Comparative Proteomics of Purified Pathogen Vacuoles Correlates Intracellular Replication ofLegionella pneumophilawith the Small GTPase Ras-related protein 1 (Rap1).
Legionella pneumophilais an opportunistic bacterial pathogen that causes a severe lung infection termed "Legionnaires' disease." The pathogen replicates in environmental protozoa as well as in macrophages within a unique membrane-bound compartment, theLegionella-containing-vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates ca. 300 "effector proteins" into host cells, where they target distinct host factors. TheL. pneumophila"pentuple" mutant (Δpentuple) lacks 5 gene clusters (31% of the effector proteins) and replicates in macrophages but not inDictyostelium discoideumamoeba. To elucidate the host factors defining a replication-permissive compartment, we compare here the proteomes of intact LCVs isolated fromD. discoideumor macrophages infected with Δpentuple or the parental strain Lp02. This analysis revealed that the majority of host proteins are shared inD. discoideumor macrophage LCVs containing the mutant or the parental strain, respectively, whereas some proteins preferentially localize to distinct LCVs. The small GTPase Rap1 was identified onD. discoideumLCVs containing strain Lp02 but not the Δpentuple mutant and on macrophage LCVs containing either strain. The localization pattern of active Rap1 onD. discoideumor macrophage LCVs was confirmed by fluorescence microscopy and imaging flow cytometry, and the depletion of Rap1 by RNA interference significantly reduced the intracellular growth ofL. pneumophilaThus, comparative proteomics identified Rap1 as a novel LCV host component implicated in intracellular replication ofL. pneumophila.
From the ‡Max von Pettenkofer Institute, Ludwig-Maximilians University, Munich, Germany.
Johanna
J
Schmölders
From the ‡Max von Pettenkofer Institute, Ludwig-Maximilians University, Munich, Germany.
Christian
C
Manske
§Institute for Microbiology, Ernst Moritz Arndt University, Greifswald, Germany.
Andreas
A
Otto
From the ‡Max von Pettenkofer Institute, Ludwig-Maximilians University, Munich, Germany.
Christine
C
Hoffmann
¶Institute of Medical Microbiology, University of Zürich, Switzerland.
Bernhard
B
Steiner
¶Institute of Medical Microbiology, University of Zürich, Switzerland.
Amanda
A
Welin
§Institute for Microbiology, Ernst Moritz Arndt University, Greifswald, Germany; dbecher@uni-greifswald.de hilbi@imm.uzh.ch.
Dörte
D
Becher
From the ‡Max von Pettenkofer Institute, Ludwig-Maximilians University, Munich, Germany; dbecher@uni-greifswald.de hilbi@imm.uzh.ch.|¶Institute of Medical Microbiology, University of Zürich, Switzerland.
Hubert
H
Hilbi
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002853:Chromatography, Liquid; D004261:DNA Replication; D004023:Dictyostelium / Q000378:metabolism* / Q000382:microbiology; D017353:Gene Deletion; D016952:Legionella pneumophila / Q000235:genetics / Q000502:physiology*; D007877:Legionnaires' Disease / Q000382:microbiology; D008264:Macrophages / Q000378:metabolism* / Q000382:microbiology; D051379:Mice; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015800:Protozoan Proteins / Q000378:metabolism; D000067996:RAW 264.7 Cells; D053719:Tandem Mass Spectrometry; D014617:Vacuoles / Q000378:metabolism / Q000382:microbiology*; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D015800:Protozoan Proteins; D020559:Monomeric GTP-Binding Proteins; C000620839:Rap1 protein, mouse; D020827:rap1 GTP-Binding Proteins