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.     

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.     

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.     

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.     

Viewing Legionella pneumophila Pathogenesis through an Immunological Lens

Legionella pneumophila is the causative agent of the severe pneumonia Legionnaires' disease. L. pneumophila is ubiquitously found in freshwater environments, where it replicates within free-living protozoa. Aerosolization of contaminated water supplies allows the bacteria to be inhaled into the human lung, where L. pneumophila can be phagocytosed by alveolar macrophages and replicate intracellularly. The Dot/Icm type IV secretion system (T4SS) is one of the key virulence factors required for intracellular bacterial replication and subsequent disease. The Dot/Icm apparatus translocates more than 300 effector proteins into the host cell cytosol. These effectors interfere with a variety of cellular processes, thus enabling the bacterium to evade phagosome–lysosome fusion and establish an endoplasmic reticulum-derived Legionella-containing vacuole, which facilitates bacterial replication. In turn, the immune system has evolved numerous strategies to recognize intracellular bacteria such as L. pneumophila, leading to potent inflammatory responses that aid in eliminating infection. This review aims to provide an overview of L. pneumophila pathogenesis in the context of the host immune response.     

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.     

Phosphorylation signalling through the Legionella quorum sensing histidine kinases LqsS and LqsT converges on the response regulator LqsR

The environmental bacterium Legionella pneumophila is the causative agent of Legionnaires' disease, a life‐threatening pneumonia. For cell–cell communication the bacteria employ the autoinducer LAI‐1 (3‐hydroxypentadecane‐4‐one), which is produced and detected by the Lqs (Legionella quorum sensing) system. The system comprises the autoinducer synthase LqsA, the putative sensor kinases LqsS and LqsT, and the prototypic response regulator LqsR. Lqs‐regulated processes include L. pneumophila‐phagocyte interactions, production of extracellular filaments, and natural competence. Using biochemical approaches we show here that LqsS and LqsT are autophosphorylated by [γ‐³²P]‐ATP at a conserved histidine residue (H₂₀₀ or H₂₀₄) located in their cytoplasmic histidine kinase domain. Pull‐down assays revealed that LqsS and LqsT are bound by LqsR or phospho‐LqsR. Dependent on the conserved receiver domain aspartate (D₁₀₈), the response regulator prevented autophosphorylation of both sensor kinases by catalysing the dephosphorylation of phospho‐LqsS or phospho‐LqsT. Moreover, LqsR formed dimers upon phosphorylation at D₁₀₈ by either acetyl‐phosphate or phospho‐LqsT. Finally, upon heterologous production in Escherichia coli, LqsT (but not LqsS) was autophosphorylated by ATP, and LqsR prevented the autophosphorylation by catalysing the dephosphorylation of phospho‐LqsT. In summary, these results indicate that phosphorylation signalling through the Legionella quorum sensing histidine kinases LqsS and LqsT converges on the response regulator LqsR.     

Identification and functional characterization of K+ transporters encoded by Legionella pneumophila kup genes

Legionnaires' disease is an emerging, severe, pneumonia‐like illness caused by the Gram‐negative intracellular bacteria Legionella pneumophila, which are able to infect and replicate intracellularly in macrophages. Little is known regarding the mechanisms used by intracellular L. pneumophila for the acquisition of specific nutrients that are essential for bacterial replication. Here, we investigate three L. pneumophila genes with high similarity to the Escherichia coli K⁺ transporters. These three genes were expressed by L. pneumophila and have been designated kupA, kupB and kupC. Investigation using the L. pneumophila kup mutants revealed that kupA is involved in K⁺ acquisition during axenic growth. The kupA mutants replicated efficiently in rich axenic media, but poorly in a chemically defined medium. The kupA mutants were defective in the recruitment of polyubiquitinated proteins to the Legionella‐containing vacuole that is formed in macrophages and displayed an intracellular multiplication defect during the replication in Acanthamoeba castellanii and in mouse macrophages. We found that bafilomycin treatment of macrophages was able to rescue the growth defects of kupA mutants, but itdid not influence the replication of wild‐type bacteria. The defects identified in kupA mutants of L. pneumophila were complemented by the expression E. coli trkD/Kup gene in trans, a bona fide K⁺ transporter encoded by E. coli. Collectively, our data indicate that KupA is a functional K⁺ transporter expressed by L. pneumophila that facilitates the bacterial replication intracellularly and in nutrient‐limited conditions.     

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.     

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires'' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae''s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.     

The Machinery at Endoplasmic Reticulum-Plasma Membrane Contact Sites Contributes to Spatial Regulation of Multiple Legionella Effector Proteins

The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIα (PI4KIIIα) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIα was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis.     

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.     

Molecular Mimicry by an F-Box Effector of Legionella pneumophila Hijacks a Conserved Polyubiquitination Machinery within Macrophages and Protozoa

The ability of Legionella pneumophila to proliferate within various protozoa in the aquatic environment and in macrophages indicates a remarkable evolution and microbial exploitation of evolutionarily conserved eukaryotic processes. Ankyrin B (AnkB) of L. pneumophila is a non-canonical F-box-containing protein, and is the only known Dot/Icm-translocated effector of L. pneumophila essential for intra-vacuolar proliferation within both macrophages and protozoan hosts. We show that the F-box domain of AnkB and the ⁹L¹⁰P conserved residues are essential for intracellular bacterial proliferation and for rapid acquisition of polyubiquitinated proteins by the Legionella-containing vacuole (LCV) within macrophages, Dictyostelium discoideum, and Acanthamoeba. Interestingly, translocation of AnkB and recruitment of polyubiquitinated proteins in macrophages and Acanthamoeba is rapidly triggered by extracellular bacteria within 5 min of bacterial attachment. Ectopically expressed AnkB within mammalian cells is localized to the periphery of the cell where it co-localizes with host SKP1 and recruits polyubiquitinated proteins, which results in restoration of intracellular growth to the ankB mutant similar to the parental strain. While an ectopically expressed AnkB-⁹L¹⁰P/AA variant is localized to the cell periphery, it does not recruit polyubiquitinated proteins and fails to trans-rescue the ankB mutant intracellular growth defect. Direct in vivo interaction of AnkB but not the AnkB-⁹L¹⁰P/AA variant with the host SKP1 is demonstrated. Importantly, RNAi-mediated silencing of expression of SKP1 renders the cells non-permissive for intracellular proliferation of L. pneumophila. The role of AnkB in exploitation of the polyubiquitination machinery is essential for intrapulmonary bacterial proliferation in the mouse model of Legionnaires'' disease. Therefore, AnkB exhibits a novel molecular and functional mimicry of eukaryotic F-box proteins that exploits conserved polyubiquitination machinery for intracellular proliferation within evolutionarily distant hosts.     

Structural and functional study of Legionella pneumophila effector RavA

Legionella pneumophila is an intracellular pathogen that causes Legionnaire''s disease in humans. This bacterium can be found in freshwater environments as a free‐living organism, but it is also an intracellular parasite of protozoa. Human infection occurs when inhaled aerosolized pathogen comes into contact with the alveolar mucosa and replicates in alveolar macrophages. Legionella enters the host cell by phagocytosis and redirects the Legionella‐containing phagosomes from the phagocytic maturation pathway. These nascent phagosomes fuse with ER‐derived secretory vesicles and membranes forming the Legionella‐containing vacuole. Legionella subverts many host cellular processes by secreting over 300 effector proteins into the host cell via the Dot/Icm type IV secretion system. The cellular function for many Dot/Icm effectors is still unknown. Here, we present a structural and functional study of L. pneumophila effector RavA (Lpg0008). Structural analysis revealed that the RavA consists of four ~85 residue long α‐helical domains with similar folds, which show only a low level of structural similarity to other protein domains. The ~90 residues long C‐terminal segment is predicted to be natively unfolded. We show that during L. pneumophila infection of human cells, RavA localizes to the Golgi apparatus and to the plasma membrane. The same localization is observed when RavA is expressed in human cells. The localization signal resides within the C‐terminal sequence C⁴⁰⁹WTSFCGLF⁴¹⁷. Yeast‐two‐hybrid screen using RavA as bait identified RAB11A as a potential binding partner. RavA is present in L. pneumophila strains but only distant homologs are found in other Legionella species, where the number of repeats varies.     

Caenorhabditis is a metazoan host for Legionella

We investigated whether nematodes contribute to the persistence, differentiation and amplification of Legionella species in soil, an emerging source for Legionnaires' disease. Here we show that Legionella spp. colonize the intestinal tracts of Caenorhabditis nematodes leading to worm death. Susceptibility to Legionella is influenced by innate immune responses governed by the p38 mitogen‐activated protein kinase and insulin/insulin growth factor‐1 receptor signalling pathways. We also show that L. pneumophila colonizes the intestinal tract of nematodes cultivated in soil. To distinguish between transient infection and persistence, plate‐fed and soil‐extracted nematodes‐fed fluorescent strains of L. pneumophila were analysed. Bacteria replicated within the nematode intestinal tract, did not invade surrounding tissue, and were excreted as differentiated forms that were transmitted to offspring. Interestingly, the ultrastructural features of the differentiated bacterial forms were similar to cyst‐like forms observed within protozoa, amoeba and mammalian cell lines. While intestinal colonization of L. pneumophila dotA and icmT mutant strains did not alter the survival rate of nematodes in comparison to wild‐type strains, nematodes colonized with the dot/icm mutant strains exhibited significantly increased levels of germline apoptosis. Taken together, these studies show that nematodes may serve as natural hosts for these organisms and thereby contribute to their dissemination in the environment and suggest that the remarkable ability of L. pneumophila to subvert host cell signalling and evade mammalian immune responses evolved through the natural selection associated with cycling between protozoan and metazoan hosts.     

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.