Rab1 Guanine Nucleotide Exchange Factor SidM Is a Major Phosphatidylinositol 4-Phosphate-binding Effector Protein of Legionella pneumophila

• DOI: 10.1074/jbc.m807505200
• Publication Date: 2008-12-19T01:17:49Z
• Authors: Eva Brombacher; Simon Urwyler; Curdin Ragaz; Stefan S. Weber; Keiichiro Kami; Michael Overduin; Hubert Hilbi
• Keywords: 0301 basic medicine; Phagocytes; Lipids and Lipoproteins: Metabolism, Regulation, and Signaling; Peptide Mapping; Cell Line; Legionella pneumophila; Protein Structure, Tertiary; rab1 GTP-Binding Proteins; 03 medical and health sciences; Bacterial Proteins; Phosphatidylinositol Phosphates; Mutation; Vacuoles; Animals; Guanine Nucleotide Exchange Factors; Humans; Drosophila; Legionnaires' Disease; Carrier Proteins; Protein Binding

The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some which subvert host GTP phosphoinositide (PI) metabolism. Icm/Dot substrate SidC anchors to membrane Legionella-containing vacuoles (LCVs) specifically binding phosphatidylinositol 4-phosphate (PtdIns(4)P). Using nonbiased screen for novel L. pneumophila PI-binding we identified Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as predominant PtdIns(4)P-binding protein. Purified SidM directly bound PtdIns(4)P, whereas SidM-interacting LidA preferentially PtdIns(3)P but also Arf1 GEF RalF did not bind any PIs. domain was mapped 12-kDa C-terminal sequence, termed “P4M” (PtdIns4P SidM/DrrA). isolated P4M is largely helical displayed higher PtdIns(4)P activity context α-helical, monomeric full-length constructs containing were Icm/Dot-proficient localized LCV membrane, indicating that on LCVs via its domain. An ΔsidM mutant strain significantly amounts LCVs, suggesting compete limiting vacuole. Finally, RNA interference revealed formed PtdIns 4-kinase IIIβ. Thus, exploits produced IIIβ anchor effectors LCVs. Gram-negative pathogen it evolved parasite various species environmental predatory protozoa, including social amoeba Dictyostelium discoideum (1Fields B.S. Benson R.F. Besser R.E. Clin. Microbiol. Rev. 2002; 15: 506-526Crossref PubMed Scopus (1306) Google Scholar, 2Hilbi H. Weber S.S. Ragaz C. Nyfeler Y. Urwyler S. 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Eberl Bacteriol. 190: 7532-7547Crossref (54) In macrophages amoebae, compartment, (LCV). 3The abbreviations used are: LCV, vacuole; AUC, analytical ultracentrifugation; Dot, defective trafficking; Icm, multiplication; GEF, factor; m.o.i., multiplicity infection; PI, phosphoinositide; PtdIns, phosphatidylinositol; T4SS, system; PI4K, 4-kinase; ER, endoplasmic reticulum; GDI, dissociation inhibitor; GDF, GDI displacement RT, reverse transcription; GST, glutathione S-transferase; dsRNA, double-stranded RNA; PH, pleckstrin homology; TGN, trans-Golgi network; GFP, green fluorescent avoid fusion with lysosomes (6Horwitz M.A. Exp. Med. 1983; 158: 2108-2126Crossref (510) Scholar), intercept vesicular traffic at reticulum (ER) exit sites (7Kagan J.C. Roy C.R. Nat. Cell 4: 945-954Crossref (363) fuse ER (8Horwitz 1319-1331Crossref (496) 9Robinson C.G. 2006; 8: 793-805Crossref (127) 10Lu Clarke M. 2005; 7: 995-1007Crossref (96) uptake formation amoebae depends (T4SS) (11Hilbi Segal G. Shuman H.A. 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These bacterial GEFs recruited activate their targets Small GTPases involved many eukaryotic transduction actin cytoskeleton (22Jaffe A.B. Hall Annu. 21: 247-269Crossref (2379) Inactive GDP inhibitor (GDI). activated removal GEFs, promotes interaction downstream such protein lipid kinases adaptor proteins. cycle closed hydrolysis GTP, mediated GTPase-activating Rab1, essential Golgi transport, additionally, acts (GDF) (23Machner 318: 974-977Crossref (176) 24Ingmundson Nature. 450: 365-369Crossref (273) function assisted LidA, localizes binds thus supporting recruitment early secretory vesicles 23Machner 25Conover G.M. Derre I. Vogel 2003; 48: 305-321Crossref (201) 26Derre Infect. Immun. 73: 4370-4380Crossref (81) Another substrate, LepB (27Chen de Lu 2004; 303: 1358-1361Crossref (240) contributes Rab1-mediated cycling inactivating through function, acting antagonist (24Ingmundson recruits activates GTPase ADP-ribosylation 1 (Arf1), retrograde transport from Dominant negative 28Kagan Stein Pypaert 199: 1201-1211Crossref (237) knockdown (29Dorer M.S. Kirton D. Bader J.S. 2: e34Crossref (177) impairs well (30Urwyler Lee Mueller L.N. Aebersold Traffic. 2009; 10: 76-87Crossref (140) paralogue SdcA localize (31Luo Z.Q. 101: 841-846Crossref (372) where proteins (PtdIns(4)P) (32Weber Reus e46Crossref (234) 33Ragaz Pietsch 2416-2433Crossref (166) Phosphoinositides (PIs) regulate receptor-mediated remodeling, dynamics (34De Matteis Godi 6: 487-492Crossref (284) 35Di Paolo De Camilli 443: 651-657Crossref (2106) present cytoplasmic network (TGN), this PI Arf-dependent kinase (PI4K IIIβ) (36Godi Pertile Meyers Marra Di Tullio Iurisci Luini Corda 1999; 1: 280-287Crossref promote along pathway. Recently, found mediate export (37Blumental-Perry Haney C.J. Weixel K.M. Watkins S.C. Weisz O.A. Aridor 671-682Abstract (94) At present, exploitation signaling remain ill defined. using different PIs coupled agarose beads, major effector. We within sequence. constructs, domain, be levels controlled PI4K Strains Media—Bacteria plasmids study listed supplemental Table S1. grown CYE agar plates AYE broth; Escherichia coli cultured LB medium. Antibiotics added following concentrations: 5 μg/ml chloramphenicol 50 kanamycin 30 ampicillin E. coli. wild-type Ax3 axenically HL-5 medium 23 °C described, adding 20 G418 if required (pSU01). Drosophila Kc167 25 Schneider's 10% heat-inactivated fetal bovine serum (Invitrogen). Cloning, Recombinant Protein Production, Purification—Translational N-terminal gst m45 fusions ralF, lidA, sidM, fragments sidM constructed PCR amplification chromosomal JR32 DNA primers S2. cloned into vectors pGEX-4T-1, pGEX-6P-1, pCR33, respectively, yielding SidC-(1–586)-DL-M9/M13 cloning generated oMBglII444fw oMBglII544fw oCR117 BglII site sidC SalI pGEX, resulting insertion two additional amino acids (Asp Leu) fragments. All sequenced. Expression M45 verified Western blot analysis monoclonal mouse anti-M45 hybridoma supernatant affinity-purified polyclonal rabbit anti-SidC antibody goat anti-mouse -rabbit secondary peroxidase-labeled (Sigma). deletions ralF performed protocol described previously (38Albers Al Alam Goyert S.M. Gangloff Microbiology. 153: 3817-3829Crossref (21) 39Wiater L.A. Plasmid. 1994; 32: 280-294Crossref (9) GST Details outlined material. Pulldown Proteins—L. OD600 3, harvested 4 °C, washed once cold W-buffer (10 mm HEPES (pH 7.4), 150 NaCl), lysed French press. After addition 1mm phenylmethylsulfonyl fluoride, debris removed centrifugation min, 3,300 × g), ultracentrifugation (1 h, 155,000 g). amount soluble estimated Bradford assay (Bio-Rad). For pulldown assays 1–2 ml lysate 10–30 mg total incubated overnight 50–100 μl PI-coated beads pm PtdIns/μl slurry; Echelon). five times W-buffer. Bound eluted Laemmli buffer (5 95 °C) analyzed SDS-PAGE/Coomassie Brilliant Blue silver staining. digested trypsin matrix-assisted laser desorption ionization-tandem mass spectrometry or, alternatively, liquid chromatography-electrospray Functional Genomics Center Zurich. same protocol, pmol purified samples, PtdIns(4)P-coated suspended supplemented 0.25% Nonidet P-40. Binding Different Proteins Vitro—The specificity tested protein-lipid overlay 40Dowler Currie Campbell Deak Kular Downes C.P. Alessi D.R. Biochem. 2000; 351: 19-31Crossref (480) 200 nm expressed pGEX-4T-1 detailed Immunofluorescence Microscopy Quantification LCVs—D. infected (m.o.i. 50) fluorescence microscopy (4Tiaden 32Weber bacteria stained rhodamine-conjugated anti-L. Philadelphia-1 serogroup (m-Tech), M45-tagged labeled anti hybridoma, Cy5-conjugated (The Jackson Laboratories). Alternatively, (Santa Cruz Biotechnology), Cy3-conjugated fluorescein isothiocyanate-labeled anti-rabbit other experiments, DsRed-labeled (41Mampel Haagensen J.A.J. Molin Appl. 72: 2885-2895Crossref (128) infect calnexin-GFP-producing discoideum, visualized Cy5-labeled quantified calnexin-positive determining intensity area covering individual after local background correction QuantityOne software Interference—RNA silencing 30Urwyler Briefly, 106 cells/ml plated without h dsRNA (20 μg/ml). transfection process terminated calf final concentration 10%, cells 4–5 days prior infections. genomic oligonucleotides S2 amplify template T7 promoter vitro transcription. products transcribed Megascript kit (Ambion), quality assessed gel electrophoresis. Gene specific confirmed RT-PCR. determine effects gene 2.5 105 24-well dishes coverslip transfected incubation days. further 15 SidC-positive immunofluorescence Analytical Ultracentrifugation—To produce (AUC), GST-SidM produced, fragment cleaved off (33Ragaz A Beckman XL-I ultracentrifuge 8-cell 50Ti rotor AUC studies. Samples prepared Tris-HCl 7.4) NaCl dithiothreitol centrifuged 20,000, 22,000, 24,000 rpm °C. absorbance sample measured wavelength 280 throughout cell. three measurements taken 1-h intervals end each run. compared ensure equilibrium had reached. Data experiment SEDPHAT (42Vistica Dam Balbo Yikilmaz Mariuzza Rouault T.A. Schuck Anal. 326: 234-256Crossref (311) Parameters partial volume calculated SEDNTERP (43Laue T.M. Shah B.D. Ridgeway Pelletier S.L. Harding S.E. Ultracentrifugation Biochemistry Polymer Royal Society Chemistry, Cambridge, UK1992: 90-125Google Far-UV Circular Dichroism Spectroscopy—The CD spectra Jasco J-810 spectropolarimeter 0.02-cm path length cuvette. solutions 0.4 mg/ml (5.4, 8.0, 16.8, 32.5 μm SidM, M7, M9, M13, respectively). scanned range 185–300 nm, collected structure content CDSSTR algorithm (44Johnson W.C. Proteins. 35: 307-312Crossref (628) reference data set 7 (which contains 48 5, denatured) DICHROWEB server (45Lobley Whitmore Wallace B.A. Bioinformatics (Oxf.). 18: 211-212Crossref (646) 46Whitmore Nucleic Acids Res. D593-D594Crossref back-calculated experimental estimate normalized root mean square deviation values, below 0.1. Thermofluor Assay—Thermofluor experiments carried out real time machine Mx3005P (Stratagene). mixed dye SYPRO Orange (Molecular Probes) Thermo-Fast 96-well plate (ABgene), concentrations μm. heated rate °C/min 93 increments. Fluorescence filtered custom excitation (492 nm) emission (568 filters. primary (relative versus temperature) fit standard equations describing thermal stability, (47Mezzasalma Kranz J.K. Chan W. Struble G.T. Schalk-Hihi Deckman I.C. Springer Todd M.J. Biomol. Screen. 12: 418-428Crossref Statistical Analysis—Homology searches packages: BLAST (www.ncbi.nlm.nih), Scansite, PHYRE (protein homology/analogy recognition engine), ELM (eukaryotic linear motif resource). Prediction coiled coils Coils Server, prediction DomPred PSIPRED (Protein Structure Server) (48McGuffin L.J. Bryson Jones D.T. 16: 404-405Crossref (2785) statistical analysis, one-tailed Student's t test used, considering p < 0.05 significant. Identification Protein—The recently discovered recognized identify coated Staining eluting Coomassie large molecular ∼75 kDa, predominantly (Fig. 1A). weaker interactions PtdIns(3,4)P2. interactor 73-kDa known Upon visualizing staining, 75-kDa eluates PtdIns(4,5)P2 PtdIns(3,4,5)P3 1B). appeared phosphorylated 4-position. No discovered. SidC, similar expected under above conditions, 50-kDa fragment, “P4C” retained (data shown). whether efficiently absence repeated lysates strain. Whereas no high detected 1C). results suggest pneumophila. recovered enrichment because proteolytic degradation (supplemental Fig. S1). stable presence least thus, proteolysis does account failure recover significant conditions used. Specifically Binds Vitro—To assess heterologously Under 99-kDa PIs, alone 2A). indicate requiring co-factors. 1), even though Possibly, strongly SidC. compare affinities localizin