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

Abstract

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 .

Proteomic identification ofCoxiella burnetiieffector proteins targeted to the host cell mitochondria during infection

“…We propose the techniques and analytical approaches developed here could be applied to the study of other organelles during C. burnetii infection, or more broadly, to the study of other intracellular bacterial pathogens. Whilst previous studies have been undertaken to look at bacterial containing vacuoles, few have attempted to look at organelles free of bacterial contamination during infection (79,80). Expanding this study spatially and temporally would provide (81,82).…”

Phosphoinositides and the Fate of Legionella in Phagocytes

“…We exploited this feature to isolate LCVs from homogenates of infected host cells by establishing a two-step procedure comprising immuno-affinity enrichment with an anti-SidC antibody, followed by Histodenz density gradient centrifugation (138,139). Using this protocol, intact LCVs were isolated from D. discoideum amoeba (28,140), murine RAW 264.7 macrophage-like cells (24,27) and bone marrow-derived primary macrophages (141). The isolated LCVs were utilized for biochemical fusion experiments (28) and proteomics analysis (24,27,140,141), which identified small GTPases and their effectors (Rab family, Rap1, Ran, RanBP1), large GTPases, components of the endosomal and late secretory trafficking pathways, as well as protein or lipid kinases and phosphatases.…”

“…Using this protocol, intact LCVs were isolated from D. discoideum amoeba (28,140), murine RAW 264.7 macrophage-like cells (24,27) and bone marrow-derived primary macrophages (141). The isolated LCVs were utilized for biochemical fusion experiments (28) and proteomics analysis (24,27,140,141), which identified small GTPases and their effectors (Rab family, Rap1, Ran, RanBP1), large GTPases, components of the endosomal and late secretory trafficking pathways, as well as protein or lipid kinases and phosphatases. LCV localization of some of these proteins was confirmed by fluorescence microscopy using D. discoideum strains producing the corresponding GFP-fusion proteins (24, 26-28, 140, 142).…”

The recycling endosome and bacterial pathogens

“…Proteomic studies of intact LCVs isolated from RAW264.7 murine macrophages revealed the presence of a number of Rabs including several associated with recycling endocytosis (Bruckert & Abu Kwaik, 2015;Schmolders et al, 2017). A recent study compared the proteome of LCVs from macrophages infected with either wild type or a pentuple deletion mutant, a strain lacking five gene clusters covering 31% of the L. pneumophila effector proteins (O'Connor, Adepoju, Boyd, & Isberg, 2011), which replicates within macrophages, but not within amoebae, their natural host.…”