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Eva Rothmeier Gudrun Pfaffinger Christine Hoffmann Christopher F. Harrison Heinrich Grabmayr Urska Repnik Mandy Hannemann Stefan W?lke Andreas Bausch Gareth Griffiths Annette Müller-Taubenberger Aymelt Itzen Hubert Hilbi 《PLoS pathogens》2013,9(9)
The causative agent of Legionnaires'' disease, Legionella pneumophila, uses the Icm/Dot type IV secretion system (T4SS) to form in phagocytes a distinct “Legionella-containing vacuole” (LCV), which intercepts endosomal and secretory vesicle trafficking. Proteomics revealed the presence of the small GTPase Ran and its effector RanBP1 on purified LCVs. Here we validate that Ran and RanBP1 localize to LCVs and promote intracellular growth of L. pneumophila. Moreover, the L. pneumophila protein LegG1, which contains putative RCC1 Ran guanine nucleotide exchange factor (GEF) domains, accumulates on LCVs in an Icm/Dot-dependent manner. L. pneumophila wild-type bacteria, but not strains lacking LegG1 or a functional Icm/Dot T4SS, activate Ran on LCVs, while purified LegG1 produces active Ran(GTP) in cell lysates. L. pneumophila lacking legG1 is compromised for intracellular growth in macrophages and amoebae, yet is as cytotoxic as the wild-type strain. A downstream effect of LegG1 is to stabilize microtubules, as revealed by conventional and stimulated emission depletion (STED) fluorescence microscopy, subcellular fractionation and Western blot, or by microbial microinjection through the T3SS of a Yersinia strain lacking endogenous effectors. Real-time fluorescence imaging indicates that LCVs harboring wild-type L. pneumophila rapidly move along microtubules, while LCVs harboring ΔlegG1 mutant bacteria are stalled. Together, our results demonstrate that Ran activation and RanBP1 promote LCV formation, and the Icm/Dot substrate LegG1 functions as a bacterial Ran activator, which localizes to LCVs and promotes microtubule stabilization, LCV motility as well as intracellular replication of L. pneumophila. 相似文献
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Rothmeier Louisa Marie Sahm René Watermann Burkard Grabow Karsten Koester Meike Cichy Anna Martens Andreas 《Hydrobiologia》2021,848(10):2569-2578
Hydrobiologia - The introduction of non-indigenous organisms in new areas in the context of host-parasite interactions is still poorly understood. This study aimed at a parasitological and... 相似文献
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Sylvia Simon Maria A. Wagner Eva Rothmeier Annette Müller‐Taubenberger Hubert Hilbi 《Cellular microbiology》2014,16(7):977-992
The environmental bacterium Legionella pneumophila causes a severe pneumonia termed Legionnaires' disease. L. pneumophila employs a conserved mechanism to replicate within a specific vacuole in macrophages or protozoa such as the social soil amoeba Dictyostelium discoideum. Pathogen–host interactions depend on the Icm/Dot type IV secretion system (T4SS), which translocates approximately 300 different effector proteins into host cells. Here we analyse the effects of L. pneumophila on migration and chemotaxis of amoebae, macrophages or polymorphonuclear neutrophils (PMN). Using under‐agarose assays, L. pneumophila inhibited in a dose‐ and T4SS‐dependent manner the migration of D. discoideum towards folate as well as starvation‐induced aggregation of the social amoebae. Similarly, L. pneumophila impaired migration of murine RAW 264.7 macrophages towards the cytokines CCL5 and TNFα, or of primary human PMN towards the peptide fMLP respectively. L. pneumophila lacking the T4SS‐translocated activator of the small eukaryotic GTPase Ran, Lpg1976/LegG1, hyper‐inhibited the migration of D. discoideum, macrophages or PMN. The phenotype was reverted by plasmid‐encoded LegG1 to an extent observed for mutant bacteria lacking a functional Icm/Dot T4SS.Similarly, LegG1 promoted random migration of L. pneumophila‐infected macrophages and A549 epithelial cells in a Ran‐dependent manner, or upon ‘microbial microinjection’ into HeLa cells by a Yersinia strain lacking endogenous effectors. Single‐cell tracking and real‐time analysis of L. pneumophila‐infected phagocytes revealed that the velocity and directionality of the cells were decreased, and cell motility as well as microtubule dynamics was impaired. Taken together, these findings indicate that the L. pneumophila Ran activator LegG1 and consequent microtubule polymerization are implicated in Icm/Dot‐dependent inhibition of phagocyte migration. 相似文献
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Christine Hoffmann Ivo Finsel Andreas Otto Gudrun Pfaffinger Eva Rothmeier Michael Hecker Dörte Becher Hubert Hilbi 《Cellular microbiology》2014,16(7):1034-1052
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. 相似文献
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