Comparative Proteomics of Purified Pathogen Vacuoles Correlates Intracellular Replication of with the Small GTPase Ras-related Protein 1 (Rap1)

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 -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 "pentuple" mutant (Δpentuple) lacks 5 gene clusters (31% of the effector proteins) and replicates in macrophages but not in amoeba. To elucidate the host factors defining a replication-permissive compartment, we compare here the proteomes of intact LCVs isolated from or macrophages infected with Δpentuple or the parental strain Lp02. This analysis revealed that the majority of host proteins are shared in or macrophage LCVs containing the mutant or the parental strain, respectively, whereas some proteins preferentially localize to distinct LCVs. The small GTPase Rap1 was identified on LCVs containing strain Lp02 but not the Δpentuple mutant and on macrophage LCVs containing either strain. The localization pattern of active Rap1 on 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 Thus, comparative proteomics identified Rap1 as a novel LCV host component implicated in intracellular replication of .