Functional analysis of novel Rab GTPases identified in the proteome of purifiedLegionella-containing vacuoles from macrophages
Year: 2013
Type: article
Abstract: The opportunistic pathogen Legionella pneumophila employs the Icm/Dot type IV secretion system and ∼300 different effector proteins to replicate in macrophages and amoebae in a distinct 'Legionella-containing vacuole' (LCV). LCVs from infected RAW 264.7 macrophages were enriched by immuno-affinity separation and density gradient centrifugation, using an antibody against the L. pneumophila effector SidC, which specifically binds to the phosphoinositide PtdIns(4)P on the pathogen vacuole membrane. The proteome of purified LCVs was determined by mass spectro-metry (data are available via ProteomeXchange with identifier PXD000647). The proteomics analysis revealed more than 1150 host proteins, including 13 small GTPases of the Rab family. Using fluorescence microscopy, 6 novel Rab proteins were confirmed to localize on pathogen vacuoles harbouring wild-type but not ΔicmT mutant L. pneumophila. Individual depletion of 20 GTPases by RNA interference indicated that endocytic GTPases (Rab5a, Rab14 and Rab21) restrict intracellular growth of L. pneumophila, whereas secretory GTPases (Rab8a, Rab10 and Rab32) implicated in Golgi-endosome trafficking promote bacterial replication. Upon silencing of Rab21 or Rab32, fewer LCVs stained positive for Rab4 or Rab9, implicated in secretory or retrograde trafficking respectively. Moreover, depletion of Rab8a, Rab14 or Rab21 significantly decreased the number of SidC-positive LCVs, suggesting that PtdIns(4)P is reduced under these conditions. L. pneumophila proteins identified in purified LCVs included proteins putatively implicated in phosphorus metabolism and as many as 60 Icm/Dot-translocated effectors, which are likely required early during infection. Taken together, the phagocyte and Legionella proteomes of purified LCVs lay the foundation for further hypothesis-driven investigations of the complex process of pathogen vacuole formation.
Source: Cellular Microbiology
DOI: 10.1111/cmi.12256
Language: English
Subfield: Endocrinology
Domain: Life Sciences
Open Access status: bronze
Funders: National Science Foundation, Imperial College London, Deutsche Forschungsgemeinschaft, Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, Bundesministerium für Bildung und Forschung, Eidgenössische Technische Hochschule Zürich, Turun Yliopisto, Universidade de Lisboa, Université de Genève
Awards: Zentrum für Innovationskompetenz, Nachwuchsgruppe: Posttranslationale Proteinmodifikationen als molekulare Schalter in Erreger-Wirt-Interaktionen, G3541515630, Medizinische Infektionsgenomik: Pathogen-Wirtsinteraktom und Signalkomplexe in bakteriellen Infektionen, Projektteil München, Hilbi, G544591541, Legionella trafficking and host phosphoinositide metabolism, G8659111823
OpenAlex ID: W1542673149