune defense responses against invading microbial pathogens. Legionella pneumophila infection of lung macrophages causes Legionnaire's disease with pneumonia symptoms. A set of NF-kappaB-controlled genes involved in inflammation and anti-apoptosis are up-regulated in macrophages upon L. pneumophila infection in a Legionella Dot/Icm type IV secretion system-dependent manner. Among approximately 100 Dot/Icm substrates screened, we identified LegK1 as the sole Legionella protein that harbors a highly potent NF-kappaB-stimulating activity. LegK1 does not affect MAPK and IFN pathways. Activation of the NF-kappaB pathway by LegK1 requires its eukaryotic-like Ser/Thr kinase activity and is independent of upstream components in the NF-kappaB pathway, including TRAFs, NIK, MEKK3, and TAK1. Cell-free reconstitution revealed that LegK1 stimulated NF-kappaB activation in the absence of IKKalpha and IKKbeta, and LegK1 efficiently phosphorylated IkappaBalpha on Ser-32 and Ser-36 both in vitro and in cells. LegK1 seems to mimic the host IKK as LegK1 also directly phosphorylated other IkappaB family of inhibitors including p100 in the noncanonical NF-kappaB pathway. Phosphorylation of p100 by LegK1 led to its maturation into p52. Thus, LegK1 is a bacterial effector that directly activates the host NF-kappaB signaling and likely plays important roles in modulating macrophage defense or inflammatory responses during L. pneumophila infection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Potent and specific activation of the NF-κB pathway by LegK1, an eukaryotic-like Ser/Thr kinase from L. pneumophila. (A) Luciferase assays of LegK1-induced NF-κB activation. HEK 293T cells were transfected with NF-κB dual-luciferase reporter plasmids together with an empty vector (Vec), or expression plasmid for one of the three Legionella Ser/Thr kinases (LegK1, LegK2, and LegK3). LegK1-KA is the kinase inactive mutant (K121A). TNFα treatment was included as a positive control for comparison. For all luciferase assays, mean relative luciferase activity from duplicate determinations is shown. Error bars indicate standard deviation. (B) Immunoblotting of IκBα phosphorylation induced by ectopic expression of LegK1. HEK 293T cells were transfected with an empty vector (Vec) or LegK1 (WT or KA) (Left), or treated with TNFα for 5 or 10 min (Right). Levels of indicated proteins were analyzed by immunoblotting of total cell lysates. pSer32-IκBα antibody recognizes endogenous IκBα phosphorylated on Ser-32. (C) Immunofluorescence assays of p65 nuclear translocation induced by LegK1. HeLa cells expressing EGFP-tagged LegK1 (WT or KA mutant) were stained with p65 specific antibody (red) or DAPI to mark the nuclei (blue). As controls, pEGFP-C1 vector-transfected HeLa cells were left untreated or treated with TNFα for 1 h. (D and E) IFNβ promoter-driven luciferase assays of the three Legionella Ser/Thr kinases. HEK 293T cells harboring indicated luciferase reporter plasmid were transfected with an empty vector (Vec), or an indicated expression construct. 5D refers to a constitutive active IRF3 mutant (S396D/S398D/S402D/T404D/S405D) serving as a positive control for IFNβ-4×PRD(III/I)-luc. IKKβ is used as a positive control for IFNβ-PRD(II)-luc.
Fig. 2.
Fig. 2.
Dot/Icm dependent translocation of LegK1 into U937 cells. Differentiated U937 cells were infected with wild-type L. pneumophila (Lp02) or the dotA mutant (Lp03) strain harboring TEM1-LegAS4, TEM1-LegK1, or TEM1-GST expression plasmids at a MOI of 10. Two hours after infection, cells were loaded with CCF2/AM dye and translocation was determined by a comparison of cleaved to uncleaved CCF2/AM that gives blue and green fluorescence, respectively. The fluorescence images shown are representatives of three separate experiments.
Fig. 3.
Fig. 3.
Signaling components upstream of the IKK complex are not required for LegK1-induced NF-κB activation. (A, B, and D) Luciferase assays of LegK1-induced NF-κB activation in TRAF2 (A), TRAF6 (B), and TAK1 (D) siRNA knockdown cells. The immunoblots (Inset) show the siRNA knockdown efficiency of the corresponding proteins, and TRAF2, TRAF6, and TAK1/TAB1 expression plasmids were used as positive controls. (C) LegK1-induced NF-κB luciferase activation in MEKK3−/− MEF cells. Wild-type (Left) or MEKK3−/− (Right) MEF cells were transfected with indicated plasmid together with the NF-κB luciferase reporter plasmid. V, WT, and KA refer to vector, LegK1-WT, and LegK1-KA mutant, respectively. (E and F) Effects of LegK1 expression on JNK activation. HEK 293T cells were transfected with indicated expression constructs together with HA-JNK (E) or the JNK luciferase reporter plasmid (F). Activation of the JNK pathway was analyzed by phospho-JNK immunoblotting (E) or luciferase reporter assays (F).
Fig. 4.
Fig. 4.
Reconstitution of LegK1-induced NF-κB activation in cell-free extracts and IKK-independent NF-κB activation by LegK1. (A) Activation of the NF-κB pathway by recombinant LegK1 in cell-free extracts. HeLa S100 were incubated with a buffer control, recombinant TRAF6, or bacterially purified LegK1 (WT or the KA mutant) in the presence of ATP regeneration system at 30 °C. Samples were analyzed by immunoblotting using antibodies recognizing IκBα or phospho-Ser-32 IκBα. Left and Right show the dose dependency and time-course analysis, respectively. (B) Activation of the NF-κB pathway by recombinant LegK1 in extracts from IKK knockout MEF cells. Experiments were performed and data are presented similarly as shown in (A) except that indicated wild-type, IKKα−/−, IKKβ−/−, or IKKα/β−/− (double knockout) MEF cells were used. (C) Luciferase assays of LegK1-induced NF-κB activation in IKKα/β−/− MEF cells. (D) Effects of LegK1 expression on phosphorylation of IKKα and IKKβ in cells. HEK 293T cells were transfected with EGFP-LegK1 (WT or the KA mutant), NIK, or TAK1/TAB1 as indicated. Cell lysates were subjected to immunoprecipitation by NEMO antibody. Total cell lysates (Input) or the immunoprecipitates (NEMO-IP) were analyzed by immunoblotting using indicated antibodies. (E) Effects of LegK1 expression on phosphorylation of IKKα and IKKβ in cell-free extracts. HeLa S100 was incubated with a buffer control, recombinant TRAF6, or LegK1 (WT or the KA mutant), and the reactions were analyzed by immunoblotting using antibodies as indicated.
Fig. 5.
Fig. 5.
Recombinant LegK1 directly phosphorylates IκBα in vitro. (A) In vitro phosphorylation of IκBα by recombinant LegK1 and its truncation mutants. Purified GST-IκBα or myelin basic protein (MBP) was used as the substrate in the in vitro kinase assay by using indicated purified kinases. 32P autoradiography (Upper) shows incorporation of phosphates into the substrate and immunoblotting using the pSer32-IκBα antibody (middle) reflects the site-specific phosphorylation. Lower shows the relative level of LegK1 added into each reaction. (B) NF-κB luciferase assays of LegK1 kinase activation loop mutant (SY/AA, LegK1 S252A/Y256A) (B) or truncation mutants of LegK1 (D). HEK 293T cells were transfected with LegK1 or its variants as indicated, and the relative NF-κB luciferase activity is shown. (C) Phosphorylation of IκBα induced by truncation mutants of LegK1 in HeLa S100 extracts.
Fig. 6.
Fig. 6.
Phosphorylation of p100 in the noncanonical NF-κB pathway by LegK1 induces its processing into p52. (A and B) Processing of p100 into p52 induced by LegK1 expression. HEK 293T (A) or indicated MEF cells (wild-type or IKKα/β knockout) (B) were transfected with Myc-p100 together with indicated expression plasmids. Shown are immunoblots of the total cell lysates using Myc or indicated antibodies. (C) Phosphorylation of endogenous p100 induced by recombinant LegK1 in cell-free extracts. Cell extracts were prepared from HeLa S3 or MEF cells and reactions were analyzed by immunoblotting using antibodies specific for p100 or p100 phosphorylated at Ser-866 and Ser-870. (D) Direct phosphorylation of purified p100 by LegK1. In vitro kinase assay was carried out as that shown in Fig. 5A except that bacterially purified GST-p100C (the C-terminal domain, amino acids 755–900) was used as the substrate.
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