Proteome analysis of Legionella vacuoles purified by magnetic immunoseparation reveals secretory and endosomal GTPases

Legionella pneumophila , the causative agent of Legionnaires' disease, replicates in macrophages and amoebae within ' Legionella -containing vacuoles' (LCVs), which communicate with the early secretory pathway and the endoplasmic reticulum. Formation of LCVs requires the bacterial Icm/Dot type IV secretion system. The Icm/Dot-translocated effector protein SidC selectively anchors to LCVs by binding the host lipid phosphatidylinositol-4-phosphate (PtdIns(4) P ). Here, we describe a novel and simple approach to purify intact vacuoles formed by L. pneumophila within Dictyostelium discoideum by using magnetic immunoseparation with an antibody against SidC, followed by density gradient centrifugation. To monitor LCV purification by fluorescence microscopy, we used Dictyostelium producing the LCV marker calnexin-GFP and L. pneumophila labeled with the red fluorescent protein DsRed. A proteome analysis of purified LCVs by liquid chromatography coupled to tandem mass spectrometry revealed 566 host proteins, including known LCV components, such as the small GTPases Arf1, Rab1 and Rab7. Rab8, an endosomal regulator of the late secretory pathway originating from the trans Golgi network, and the endosomal GTPase Rab14 were identified as novel LCV components, which were found to be present on vacuoles harboring wild-type but not Icm/Dot-deficient L. pneumophila . Thus, LCVs also communicate with the late secretory and endosomal pathways. Depletion of Rab8 or Arf1 by RNA interference reduced the amount of SidC on LCVs, indicating that the GTPases promote the recruitment of Legionella effectors by regulating the level of PtdIns(4) P .     

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.     

The Legionella pneumophila phosphatidylinositol-4 phosphate-binding type IV substrate SidC recruits endoplasmic reticulum vesicles to a replication-permissive vacuole

Legionella pneumophila, the causative agent of Legionnaires' disease, uses the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system to establish within amoebae and macrophages an endoplasmic reticulum (ER)-derived replication-permissive compartment, the Legionella-containing vacuole (LCV). The Icm/Dot substrate SidC and its paralogue SdcA anchor to LCVs via phosphatidylinositol-4 phosphate [PtdIns(4)P]. Here we identify the unique 20 kDa PtdIns(4)P-binding domain of SidC, which upon heterologous expression in Dictyostelium binds to LCVs and thus is useful as a PtdIns(4)P-specific probe. LCVs harbouring L. pneumophilaDeltasidC-sdcA mutant bacteria recruit ER and ER-derived vesicles less efficiently and carry endosomal but not lysosomal markers. The phenotypes are complemented by supplying sidC on a plasmid. L. pneumophilaDeltasidC-sdcA grows at wild-type rate in calnexin-negative LCVs, suggesting that communication with the ER is dispensable for establishing a replicative compartment. The amount of SidC and calnexin is directly proportional on isolated LCVs, and in a cell-free system, the recruitment of calnexin-positive vesicles to LCVs harbouring DeltasidC-sdcA mutant bacteria is impaired. Beads coated with purified SidC or its 70 kDa N-terminal fragment recruit ER vesicles in Dictyostelium and macrophage lysates. Our results establish SidC as an L. pneumophila effector protein, which anchors to PtdIns(4)P on LCVs and recruits ER vesicles to a replication-permissive vacuole.     

Soluble NSF attachment protein receptor molecular mimicry by a Legionella pneumophila Dot/Icm effector

Upon infection, Legionella pneumophila uses the Dot/Icm type IV secretion system to translocate effector proteins from the Legionella‐containing vacuole (LCV) into the host cell cytoplasm. The effectors target a wide array of host cellular processes that aid LCV biogenesis, including the manipulation of membrane trafficking. In this study, we used a hidden Markov model screen to identify two novel, non‐eukaryotic s oluble N SF a ttachment protein re ceptor (SNARE) homologs: the bacterial Legionella SNARE effector A (LseA) and viral SNARE homolog A proteins. We characterized LseA as a Dot/Icm effector of L. pneumophila, which has close homology to the Qc‐SNARE subfamily. The lseA gene was present in multiple sequenced L. pneumophila strains including Corby and was well distributed among L. pneumophila clinical and environmental isolates. Employing a variety of biochemical, cell biological and microbiological techniques, we found that farnesylated LseA localized to membranes associated with the Golgi complex in mammalian cells and LseA interacted with a subset of Qa‐, Qb‐ and R‐SNAREs in host cells. Our results suggested that LseA acts as a SNARE protein and has the potential to regulate or mediate membrane fusion events in Golgi‐associated pathways.     

Rab1 Guanine Nucleotide Exchange Factor SidM Is a Major Phosphatidylinositol 4-Phosphate-binding Effector Protein of Legionella pneumophila

The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed “P4M” (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the α-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila ΔsidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase IIIβ. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIβ to anchor the effectors SidC and SidM to LCVs.The Gram-negative pathogen Legionella pneumophila is the causative agent of Legionnaires disease, but it evolved as a parasite of various species of environmental predatory protozoa, including the social amoeba Dictyostelium discoideum (1, 2). The human disease is linked to the inhalation of contaminated aerosols, followed by replication in alveolar macrophages. To accommodate the transfer between host cells, L. pneumophila alternates between replicative and transmissive phases, the regulation of which includes an apparent quorum-sensing system (3–5).In macrophages and amoebae, L. pneumophila forms a replicative compartment, the Legionella-containing vacuole (LCV).³ LCVs avoid fusion with lysosomes (6), intercept vesicular traffic at endoplasmic reticulum (ER) exit sites (7), and fuse with the ER (8–10). The uptake of L. pneumophila and formation of LCVs in macrophages and amoebae depends on the Icm/Dot type IV secretion system (T4SS) (11–14). Although more than 100 Icm/Dot substrates (“effector” proteins) have been identified to date, only few are functionally characterized, including effectors that interfere with host cell signal transduction, vesicle trafficking, or apoptotic pathways (15–18).Two Icm/Dot-translocated substrates, SidM/DrrA (19, 20) and RalF (21), have been characterized as guanine nucleotide exchange factors (GEFs) for the Rho subfamily of small GTPases. These bacterial GEFs are recruited to and activate their targets on LCVs. Small GTPases of the Rho subfamily are involved in many eukaryotic signal transduction pathways and in actin cytoskeleton regulation (22). Inactive Rho GTPases bind GDP and a guanine nucleotide dissociation inhibitor (GDI). The GTPases are activated by removal of the GDI and the exchange of GDP with GTP by GEFs, which promotes the interaction with downstream effector proteins, such as protein or lipid kinases and various adaptor proteins. The cycle is closed by hydrolysis of the bound GTP, which is mediated by GTPase-activating proteins.SidM is a GEF for Rab1, which is essential for ER to Golgi vesicle transport, and additionally, SidM acts as a GDI displacement factor (GDF) to activate Rab1 (23, 24). The function of SidM is assisted by the Icm/Dot substrate LidA, which also localizes to LCVs. LidA preferentially binds to activated Rab1, thus supporting the recruitment of early secretory vesicles by SidM (19, 20, 23, 25, 26). Another Icm/Dot substrate, LepB (27), contributes to Rab1-mediated membrane cycling by inactivating Rab1 through its GTPase-activating protein function, thus acting as an antagonist of SidM (24).The Icm/Dot substrate RalF recruits and activates the small GTPase ADP-ribosylation factor 1 (Arf1), which is involved in retrograde vesicle transport from Golgi to ER (21). Dominant negative Arf1 (7, 28) or knockdown of Arf1 by RNA interference (29) impairs the formation of LCVs, as well as the recruitment of the Icm/Dot substrate SidC to the LCV (30).SidC and its paralogue SdcA localize to the LCV membrane (31), where the proteins specifically bind to the host cell lipid phosphatidylinositol 4-phosphate (PtdIns(4)P) (32, 33). Phosphoinositides (PIs) regulate eukaryotic receptor-mediated signal transduction, actin remodeling, and membrane dynamics (34, 35). PtdIns(4)P is present on the cytoplasmic membrane, but localizes preferentially to the trans-Golgi network (TGN), where this PI is produced by an Arf-dependent recruitment of PtdIns(4)P kinase IIIβ (PI4K IIIβ) (36) to promote trafficking along the secretory pathway. Recently, PtdIns(4)P was found to also mediate the export of early secretory vesicles from ER exit sites (37). At present, the L. pneumophila effector proteins that mediate exploitation of host PI signaling remain ill defined.In a nonbiased screen for L. pneumophila PI-binding proteins using different PIs coupled to agarose beads, we identified SidM as a major PtdIns(4)P-binding effector. We mapped its PtdIns(4)P binding activity to a novel P4M domain within a 12-kDa C-terminal sequence. SidM constructs, including the P4M domain, were found to be translocated and bind the LCV membrane, where the levels of PtdIns(4)P are controlled by PI4K IIIβ.     

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 Leishmania⁵, Listeria⁶, Mycobacterium⁷, Rhodococcus⁸, Salmonella⁹ 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 before¹³. 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.     

ER remodeling by the large GTPase atlastin promotes vacuolar growth of Legionella pneumophila

The pathogenic bacterium Legionella pneumophila replicates in host cells within a distinct ER‐associated compartment termed the Legionella‐containing vacuole (LCV). How the dynamic ER network contributes to pathogen proliferation within the nascent LCV remains elusive. A proteomic analysis of purified LCVs identified the ER tubule‐resident large GTPase atlastin3 (Atl3, yeast Sey1p) and the reticulon protein Rtn4 as conserved LCV host components. Here, we report that Sey1/Atl3 and Rtn4 localize to early LCVs and are critical for pathogen vacuole formation. Sey1 overproduction promotes intracellular growth of L. pneumophila, whereas a catalytically inactive, dominant‐negative GTPase mutant protein, or Atl3 depletion, restricts pathogen replication and impairs LCV maturation. Sey1 is not required for initial recruitment of ER to PtdIns(4)P‐positive LCVs but for subsequent pathogen vacuole expansion. GTP (but not GDP) catalyzes the Sey1‐dependent aggregation of purified, ER‐positive LCVs in vitro. Thus, Sey1/Atl3‐dependent ER remodeling contributes to LCV maturation and intracellular replication of L. pneumophila.     

Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella‐containing vacuole

Legionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high‐throughput RNAi screens against host membrane trafficking genes to identify factors that antagonise vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the ΔsdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilise the replication vacuole, EEA1 and Rab11FIP1 showed increased density about the sdhA mutant vacuole compared with the wild type (WT) vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant redistribution. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilise vacuole integrity during infection.     

Activation of Ran GTPase by a Legionella Effector Promotes Microtubule Polymerization,Pathogen Vacuole Motility and Infection

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.     

Icm/Dot‐dependent inhibition of phagocyte migration by Legionella is antagonized by a translocated Ran GTPase activator

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.     

The natural alternative: protozoa as cellular models for Legionella infection

The severe pneumonia known as Legionnaires' disease occurs following infection by the Gram‐negative bacterium Legionella pneumophila. Normally resident in fresh‐water sources, Legionella are subject to predation by eukaryotic phagocytes such as amoeba and ciliates. To counter this, L. pneumophila has evolved a complex system of effector proteins which allow the bacteria to hijack the phagocytic vacuole, hiding and replicating within their erstwhile killers. These same mechanisms allow L. pneumophila to hijack another phagocyte, lung‐based macrophages, which thus avoids a vital part of the immune system and leads to infection. The course of infection can be divided into five main categories: pathogen uptake, formation of the replication‐permissive vacuole, intracellular replication, host cell response, and bacterial exit. L. pneumophila effector proteins target every stage of this process, interacting with secretory, endosomal, lysosomal, retrograde and autophagy pathways, as well as with mitochondria. Each of these steps can be studied in protozoa or mammalian cells, and the knowledge gained can be readily applied to human pathogenicity. Here we describe the manner whereby L. pneumophila infects host protozoa, the various techniques which are available to analyse these processes and the implications of this model for Legionella virulence and the pathogenesis of Legionnaires' disease.     

Novel export control of a Legionella Dot/Icm substrate is mediated by dual,independent signal sequences

The Legionella pneumophila Dot/Icm T4SS injects ～ 300 protein effector proteins into host cells. Dot/Icm substrates have been proposed to contain a carboxy‐terminal signal sequence that is necessary and sufficient for export, although both traits have been demonstrated for only a small fraction of these proteins. In this study, we discovered that export of the substrate SidJ is mediated by dual signal sequences that include a conventional C‐terminal domain and a novel internal motif. The C‐terminal signal sequence facilitates secretion of SidJ into host cells at early points of infection, whereas the internal signal sequence mediates secretion at later time points. Interestingly, only the internal signal sequence is necessary for complementation of the intracellular growth defect of a ΔsidJ mutant. Although this is the first report of a Dot/Icm substrate being secreted by an internal signal sequence, many other substrates may be exported in a similar manner. In addition, efficient translocation of SidJ is dependent on the chaperone‐like type IV adaptors IcmS/IcmW. Five IcmS/IcmW binding domains that are distinct from both signal sequences were elucidated and, interestingly, only secretion mediated by the internal signal sequence requires IcmS/IcmW. Thus, Legionella employs multiple sophisticated molecular mechanisms to regulate the export of SidJ.     

The inositol polyphosphate 5-phosphatase OCRL1 restricts intracellular growth of Legionella, localizes to the replicative vacuole and binds to the bacterial effector LpnE

Legionella pneumophila , the causative agent of Legionnaires' disease, replicates within a specific vacuole in amoebae and macrophages. To form these ' Legionella -containing vacuoles' (LCVs), the bacteria employ the Icm/Dot type IV secretion system and effector proteins, some of which anchor to the LCV membrane via the host glycolipid phosphatidylinositol 4-phosphate [PtdIns(4) P ]. Here we analysed the role of inositol polyphosphate 5-phosphatases (IP5Ps) during L. pneumophila infections. Bacterial replication and LCV formation occurred more efficiently in Dictyostelium discoideum amoebae lacking the IP5P Dd5P4, a homologue of human OCRL1 (Oculocerebrorenal syndrome of Lowe), implicated in retrograde endosome to Golgi trafficking. The phenotype was complemented by Dd5P4 but not the catalytically inactive 5-phosphatase. Ectopically expressed Dd5P4 or OCRL1 localized to LCVs in D. discoideum via an N-terminal domain previously not implicated in membrane targeting, and OCRL1 was also identified on LCVs in macrophages. Dd5P4 was catalytically active on LCVs and accumulated on LCVs harbouring wild-type but not Δ icmT mutant L. pneumophila . The N-terminal domain of OCRL1 bound L. pneumophila LpnE, a Sel1-like repeat protein involved in LCV formation, which localizes to LCVs and selectively binds PtdIns(3) P . Our results indicate that OCRL1 restricts intracellular growth of L. pneumophila and binds to LCVs in association with LpnE.     

Legionella pneumophila Subversion of Host Vesicular Transport by SidC Effector Proteins

Tethering proteins play a key role in vesicular transport, ensuring that cargo arrives at a specific destination. The bacterial effector protein SidC and its paralog SdcA have been described as tethering factors encoded by the intracellular pathogen Legionella pneumophila. Here, we demonstrate that SidC proteins are important for early events unique to maturation of vacuoles containing Legionella and discover monoubiquitination of Rab1 as a new SidC‐dependent activity. The crystal structure of the SidC N‐terminus revealed a novel fold that is important for function and could be involved in Legionella adaptations to evolutionarily divergent host cells it encounters in natural environments.      

Legionella hijacks the host Golgi-to-ER retrograde pathway for the association of Legionella-containing vacuole with the ER

Legionella pneumophila (L. pneumophila) is a gram-negative bacterium that replicates in a compartment that resembles the host endoplasmic reticulum (ER). To create its replicative niche, L. pneumophila manipulates host membrane traffic and fusion machineries. Bacterial proteins called Legionella effectors are translocated into the host cytosol and play a crucial role in these processes. In an early stage of infection, Legionella subverts ER-derived vesicles (ERDVs) by manipulating GTPase Rab1 to facilitate remodeling of the Legionella-containing vacuole (LCV). Subsequently, the LCV associates with the ER in a mechanism that remains elusive. In this study, we show that L. pneumophila recruits GTPases Rab33B and Rab6A, which regulate vesicle trafficking from the Golgi to the ER, to the LCV to promote the association of LCV with the ER. We found that recruitment of Rab6A to the LCV depends on Rab33B. Legionella effector SidE family proteins, which phosphoribosyl-ubiquitinate Rab33B, were found to be necessary for the recruitment of Rab33B to the LCV. Immunoprecipitation experiments revealed that L. pneumophila facilitates the interaction of Rab6 with ER-resident SNAREs comprising syntaxin 18, p31, and BNIP1, but not tethering factors including NAG, RINT-1, and ZW10, which are normally required for syntaxin 18-mediated fusion of Golgi-derived vesicles with the ER. Our results identified a Rab33B-Rab6A cascade on the LCV and the interaction of Rab6 with ER-resident SNARE proteins for the association of LCV with the ER and disclosed the unidentified physiological role of SidE family proteins.     

A Legionella pneumophila Effector Protein Encoded in a Region of Genomic Plasticity Binds to Dot/Icm-Modified Vacuoles

Legionella pneumophila is an opportunistic pathogen that can cause a severe pneumonia called Legionnaires'' disease. In the environment, L. pneumophila is found in fresh water reservoirs in a large spectrum of environmental conditions, where the bacteria are able to replicate within a variety of protozoan hosts. To survive within eukaryotic cells, L. pneumophila require a type IV secretion system, designated Dot/Icm, that delivers bacterial effector proteins into the host cell cytoplasm. In recent years, a number of Dot/Icm substrate proteins have been identified; however, the function of most of these proteins remains unknown, and it is unclear why the bacterium maintains such a large repertoire of effectors to promote its survival. Here we investigate a region of the L. pneumophila chromosome that displays a high degree of plasticity among four sequenced L. pneumophila strains. Analysis of GC content suggests that several genes encoded in this region were acquired through horizontal gene transfer. Protein translocation studies establish that this region of genomic plasticity encodes for multiple Dot/Icm effectors. Ectopic expression studies in mammalian cells indicate that one of these substrates, a protein called PieA, has unique effector activities. PieA is an effector that can alter lysosome morphology and associates specifically with vacuoles that support L. pneumophila replication. It was determined that the association of PieA with vacuoles containing L. pneumophila requires modifications to the vacuole mediated by other Dot/Icm effectors. Thus, the localization properties of PieA reveal that the Dot/Icm system has the ability to spatially and temporally control the association of an effector with vacuoles containing L. pneumophila through activities mediated by other effector proteins.     

In situ structure of the Legionella Dot/Icm type IV secretion system by electron cryotomography

Type IV secretion systems (T4SSs) are large macromolecular machines that translocate protein and DNA and are involved in the pathogenesis of multiple human diseases. Here, using electron cryotomography (ECT), we report the in situ structure of the Dot/Icm type IVB secretion system (T4BSS) utilized by the human pathogen Legionella pneumophila. This is the first structure of a type IVB secretion system, and also the first structure of any T4SS in situ. While the Dot/Icm system shares almost no sequence similarity with type IVA secretion systems (T4ASSs), its overall structure is seen here to be remarkably similar to previously reported T4ASS structures (those encoded by the R388 plasmid in Escherichia coli and the cag pathogenicity island in Helicobacter pylori). This structural similarity suggests shared aspects of mechanism. However, compared to the negative‐stain reconstruction of the purified T4ASS from the R388 plasmid, the L. pneumophila Dot/Icm system is approximately twice as long and wide and exhibits several additional large densities, reflecting type‐specific elaborations and potentially better structural preservation in situ.     

Fusion of Legionella pneumophila outer membrane vesicles with eukaryotic membrane systems is a mechanism to deliver pathogen factors to host cell membranes

The formation and release of outer membrane vesicles (OMVs) is a phenomenon observed in many bacteria, including Legionella pneumophila. During infection, this human pathogen primarily invades alveolar macrophages and replicates within a unique membrane‐bound compartment termed Legionella‐containing vacuole. In the current study, we analysed the membrane architecture of L. pneumophila OMVs by small‐angle X‐ray scattering and biophysically characterized OMV membranes. We investigated the interaction of L. pneumophila OMVs with model membranes by Förster resonance energy transfer and Fourier transform infrared spectroscopy. These experiments demonstrated the incorporation of OMV membrane material into liposomes composed of different eukaryotic phospholipids, revealing an endogenous property of OMVs to fuse with eukaryotic membranes. Cellular co‐incubation experiments showed a dose‐ and time‐dependent binding of fluorophore‐labelled OMVs to macrophages. Trypan blue quenching experiments disclosed a rapid internalization of OMVs into macrophages at 37 and 4°C. Purified OMVs induced tumour necrosis factor‐α production in human macrophages at concentrations starting at 300 ng ml^?1. Experiments on HEK293‐TLR2 and TLR4/MD‐2 cell lines demonstrated a dominance of TLR2‐dependent signalling pathways. In summary, we demonstrate binding, internalization and biological activity of L. pneumophila OMVs on human macrophages. Our data support OMV membrane fusion as a mechanism for the remote delivery of virulence factors to host cells.     

The Legionella pneumophila effector protein DrrA is a Rab1 guanine nucleotide-exchange factor

The intracellular pathogen Legionella pneumophila avoids fusion with lysosomes and subverts membrane transport from the endoplasmic reticulum to create an organelle that supports bacterial replication. Transport of endoplasmic reticulum-derived vesicles to the Legionella-containing vacuole (LCV) requires bacterial proteins that are translocated into host cells by a type IV secretion apparatus called Dot/Icm. Recent observations have revealed recruitment of the host GTPase Rab1 to the LCV by a process requiring the Dot/Icm system. Here, a visual screen was used to identify L. pneumophila mutants with defects in Rab1 recruitment. One of the factors identified in this screen was DrrA, a new Dot/Icm substrate protein translocated into host cells. We show that DrrA is a potent and highly specific Rab1 guanine nucleotide-exchange factor (GEF). DrrA can disrupt Rab1-mediated secretory transport to the Golgi apparatus by competing with endogenous exchange factors to recruit and activate Rab1 on plasma membrane-derived organelles. These data establish that intracellular pathogens have the capacity to directly modulate the activation state of a specific member of the Rab family of GTPases and thus further our understanding of the mechanisms used by bacterial pathogens to manipulate host vesicular transport.