Comparative proteomics of purified pathogen vacuoles correlates intracellular replication of Legionella pneumophila with the small GTPase Rap1

ABSTRACT: Legionella pneumophila is 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, the Legionella-containing-vacuole (LCV). Formation of the pathogen vacuole requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates ca. 300 effector proteins into host cells, where they subvert pivotal host processes and govern LCV formation. The L. pneumophila “pentuple” mutant lacks 5 gene clusters comprising 12% of the genome and 30% of the effector proteins. The mutant strain replicates in murine bone marrow-derived macrophages but not in Dictyostelium discoideum amoeba. In order to elucidate the host cell proteome defining a replication permissive compartment, we compare here the proteomes of intact LCVs isolated from D. discoideum or murine RAW 264.7 macrophages infected with the L. pneumophila parental strain Lp02 or the “pentuple” mutant. Proteome analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed host proteins specifically localizing to LCVs harbouring strain Lp02 or the “pentuple” mutant, respectively. The small GTPase Rap1 was identified on D. discoideum LCVs containing strain Lp02 but not the “pentuple” mutant and on macrophage LCVs containing either strain. The localization pattern of Rap1 on D. discoideum LCVs was confirmed by fluorescence microscopy, and depletion of Rap1 in A549 epithelial cells by RNA interference significantly reduced the intracellular growth of L. pneumophila. Thus, comparative proteomics identified Rap1 as a novel LCV host component implicated in intracellular replication of L. pneumophila.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Legionella

SUBMITTER: Andreas Otto  

LAB HEAD: Andreas Otto

PROVIDER: PXD004797 | Pride | 2017-02-10

REPOSITORIES: Pride