Abstract
By means of the Icm/Dot type IV secretion system Legionella pneumophila translocates several effector proteins into host cells, where they anchor to the cytoplasmic face of the LCV membrane by binding to phosphoinositide (PI) lipids. Thus, phosphatidylinositol-4-phosphate anchors the effector proteins SidC and SidM, which promote the interaction of LCVs with the ER and the secretory vesicle trafficking pathway. In this chapter, we describe protocols to (1) identify PI-binding proteins in Legionella lysates using PI-beads, (2) determine PI-binding specificities and affinities of recombinant Legionella effector proteins by protein–lipid overlays, and (3) use Legionella effectors to identify cellular PI lipids.
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Abbreviations
- ACES:
-
N-(2-Acetamido)-2-aminoethanesulfonic acid
- HEPES:
-
N-2-Hydroxyethylpiperazine-N ¢-2-ethanesulfonic acid
- Icm/Dot:
-
Intracellular multiplication/defective organelle trafficking
- PI:
-
Phosphoinositide
- PtdIns(4)P :
-
Phosphatidylinositol-4-phosphate
- T4SS:
-
Type IV secretion system
References
Di Paolo G, De Camilli P (2006) Phosphoinositides in cell regulation and membrane dynamics. Nature 443:651–657
Michell RH (2008) Inositol derivatives: evolution and functions. Nat Rev Mol Cell Biol 9:151–161
Shin HW, Nakayama K (2004) Dual control of membrane targeting by PtdIns(4)P and ARF. Trends Biochem Sci 29:513–515
Behnia R, Munro S (2005) Organelle identity and the signposts for membrane traffic. Nature 438:597–604
Christoforidis S, Miaczynska M, Ashman K, Wilm M, Zhao L, Yip SC, Waterfield MD, Backer JM, Zerial M (1999) Phosphatidylinositol-3-OH kinases are Rab5 effectors. Nat Cell Biol 1:249–252
Godi A, Pertile P, Meyers R, Marra P, Di Tullio G, Iurisci C, Luini A, Corda D, De Matteis MA (1999) ARF mediates recruitment of PtdIns-4-OH kinase-beta and stimulates synthesis of PtdIns(4,5)P2 on the Golgi complex. Nat Cell Biol 1:280–287
Pizarro-Cerda J, Cossart P (2004) Subversion of phosphoinositide metabolism by intracellular bacterial pathogens. Nat Cell Biol 6:1026–1033
Hilbi H (2006) Modulation of phosphoinositide metabolism by pathogenic bacteria. Cell Microbiol 8:1697–1706
Weber SS, Ragaz C, Hilbi H (2009) Pathogen trafficking pathways and host phosphoinositide metabolism. Mol Microbiol 71:1341–1352
Isberg RR, O’Connor TJ, Heidtman M (2009) The Legionella pneumophila replication vacuole: making a cosy niche inside host cells. Nat Rev Microbiol 7:13–24
Hilbi H, Hoffmann C, Harrison CF (2011) Legionella spp. outdoors: colonization, communication and persistence. Environ Microbiol Rep 3:286–296
Urwyler S, Brombacher E, Hilbi H (2009) Endosomal and secretory markers of the Legionella-containing vacuole. Commun Integr Biol 2:107–109
Hubber A, Roy CR (2010) Modulation of host cell function by Legionella pneumophila type IV effectors. Annu Rev Cell Dev Biol 26:261–283
Hilbi H, Weber S, Finsel I (2011) Anchors for effectors: subversion of phosphoinositide lipids by Legionella. Front Microbiol 2:91
Weber SS, Ragaz C, Reus K, Nyfeler Y, Hilbi H (2006) Legionella pneumophila exploits PI(4)P to anchor secreted effector proteins to the replicative vacuole. PLoS Pathog 2:e46
Ragaz C, Pietsch H, Urwyler S, Tiaden A, Weber SS, Hilbi H (2008) The Legionella pneumophila phosphatidylinositol-4 phosphate-binding type IV substrate SidC recruits endoplasmic reticulum vesicles to a replication-permissive vacuole. Cell Microbiol 10:2416–2433
Brombacher E, Urwyler S, Ragaz C, Weber SS, Kami K, Overduin M, Hilbi H (2009) Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila. J Biol Chem 284:4846–4856
Weber SS, Ragaz C, Hilbi H (2009) The inositol polyphosphate 5-phosphatase OCRL1 restricts intracellular growth of Legionella, localizes to the replicative vacuole and binds to the bacterial effector LpnE. Cell Microbiol 11:442–460
Dowler S, Currie RA, Campbell DG, Deak M, Kular G, Downes CP, Alessi DR (2000) Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities. Biochem J 351:19–31
Horwitz MA (1983) Formation of a novel phagosome by the Legionnaires’ disease bacterium (Legionella pneumophila) in human monocytes. J Exp Med 158:1319–1331
Feeley JC, Gibson RJ, Gorman GW, Langford NC, Rasheed JK, Mackel DC, Baine WB (1979) Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J Clin Microbiol 10:437–441
Müller-Taubenberger A, Lupas AN, Li H, Ecke M, Simmeth E, Gerisch G (2001) Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis. EMBO J 20:6772–6782
Cocucci SM, Sussman M (1970) RNA in cytoplasmic and nuclear fractions of cellular slime mold amebas. J Cell Biol 45:399–407
Malchow D, Nagele B, Schwarz H, Gerisch G (1972) Membrane-bound cyclic AMP phosphodiesterase in chemotactically responding cells of Dictyostelium discoideum. Eur J Biochem 28:136–142
Acknowledgment
This work was supported by the Max von Pettenkofer Institute, Ludwig-Maximilians University Munich, and the German Research Foundation (HI 1511/1-1, HI 1511/3-1).
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Weber, S., Dolinsky, S., Hilbi, H. (2013). Interactions of Legionella Effector Proteins with Host Phosphoinositide Lipids. In: Buchrieser, C., Hilbi, H. (eds) Legionella. Methods in Molecular Biology, vol 954. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-161-5_23
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DOI: https://doi.org/10.1007/978-1-62703-161-5_23
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