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

SUBMITTER: Schmolders J

PROVIDER: S-EPMC5383783 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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

Schmölders Johanna J Manske Christian C Otto Andreas A Hoffmann Christine C Steiner Bernhard B Welin Amanda A Becher Dörte D Hilbi Hubert H

Molecular & cellular proteomics : MCP 20170209 4

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. pneumophi ...[more]

PMID: 28183814

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Project description:UnlabelledLegionella pneumophila is an intracellular pathogen that resides within a membrane-bound compartment that is derived from vesicles exiting the endoplasmic reticulum (ER). To create this compartment, these bacteria use a type IV secretion system to deliver effector proteins that subvert host cell functions. Several Legionella effector proteins modulate the function of the host protein Rab1, which is a GTPase that is recruited to the Legionella-containing vacuole (LCV). Here, we examined which of the Rab1-directed enzymatic activities displayed by Legionella effectors are important for localizing the Rab1 protein to the LCV membrane. The guanine nucleotide exchange factor (GEF) domain in the effector protein DrrA (SidM) was essential for Rab1 recruitment to the LCV and Rab1 AMPylation by the nucleotidyltransferase domain in DrrA was important for Rab1 retention. Legionella organisms producing mutant DrrA proteins that were severely attenuated for GEF activity in vitro retained the ability to localize Rab1 to the LCV. Rab1 localization to the LCV mediated by these GEF-defective mutants required AMPylation. Importantly, we found that efficient localization of Rab1 to the LCV occurred when Rab1 GEF activity and Rab1 AMPylation activity were provided by separate proteins. Rab1 phosphocholination (PCylation) by the effector protein AnkX, however, was unable to substitute for Rab1 AMPylation. Lastly, the defect in Rab1 localization to the LCV in AMPylation-deficient strains of Legionella was partially suppressed if the GTPase-activating protein (GAP) LepB was eliminated. Thus, our data indicate that AMPylation of Rab1 is an effective strategy to maintain this GTPase on the LCV membrane.ImportanceActivities that enable the intracellular pathogen Legionella pneumophila to subvert the function of the host protein Rab1 were investigated. Our data show that a posttranslational modification called AMPylation is critical for maintaining a pool of Rab1 on the LCV membrane. AMPylation of Rab1 led to the accumulation of GTP-bound Rab1 on the LCV membrane by protecting the protein from inactivation by GAPs. Importantly, PCylation of Rab1 by the Legionella effector protein AnkX was neither necessary nor sufficient to maintain Rab1 on the LCV, indicating that AMPylation and PCylation represent functionally distinct activities. We conclude that modification of Rab1 by AMPylation is an effective strategy to spatially and temporally regulate the function of this GTPase on a membrane-bound organelle.

Dataset Information

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

Publications

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

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