Formation of a pathogen vacuole according to Legionella pneumophila: how to kill one bird with many stones.

Legionella species are ubiquitous, waterborne bacteria that thrive in numerous ecological niches. Yet, in contrast to many other environmental bacteria, Legionella spp. are also able to grow intracellularly in predatory protozoa. This feature mainly accounts for the pathogenicity of Legionella pneumophila, which causes the majority of clinical cases of a severe pneumonia termed Legionnaires' disease. The pathomechanism underlying L. pneumophila infection is based on macrophage resistance, which in turn is largely defined by the opportunistic pathogen's resistance towards amoebae. L. pneumophila replicates in macrophages or amoebae in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial intracellular multiplication/defective for organelle trafficking (Icm/Dot) type IV secretion system and involves a plethora of translocated effector proteins, which subvert pivotal processes in the host cell. Of the ca. 300 different experimentally validated Icm/Dot substrates, about 50 have been studied and attributed a cellular function to date. The versatility and ingenuity of these effectors' mode of actions is striking. In this review, we summarize insight into the cellular functions and biochemical activities of well-characterized L. pneumophila effector proteins and the host pathways they target. Recent studies not only substantially increased our knowledge about pathogen-host interactions, but also shed light on novel biological mechanisms.

Adrenergic antagonists restrict replication of Legionella.

Legionella pneumophila is a facultative intracellular bacterium, which upon inhalation can cause a potentially fatal pneumonia termed Legionnaires' disease. The opportunistic pathogen grows in environmental amoebae and mammalian macrophages within a unique membrane-bound compartment, the 'Legionella-containing vacuole'. Bacteria are exposed to many environmental cues including small signalling molecules from eukaryotic cells. A number of pathogenic bacteria sense and respond to catecholamine hormones, such as adrenalin and noradrenalin, a process mediated via the QseBC two-component system in some bacteria. In this study, we examined the effect of adrenergic compounds on L. pneumophila, and discovered that the adrenergic receptor antagonists benoxathian, naftopidil, propranolol and labetalol, as well as the QseC sensor kinase inhibitor LED209, reduced the growth of L. pneumophila in broth or amoebae, while replication in macrophages was enhanced. Growth restriction was common to members of the genus Legionella and Mycobacterium, and was observed for L. pneumophila in the replicative but not stationary phase of the biphasic life cycle. Deletion of the L. pneumophila qseBC genes indicated that growth inhibition by adrenergics or LED209 is mediated only to a minor extent by this two-component system, implying the presence of other adrenergic sensing systems. This study identifies adrenergic molecules as novel inhibitors of extra- and intracellular growth of Legionella and reveals LED209 as a potential lead compound to combat infections with Legionella or Mycobacterium spp.

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.

Analysis of Legionella Metabolism by Pathogen Vacuole Proteomics.

The causative agent of Legionnaires' disease, Legionella pneumophila, replicates in free-living amoebae as well as in macrophages of the innate immune system within a distinct membrane-bound compartment, the "Legionella-containing-vacuole" (LCV). LCV formation is a complex process and requires the bacterial Icm/Dot type IV secretion system, which translocates approximately 300 different "effector" proteins. Intact LCVs from infected Dictyostelium discoideum amoebae or RAW 264.7 murine macrophages can be purified using a straightforward protocol. In the first step, the LCVs in cell homogenates are tagged with an antibody directed against an L. pneumophila effector protein specifically localizing to the pathogen vacuole membrane and isolated by immunomagnetic separation using a secondary antibody coupled to magnetic beads. In the second step, the LCVs are further enriched by density gradient centrifugation through a Histodenz cushion. LCVs thus purified are analyzed by mass spectrometry-based proteomics and characterized by biochemical and cell biological approaches.

Subversion of Retrograde Trafficking by Translocated Pathogen Effectors.

Intracellular bacterial pathogens subvert the endocytic bactericidal pathway to form specific replication-permissive compartments termed pathogen vacuoles or inclusions. To this end, the pathogens employ type III or type IV secretion systems, which translocate dozens, if not hundreds, of different effector proteins into their host cells, where they manipulate vesicle trafficking and signaling pathways in favor of the intruders. While the distinct cocktail of effectors defines the specific processes by which a pathogen vacuole is formed, the different pathogens commonly target certain vesicle trafficking routes, including the endocytic or secretory pathway. Recently, the retrograde transport pathway from endosomal compartments to the trans-Golgi network emerged as an important route affecting pathogen vacuole formation. Here, we review current insight into the host cell's retrograde trafficking pathway and how vacuolar pathogens of the genera Legionella, Coxiella, Salmonella, Chlamydia, and Simkania employ mechanistically distinct strategies to subvert this pathway, thus promoting intracellular survival and replication.

Amoebae-Based Screening Reveals a Novel Family of Compounds Restricting Intracellular Legionella pneumophila.

The causative agent of Legionnaires' disease, Legionella pneumophila, grows in environmental amoebae and mammalian macrophages within a distinct compartment, the 'Legionella-containing vacuole' (LCV). Intracellular bacteria are protected from many antibiotics, and thus are notoriously difficult to eradicate. To identify novel compounds that restrict intracellular bacterial replication, we previously developed an assay based on a coculture of amoebae and GFP-producing L. pneumophila. This assay was used to screen a pathway-based, highly diverse chemical library, referred to as the Sinergia library. In this work, we chose to focus on a group of 11 hit compounds, the majority of which originated from the query molecule CN585, a compound that targets the protein phosphatase calcineurin. Further studies on 78 related compound variants revealed crucial structural attributes, namely a triple-ring scaffold with a central triazine moiety, substituted in positions 3 and 5 by two piperidine or pyrrolidine rings, and in position 1 by an amine group bearing a single aliphatic chain moiety. The most effective compound, ZINC00615682, inhibited intracellular replication of L. pneumophila with an IC50 of approximately 20 nM in Acanthamoeba castellanii and slightly less efficiently in Dictyostelium discoideum or macrophages. Pharmacological and genetic attempts to implicate calcineurin in the intracellular replication of L. pneumophila failed. Taken together, these results show that the amoebae-based screen and structure-activity relationship analysis is suitable for the identification of novel inhibitors of the intracellular replication of L. pneumophila. The most potent compound identified in this study targets (an) as yet unidentified host factor(s).

Pathogen vacuole purification from legionella-infected amoeba and macrophages.

Legionella pneumophila replicates intracellularly in environmental and immune phagocytes within a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). Formation of LCVs is strictly dependent on the Icm/Dot type IV secretion system and the translocation of "effector" proteins into the cell. Some effector proteins decorate the LCV membrane and subvert host cell vesicle trafficking pathways. Here we describe a method to purify intact LCVs from Dictyostelium discoideum amoebae and RAW 264.7 murine macrophages. The method comprises a two-step protocol: first, LCVs are enriched by immuno-magnetic separation using an antibody against a bacterial effector protein specifically localizing to the LCV membrane, and second, the LCVs are further purified by density gradient centrifugation. The purified LCVs can be characterized by proteomics and other biochemical approaches.

Immunomagnetic purification of fluorescent Legionella-containing vacuoles.

Protozoa are natural reservoirs of the environmental bacterium Legionella pneumophila. Upon inhalation of Legionella-laden aerosols, the amoeba-resistant bacteria replicate within human alveolar macrophages causing the severe pneumonia "Legionnaires' disease." Within host cells, including Dictyostelium discoideum, L. pneumophila establishes a custom-tailored compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system and involves a plethora of "effector" proteins, some of which specifically decorate the LCV membrane. This unique feature of LCVs is exploited to isolate the pathogen vacuole by immunomagnetic separation using an antibody against the effector protein SidC. LCV purity is further increased by a subsequent density gradient centrifugation step. The use of red fluorescent L. pneumophila and D. discoideum producing the LCV marker calnexin-GFP allows following the purification by fluorescence microscopy.

The Legionella effector RidL inhibits retrograde trafficking to promote intracellular replication.

The bacteria causing Legionnaires' disease, Legionella pneumophila, replicate intracellularly within unique Legionella-containing vacuoles (LCVs). LCV formation involves a type IV secretion system (T4SS) that translocates effector proteins into host cells. We show that the T4SS effector RidL localizes to LCVs, supports intracellular bacterial growth, and alters retrograde trafficking, in which selected proteins are transported from endosomes to the Golgi. The retromer complex that mediates retrograde trafficking localizes to LCVs independently of RidL and restricts intracellular bacterial growth. RidL binds the Vps29 retromer subunit and the lipid PtdIns(3)P, which localizes retromer components to membranes. Additionally, specific retromer cargo receptors and sorting nexins that mediate protein capture and membrane remodeling preferentially localize to LCVs in the absence of ridL. Ectopic RidL production inhibits retrograde trafficking, and L. pneumophila blocks retrograde transport at endosome exit sites in a ridL-dependent manner. Collectively, these findings suggest that RidL inhibits retromer function to promote intracellular bacterial replication.

Purification of pathogen vacuoles from Legionella-infected phagocytes.

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"(1). In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles(2-4). The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics. The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania(5), Listeria(6), Mycobacterium(7), Rhodococcus(8), Salmonella(9) or Legionella spp.(10). However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment. The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)(3,11) . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation(12,13) (Fig. 1). A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before(13). LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Anchors for effectors: subversion of phosphoinositide lipids by legionella.

The facultative intracellular pathogen Legionella pneumophila replicates in free-living amoebae and macrophages within a distinct compartment, the "Legionella-containing vacuole" (LCV). LCV formation involves phosphoinositide (PI) glycerolipids, which are key factors controlling vesicle trafficking pathways and membrane dynamics of eukaryotic cells. To govern the interactions with host cells, L. pneumophila employs the Icm/Dot type IV secretion system and more than 250 translocated "effector proteins" that presumably subvert host signaling and vesicle trafficking pathways. Some of the effector proteins anchor through distinct PIs to the cytosolic face of LCVs and promote the interaction with host vesicles and organelles, catalyze guanine nucleotide exchange of small GTPases, or bind to PI-metabolizing enzymes, such as OCRL1. The PI 5-phosphatase OCRL1 and its Dictyostelium homologue Dd5P4 restrict intracellular growth of L. pneumophila. Moreover, OCRL1/Dd5P4, PI 3-kinases (PI3Ks), and PI4KIIIß regulate LCV formation and localization of the effector protein SidC, which selectively decorates the LCV membrane. SidC and its 20-kDa "P4C" fragment are robust and specific probes for phosphatidylinositol-4-phosphate, and SidC can be targeted to purify intact LCVs by immuno-magnetic separation. Taken together, bacterial PI-binding effectors as well as host PIs and PI-modulating enzymes play a pivotal role for intracellular replication of L. pneumophila, and the PI-binding effectors are valuable tools for the analysis of eukaryotic PI lipids.

Isolation of Legionella-containing vacuoles by immuno-magnetic separation.

The environmental bacterium Legionella pneumophila naturally parasitizes free-living amoebae. L. pneumophila is an opportunistic human pathogen that grows in macrophages, thus causing a life-threatening pneumonia termed Legionnaires' disease. The bacteria replicate intracellularly in environmental and immune phagocytes within a unique compartment, the Legionella-containing vacuole (LCV). Formation of LCVs is a complex and robust process involving >150 secreted bacterial effector proteins, which are believed to subvert host cell signaling and vesicle trafficking pathways. This unit describes a simple approach to purify intact LCVs from Dictyostelium discoideum amoebae. The method comprises a two-step purification protocol that includes immuno-magnetic separation by means of an antibody against an effector protein specifically binding to LCVs, followed by density gradient centrifugation. The use of D. discoideum producing a fluorescent LCV marker and fluorescently labeled L. pneumophila allow tracking the enrichment of LCVs by light microscopy.