Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila

Abstract

The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed "P4M" (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the alpha-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila DeltasidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase IIIbeta. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.

FIGURE 1.

Identification of SidM in a screen for PI-binding L. pneumophila proteins. Pulldown of lysate from L. pneumophila wild-type (A and B), and ΔsidC or ΔsidM mutant strains (C) using agarose beads coated with different PIs or PtdIns. Bacterial proteins retained by washed beads were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue (A and C) or Silver (B). The dominant protein with an apparent molecular mass of ∼75 kDa eluting from beads coated with PtdIns(4)P or PtdIns(3,4)P₂ and to a smaller extent from beads coated with PtdIns(4,5)P₂ or PtdIns(3,4,5)P₃ was identified by mass spectrometry as the Rab1 GEF SidM/DrrA.

FIGURE 2.

SidM specifically binds to PtdIns(4)P in vitro. SDS gels stained with Coomassie Brilliant Blue of pulldown of affinity-purified GST-SidM with agarose beads coated with different PIs or PtdIns (A), or GST-SidM, GST-SidC, GST-RalF, and GST-LidA (3 μg) (left panel) and eluate from PtdIns(4)P-coated agarose beads incubated with the GST fusion proteins (right panel) (B). Protein-lipid overlay assay of 100 pmol (C) or serial 2-fold dilutions of the lipids indicated (D). The binding of affinity-purified GST fusion proteins to lipids immobilized on nitrocellulose membranes was analyzed using an anti-GST antibody. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine 1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine; PtdIns, phosphatidylinositol. Similar results were obtained in at least two independent experiments.

FIGURE 3.

Identification of the PtdIns(4)P-binding domain of SidM. A, SidM fragments fused to GST were affinity-purified and used in protein-lipid overlay assay to test binding to 100 pmol (B) or serial 2-fold dilutions of PtdIns(4)P and PtdIns(4,5)P₂ spotted onto nitrocellulose membranes (C).

FIGURE 4.

Structural analysis of SidM and fragments. A, sedimentation equilibrium analysis of full-length SidM revealed an ∼71-kDa species corresponding to a monomeric state. B, far-UV CD spectra of the full-length protein SidM (red) and the fragments M7 (blue), M9 (green), and M13 (black). The signal unit is converted into mean residue ellipticity (MRE). The helical structure is evidenced by strong negative ellipticities at around 220 and 208 nm. C, thermofluor assay for the full-length SidM protein and the fragments M7, M9, and M13. The estimated unfolding transition temperatures of full-length SidM and the fragment M7 were 62.3 and 71.4 °C, respectively, whereas the M9 and M13 fragments did not display cooperative unfolding transitions.

FIGURE 5.

C-terminal fragments of SidM localize to LCVs. A, confocal laser scanning micrographs of calnexin-GFP-labeled D. discoideum Ax3 (green), infected at an m.o.i. of 50 for 1 h with L. pneumophila labeled with a serogroup 1-specific antibody (red) and immunostained for M45-SidM, M45-M7, and M45-SidC with an anti-M45 antibody (blue). B, D. discoideum was infected at an m.o.i. of 50 for 1 h with L. pneumophila ΔsidC-sdcA harboring plasmid pCR34 (M45-SidC), pCR52 (M45-SidC-(1–608)), or pEB216 (SidC-(1–586)-M9) and immunostained using antibodies against L. pneumophila serogroup 1 (red) and SidC (green). The experiments were reproduced three (A) or two (B) independent times with similar results.

FIGURE 6.

Competition of SidM and SidC for PtdIns(4)P on LCVs. A, confocal laser scanning micrographs; B, dot plot of SidC fluorescence on LCVs in calnexin-GFP-producing D. discoideum Ax3 (green), infected with DsRed-labeled L. pneumophila (red) wild-type JR32, ΔsidM, ΔralF, or ΔsidC-sdcA and immunostained for SidC (blue). The data and the median (*, p < 10^–4) are derived from three independent experiments (n > 200), which were normalized to the median of SidC fluorescence of wild-type JR32. C, confocal laser scanning micrographs; D, dot plot of GFP-SidC_P4C fluorescence (green) on LCVs in D. discoideum Ax3 harboring the plasmid pSU01, infected with DsRed-labeled L. pneumophila (red) wild-type JR32, ΔsidM, ΔralF, or ΔsidC-sdcA. The data are combined from three independent experiments (n > 143), each normalized to the median fluorescence obtained with JR32 (*, p < 3 × 10^–2; **, p < 5 × 10^–4).

FIGURE 7.

Production of PtdIns(4)P on LCVs involves PI4K IIIβ. A, confocal laser scanning micrographs of Drosophila Kc167 phagocytes treated with the dsRNA indicated and infected at an m.o.i. of 50 for 15 min with DsRed-labeled wild-type L. pneumophila (red). Recruitment of the PtdIns(4)P-binding Icm/Dot substrate SidC was analyzed by immunofluorescence microscopy using an affinity-purified antibody against SidC (green). Bar, 2 μm. B, quantification of SidC recruitment to LCVs. Means and standard deviations of three independent experiments are shown (n = 303–762, *, p < 2 × 10^–2). RNAi, RNA interference.