Christine Hoffmann

Functional analysis of novel Rab GTPases identified in the proteome of purified Legionella‐containing vacuoles from macrophages

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.

IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid

Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.

Beyond Rab GTPases Legionella activates the small GTPase Ran to promote microtubule polymerization, pathogen vacuole motility, and infection.

Legionella spp. are amoebae-resistant environmental bacteria that replicate in free-living protozoa in a distinct compartment, the Legionella-containing vacuole (LCV). Upon transmission of Legionella pneumophila to the lung, the pathogens employ an evolutionarily conserved mechanism to grow in LCVs within alveolar macrophages, thus triggering a severe pneumonia termed Legionnaires’ disease. LCV formation is a complex and robust process, which requires the bacterial Icm/Dot type IV secretion system and involves the amazing number of 300 different translocated effector proteins. LCVs interact with the host cells endosomal and secretory vesicle trafficking pathway. Accordingly, in a proteomics approach as many as 12 small Rab GTPases implicated in endosomal and secretory vesicle trafficking were identified and validated as LCV components. Moreover, the small GTPase Ran and its effector protein RanBP1 have been found to decorate the pathogen vacuole. Ran regulates nucleo-cytoplasmic transport, spindle assembly, and cytokinesis, as well as the organization of non-centrosomal microtubules. In L. pneumophila-infected amoebae or macrophages, Ran and RanBP1 localize to LCVs, and the small GTPase is activated by the Icm/Dot substrate LegG1. Ran activation by LegG1 leads to microtubule stabilization and promotes intracellular pathogen vacuole motility and bacterial growth, as well as chemotaxis and migration of Legionella-infected cells.