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

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). 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. The L. pneumophila pentuple mutant (pentuple) lacks 5 gene clusters (31% of the effector proteins) and replicates in macrophages but not in Dictyostelium discoideum amoeba. To elucidate the host factors defining a replication-permissive compartment, we compare here the proteomes of intact LCVs isolated from D. discoideum or macrophages infected with Delta_pentuple or the parental strain Lp02. This analysis revealed that the majority of host proteins are shared in D. discoideum or macrophage LCVs containing the mutant or the parental strain, respectively, while some proteins preferentially localize to distinct LCVs. 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 active Rap1 on D. discoideum or macrophage LCVs was confirmed by fluorescence microscopy and imaging flow cytometry, and the depletion of Rap1 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.

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