Legionella-containing Vacuole Research Articles

The intracellular growth of the vacuolar pathogen Legionella pneumophila is dependent on the acyl chain composition of host membranes

Legionella pneumophila is an accidental human bacterial pathogen that infects and replicates within alveolar macrophages causing a severe atypical pneumonia known as Legionnaires’ disease. As a prototypical vacuolar pathogen L. pneumophila establishes a unique endoplasmic reticulum (ER)-derived organelle within which bacterial replication takes place. Bacteria-derived proteins are deposited in the host cytosol and in the lumen of the pathogen-occupied vacuole via a type IVb (T4bSS) and a type II (T2SS) secretion system respectively. These secretion system effector proteins manipulate multiple host functions to facilitate intracellular survival of the bacteria. Subversion of host membrane glycerophospholipids (GPLs) by the internalized bacteria via distinct mechanisms feature prominently in trafficking and biogenesis of the Legionella-containing vacuole (LCV). Conventional GPLs composed of a glycerol backbone linked to a polar headgroup and esterified with two fatty acids constitute the bulk of membrane lipids in eukaryotic cells. The acyl chain composition of GPLs dictates phase separation of the lipid bilayer and therefore determines the physiochemical properties of biological membranes - such as membrane disorder, fluidity and permeability. In mammalian cells, fatty acids esterified in membrane GPLs are sourced endogenously from de novo synthesis or via internalization from the exogenous pool of lipids present in serum and other interstitial fluids. Here, we exploited the preferential utilization of exogenous fatty acids for GPL synthesis by macrophages to reprogram the acyl chain composition of host membranes and investigated its impact on LCV homeostasis and L. pneumophila intracellular replication. Using saturated fatty acids as well as cis- and trans- isomers of monounsaturated fatty acids we discovered that under conditions promoting lipid packing and membrane rigidification L. pneumophila intracellular replication was significantly reduced. Palmitoleic acid – a C16:1 monounsaturated fatty acid – that promotes membrane disorder when enriched in GPLs significantly increased bacterial replication within human and murine macrophages but not in axenic growth assays. Lipidome analysis of infected macrophages showed that treatment with exogenous palmitoleic acid resulted in membrane acyl chain reprogramming in a manner that promotes membrane disorder and live-cell imaging revealed that the consequences of increasing membrane disorder impinge on several LCV homeostasis parameters. Collectively, we provide experimental evidence that L. pneumophila replication within its intracellular niche is a function of the lipid bilayer disorder and hydrophobic thickness.

Dual Control of Host Actin Polymerization by a Legionella Effector Pair

Host actin cytoskeleton is often targeted by pathogenic bacteria through the secretion of effectors. Legionella pneumophila virulence relies on the injection of the largest known arsenal of bacterial proteins, over 300 Dot/Icm type 4 secretion system effectors, into the host cytosol. Here, we define the functional interactions between VipA and LegK2, two effectors with antagonistic activities towards actin polymerization that have been proposed to interfere with the endosomal pathway. We confirmed the prominent role of LegK2 effector in Legionella infection, as the deletion of legK2 results in defects in the inhibition of actin polymerization at the Legionella-containing vacuole, as well as in endosomal escape of bacteria and subsequent intracellular replication. More importantly, we observed the restoration of the ΔlegK2 mutant defects, upon deletion of vipA gene, making LegK2/VipA a novel example of effector-effector suppression pair that targets the actin cytoskeleton and whose functional interaction impacts L. pneumophila virulence. We demonstrated that LegK2 and VipA do not modulate each other’s activity in a “metaeffector” relationship. Instead, the antagonistic activities of the LegK2/VipA effector pair would target different substrates, Arp2/3 for LegK2 and G-actin for VipA, to temporally control actin polymerization at the LCV and interfere with phagosome maturation and endosome recycling, thus contributing to the intracellular life cycle of the bacterium. Strikingly, the functional interaction between LegK2 and VipA is consolidated by an evolutionary history that has refined the best effector repertoire for the benefit of L. pneumophila virulence.

Cross-family small GTPase ubiquitination by the intracellular pathogen Legionella pneumophila.

The intracellular bacterial pathogen Legionella pneumophila (L.p.) manipulates eukaryotic host ubiquitination machinery to form its replicative vacuole. While nearly 10% of L.p.'s ∼330 secreted effector proteins are ubiquitin ligases or deubiquitinases, a comprehensive measure of temporally resolved changes in the endogenous host ubiquitinome during infection has not been undertaken. To elucidate how L.p. hijacks host cell ubiquitin signaling, we generated a proteome-wide analysis of changes in protein ubiquitination during infection. We discover that L.p. infection increases ubiquitination of host regulators of subcellular trafficking and membrane dynamics, most notably ∼40% of mammalian Ras superfamily small GTPases. We determine that these small GTPases undergo nondegradative ubiquitination at the Legionella-containing vacuole (LCV) membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central role in cross-family small GTPase ubiquitination, and that these effectors function upstream of SidE family ligases in the polyubiquitination and retention of GTPases in the LCV membrane. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. Our findings position L.p. as a tool to better understand how small GTPases can be regulated by ubiquitination in uninfected contexts.

Ubiquitination of Sec22b by a novel Legionella pneumophila ubiquitin E3 ligase.

Legionella pneumophila is a facultative intracellular pathogen that causes legionellosis. The key to its virulence is the delivery of hundreds of effector proteins into host cells via the defective in organelle trafficking/intracellular multiplication type IV secretion system. These effectors modulate numerous host signaling pathways to create a niche called the Legionella-containing vacuole (LCV) permissive for its intracellular replication. Previous investigation revealed that exploitation of the host ubiquitin system is among the most important strategies used by L. pneumophila to coopt host processes for its benefit. Here, we show that the effector Legionella ubiquitin ligase gene 15 (Lug15) (Lpg2327), which has no detectable homology with any enzyme involved in ubiquitin signaling, is an E3 ligase. In L. pneumophila-infected cells, Lug15 is localized on the LCV and impacts its association with polyubiquitinated proteins. We also demonstrate that Sec22b is ubiquitinated and recruited to the LCV by Lug15. Thus, our results establish Lug15 as a novel E3 ligase that functions to recruit a SNARE protein to remodel the L. pneumophila phagosome.IMPORTANCEProtein ubiquitination is one of the most important post-translational modifications that plays critical roles in the regulation of a wide range of eukaryotic signaling pathways. Many successful intracellular bacterial pathogens can hijack host ubiquitination machinery through the action of effector proteins that are injected into host cells by secretion systems. Legionella pneumophila is the etiological agent of legionellosis that is able to survive and replicate in various host cells. The defective in organelle trafficking (Dot)/intracellular multiplication (Icm) type IV secretion system of L. pneumophila injects over 330 effectors into infected cells to create an optimal environment permissive for its intracellular proliferation. To date, at least 26 Dot/Icm substrates have been shown to manipulate ubiquitin signaling via diverse mechanisms. Among these, 14 are E3 ligases that either cooperate with host E1 and E2 enzymes or adopt E1/E2-independent catalytic mechanisms. In the present study, we demonstrate that the L. pneumophila effector Legionella ubiquitin ligase gene 15 (Lug15) is a novel ubiquitin E3 ligase. Lug15 is involved in the remodeling of LCV with polyubiquitinated species. Moreover, Lug15 catalyzes the ubiquitination of host SNARE protein Sec22b and mediates its recruitment to the LCV. Ubiquitination of Sec22b by Lug15 promotes its noncanonical pairing with plasma membrane-derived syntaxins (e.g., Stx3). Our study further reveals the complexity of strategies utilized by L. pneumophila to interfere with host functions by hijacking host ubiquitin signaling.

Human GBP1 facilitates the rupture of the Legionella-containing vacuole and inflammasome activation.

Inflammasomes are essential for host defense against intracellular bacterial pathogens like Legionella, as they activate caspases, which promote cytokine release and cell death to control infection. In mice, interferon (IFN) signaling promotes inflammasome responses against bacteria by inducing a family of IFN-inducible GTPases known as guanylate-binding proteins (GBPs). Within murine macrophages, IFN promotes the rupture of the Legionella-containing vacuole (LCV), while GBPs are dispensable for this process. Instead, GBPs facilitate the lysis of cytosol-exposed Legionella. In contrast, the functions of IFN and GBPs in human inflammasome responses to Legionella are poorly understood. We show that IFN-γ enhances inflammasome responses to Legionella in human macrophages. Human GBP1 is required for these IFN-γ-driven inflammasome responses. Furthermore, GBP1 co-localizes with Legionella and/or LCVs in a type IV secretion system (T4SS)-dependent manner and promotes damage to the LCV, which leads to increased exposure of the bacteria to the host cell cytosol. Thus, our findings reveal species- and pathogen-specific differences in how GBPs function to promote inflammasome responses.

The Sde phosphoribosyl–linked ubiquitin transferases protect the Legionella pneumophila vacuole from degradation by the host

Legionella pneumophila grows intracellularly within the membrane-bound Legionella-containing vacuole (LCV) established by proteins translocated via the bacterial type IV secretion system (T4SS). The Sde family, one such group of translocated proteins, catalyzes phosphoribosyl-ubiquitin (pR-Ub) modification of target substrates. Mutational loss of the entire Sde family results in small defects in intracellular growth, making it difficult to identify a clear role for this posttranslational modification in supporting the intracellular lifestyle. Therefore, mutations that aggravate the loss of sde genes and caused intracellular growth defects were identified, providing a mechanistic connection between Sde function and vacuole biogenesis. These double mutants drove the formation of LCVs that showed vacuole disintegration within 2 h of bacterial contact. Sde proteins appeared critical for blocking access of membrane-disruptive early endosomal membrane material to the vacuole, as RNAi depletion of endosomal pathway components partially restored LCV integrity. The role of Sde proteins in preventing host degradation of the LCV was limited to the earliest stages of infection. The time that Sde proteins could prevent vacuole disruption, however, was extended by deletion of sidJ, which encodes a translocated protein that inactivates Sde protein active sites. These results indicate that Sde proteins act as temporally regulated vacuole guards during the establishment of the replication niche, possibly by constructing a physical barrier that blocks access of disruptive host compartments during the earliest steps of LCV biogenesis.

Phosphoribosyl-linked serine ubiquitination of USP14 by the SidE family effectors of Legionella excludes p62 from the bacterial phagosome

Xenophagy is an evolutionarily conserved host defensive mechanism to eliminate invading microorganisms through autophagic machinery. The intracellular bacterial pathogen Legionella pneumophila can avoid clearance by the xenophagy pathway via the actions of multiple Dot/Icm effector proteins. Previous studies have shown that p62, an adaptor protein involved in xenophagy signaling, is excluded from Legionella-containing vacuoles (LCVs). Such defects are attributed to the multifunctional SidE family effectors (SidEs) that exhibit classic deubiquitinase (DUB) and phosphoribosyl ubiquitination (PR-ubiquitination) activities, yet the mechanism remains elusive. In the present study, we demonstrate that the host DUB USP14 is PR-ubiquitinated by SidEs at multiple serine residues, which impairs its DUB activity and its interactions with p62. The exclusion of p62 from the bacterial phagosome requires the ubiquitin ligase but not the DUB activity of SidEs. These results reveal that PR-ubiquitination of USP14 by SidEs contributes to the evasion of xenophagic clearance by L.pneumophila.

Membrane-dependent actin polymerization mediated by the Legionella pneumophila effector protein MavH.

L. pneumophila propagates in eukaryotic cells within a specialized niche, the Legionella-containing vacuole (LCV). The infection process is controlled by over 330 effector proteins delivered through the type IV secretion system. In this study, we report that the Legionella MavH effector localizes to endosomes and remodels host actin cytoskeleton in a phosphatidylinositol 3-phosphate (PI(3)P) dependent manner when ectopically expressed. We show that MavH recruits host actin capping protein (CP) and actin to the endosome via its CP-interacting (CPI) motif and WH2-like actin-binding domain, respectively. In vitro assays revealed that MavH stimulates actin assembly on PI(3)P-containing liposomes causing their tubulation. In addition, the recruitment of CP by MavH negatively regulates F-actin density at the membrane. We further show that, in L. pneumophila-infected cells, MavH appears around the LCV at the very early stage of infection and facilitates bacterium entry into the host. Together, our results reveal a novel mechanism of membrane tubulation induced by membrane-dependent actin polymerization catalyzed by MavH that contributes to the early stage of L. pneumophila infection by regulating host actin dynamics.

Effector-mediated subversion of proteasome activator (PA)28αβ enhances host defense against Legionella pneumophila under inflammatory and oxidative stress conditions.

Legionella pneumophila is a natural pathogen of amoebae that causes Legionnaires' Disease in immunocompromised individuals via replication within macrophages. L. pneumophila virulence and intracellular replication hinges on hundreds of Dot/Icm-translocated effector proteins, which are essential for biogenesis of the replication-permissive Legionella-containing vacuole (LCV). However, effector activity can also enhance mammalian host defense via effector-triggered immunity. The L. pneumophila effector LegC4 is important for virulence in amoebae but enhances host defense against L. pneumophila in the mouse lung and, uniquely, within macrophages activated with either tumor necrosis factor (TNF) or interferon (IFN)-γ. The mechanism by which LegC4 potentiates cytokine-mediated host defense in macrophages is unknown. Here, we found that LegC4 enhances cytokine-mediated phagolysosomal fusion with Legionella-containing vacuole (LCV) and binds host proteasome activator (PA)28α, which forms a heterooligomer with PA28β to facilitate ubiquitin-independent proteasomal degradation of oxidant-damaged (carbonylated) proteins. We found that oxidative stress was sustained in the presence of LegC4 and that the LegC4 restriction phenotype was relieved in PA28αβ-deficient macrophages and in the lungs of mice in vivo. Our data also show that oxidative stress is sufficient for LegC4-mediated restriction in macrophages producing PA28αβ. PA28αβ has been traditionally associated with antigen presentation; however, our data support a novel mechanism whereby effector-mediated subversion of PA28αβ enhances cell-autonomous host defense against L. pneumophila under inflammatory and oxidative stress conditions. This work provides a solid foundation to evaluate induced proteasome regulators as mediators of innate immunity.

The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism of Legionella pneumophila.

The amoeba-resistant bacterium Legionella pneumophila causes Legionnaires' disease and employs a type IV secretion system (T4SS) to replicate in the unique, ER-associated Legionella-containing vacuole (LCV). The large fusion GTPase Sey1/atlastin is implicated in ER dynamics, ER-derived lipid droplet (LD) formation, and LCV maturation. Here, we employ cryo-electron tomography, confocal microscopy, proteomics, and isotopologue profiling to analyze LCV-LD interactions in the genetically tractable amoeba Dictyostelium discoideum. Dually fluorescence-labeled D. discoideum producing LCV and LD markers revealed that Sey1 as well as the L. pneumophila T4SS and the Ran GTPase activator LegG1 promote LCV-LD interactions. In vitro reconstitution using purified LCVs and LDs from parental or Δsey1 mutant D. discoideum indicated that Sey1 and GTP promote this process. Sey1 and the L. pneumophila fatty acid transporter FadL were implicated in palmitate catabolism and palmitate-dependent intracellular growth. Taken together, our results reveal that Sey1 and LegG1 mediate LD- and FadL-dependent fatty acid metabolism of intracellular L. pneumophila.

Investigating the Role of Bacterial Long-Chain Polyphosphates in Macrophages during Legionella pneumophilaInfection

Abstract Up to 10% of community-acquired pneumonia cases are identified as Legionnaires’ Disease, caused by the pathogen Legionella pneumophila. L. pneumophila naturally resides in freshwater systems and becomes pathogenic when man-made plumbing systems grow biofilm and amoeba if not maintained. Hosts are accidentally infected by L. pneumophilavia aerosol inhalation into the respiratory system. L. pneumophila primarily reside in human alveolar macrophages, entering and replicating via the Legionella-containing vacuole. This vacuole will also release and recruit hundreds of effector molecules to prevent itself from being phagocytosed and disguise itself as a host cellular component. Polyphosphates are highly conserved molecules found in essentially every cell performing essential functions. Long-chain polyphosphates, containing hundreds of phosphate units, are produced by bacteria to perform functions critical to their survival in hosts. We have previously found that long-chain polyphosphates produced by E. coli interfere with the host innate response against infection. Here, we show that the infection of L. pneumophila and synthetic long-chain bacterial polyphosphates interfere with the inflammatory response in macrophages. We utilized a mouse model for in vitro infection of L. pneumophila in bone-marrow-derived macrophages, showing a modulated inflammatory cytokine response and cell death. Additionally, the addition of S. cerevisiae derived exopolyphosphatase, which degrades long-chain polyphosphates, decreases inflammation in vitro.

Eat or Be Eaten: Strategies Used by Legionella to Acquire Host-Derived Nutrients and Evade Lysosomal Degradation.

To replicate within host cells, bacterial pathogens must acquire host-derived nutrients while avoiding degradative antimicrobial pathways. Fundamental insights into bacterial pathogenicity have been revealed by bacteria of the genus Legionella, which naturally parasitize free-living protozoa by establishing a membrane-bound replicative niche termed the Legionella-containing vacuole (LCV). Biogenesis of the LCV and intracellular replication rely on rapid evasion of the endocytic pathway and acquisition of host-derived nutrients, much of which is mediated by bacterial effector proteins translocated into host cells by a Dot/Icm type IV secretion system. Billions of years of co-evolution with eukaryotic hosts and broad host tropism have resulted in expansion of the Legionella genome to accommodate a massive repertoire of effector proteins that promote LCV biogenesis, safeguard the LCV from endolysosomal maturation, and mediate the acquisition of host nutrients. This minireview is focused on the mechanisms by which an ancient intracellular pathogen leverages effector proteins and hijacks host cell biology to obtain essential host-derived nutrients and prevent lysosomal degradation.

Pathogen-defending deubiquitinase possesses distinct specificity towards K6-polyubiquitination.

The bacterial pathogen Legionella pneumophila encodes numerous effectors to manipulate host ubiquitin signaling. Recently, Warren et al. revealed the structural basis of K6-polyubiquitination recognition by Legionella deubiquitinase LotA, while validating its potential as an enzymatic tool to study linkage-specific ubiquitination. During Legionella infection, LotA counteracts valosin-containing protein (VCP) recruitment to the Legionella-containing vacuole.

Legionella longbeachae Regulates the Association of Polyubiquitinated Proteins on Bacterial Phagosome with Multiple Deubiquitinases.

Legionella spp. are the causative agents of a severe pneumonia known as Legionnaires' disease. Upon being engulfed by host cells, these environmental bacteria replicate intracellularly in a plasma membrane-derived niche termed Legionella-containing vacuole (LCV) in a way that requires the defective in organelle trafficking/intracellular multiplication (Dot/Icm) protein transporter. Our understanding of interactions between Legionella and its hosts was mostly based on studies of Legionella pneumophila. In this study, we found that the LCVs created by virulent Legionella longbeachae are similarly decorated by polyubiquitinated proteins to those formed by L. pneumophila and that the ubiquitin-proteasome system (UPS) is required for optimal intracellular growth of L. longbeachae. Furthermore, we utilized bioinformatics methods and the ubiquitin-vinylmethyl ester probe to obtain potential deubiquitinases (DUBs) encoded by L. longbeachae. These efforts led to the identification of 9 L. longbeachae DUBs that displayed distinct specificity toward ubiquitin chain types. Among these, LLO_1014 and LLO_2238 are associated with the LCVs and impact the accumulation of polyubiquitinated species on the bacterial phagosome. Moreover, LLO_1014 and LLO_2238 could fully restore the phenotypes associated with Δceg23 (lotB) and Δlem27 (lotC) mutants of L. pneumophila, indicating that these DUBs have similar functions. Together, these results reveal that L. longbeachae uses multiple DUBs to construct an intracellular niche for its replication. IMPORTANCE Legionella spp. are opportunistic intracellular bacterial pathogens that cause Legionnaires' disease. Legionella utilizes the Dot/Icm type IV secretion system to deliver effector protein into host cells to modulate various cellular functions. At least 26 L. pneumophila effectors are known to hijack the host ubiquitin system via diverse mechanisms. L. longbeachae is the second leading cause of Legionnaires' disease worldwide. However, our knowledge about the interactions between L. longbeachae and its hosts is very limited. Here, we found that, similar to L. pneumophila infection, the host ubiquitin proteasome system is also important for the intracellular replication of L. longbeachae. In addition, the bacterial phagosomes harboring L. longbeachae are enriched with polyubiquitinated proteins in a Dot/Icm system-dependent manner. We further identified 9 L. longbeachae proteins that function as DUBs with distinct ubiquitin chain specificity. Of note, several of the phagosome-associated L. longbeachae DUBs regulate the recruitment of polyubiquitinated proteins to the LCV.

Legionella longbeachae effector protein RavZ inhibits autophagy and regulates phagosome ubiquitination during infection.

Legionella organisms are ubiquitous environmental bacteria that are responsible for human Legionnaires' disease, a fatal form of severe pneumonia. These bacteria replicate intracellularly in a wide spectrum of host cells within a distinct compartment termed the Legionella-containing vacuole (LCV). Effector proteins translocated by the Dot/Icm apparatus extensively modulate host cellular functions to aid in the biogenesis of the LCV and intracellular proliferation. RavZ is an L. pneumophila effector that functions as a cysteine protease to hydrolyze lipidated LC3, thereby compromising the host autophagic response to bacterial infection. In this study, we characterized the RavZ (RavZLP) ortholog in L. longbeachae (RavZLLO), the second leading cause of Legionella infections in the world. RavZLLO and RavZLP share approximately 60% sequence identity and a conserved His-Asp-Cys catalytic triad. RavZLLO is recognized by the Dot/Icm systems of both L. pneumophila and L. longbeachae. Upon translocation into the host, it suppresses autophagy signaling in cells challenged with both species, indicating the functional redundancy of RavZLLO and RavZLP. Additionally, ectopic expression of RavZLLO but not RavZLP in mammalian cells reduces the levels of cellular polyubiquitinated and polyneddylated proteins. Consistent with this process, RavZLLO regulates the accumulation of polyubiquitinated species on the LCV during L. longbeachae infection.

Proteomic analysis of Acanthamoeba castellanii response to Legionella pneumophila infection.

Legionella pneumophila is an opportunistic pathogen responsible for Legionnaires' disease or Legionellosis. This bacterium is found in the environment interacting with free-living amoebae such as Acanthamoeba castellanii. Until now, proteomic analyses have been done in amoebae infected with L. pneumophila but focused on the Legionella-containing vacuole. In this study, we propose a global proteomic analysis of the A. castellanii proteome following infection with L. pneumophila wild-type (WT) or with an isogenic ΔdotA mutant strain, which is unable to replicate intracellularly. We found that infection with L. pneumophila WT leads to reduced levels of A. castellanii proteins associated with lipid homeostasis/metabolism, GTPase regulation and kinase. The levels of organelle-associated proteins were also decreased during infection. L. pneumophila WT infection leads to increase levels of proteins associated with proteins polyubiquitination, folding or degradation and antioxidant activities. This study reinforces our knowledge of this too-little-explored but so fundamental interaction between L. pneumophila and A. castellanii, to understand how the bacterium could resist amoeba digestion.

Legionella- and host-driven lipid flux at LCV-ER membrane contact sites promotes vacuole remodeling.

Legionella pneumophila replicates in macrophages and amoeba within a unique compartment, the Legionella-containing vacuole (LCV). Hallmarks of LCV formation are the phosphoinositide lipid conversion from PtdIns(3)P to PtdIns(4)P, fusion with ER-derived vesicles and a tight association with the ER. Proteomics of purifiedLCVs indicate the presence of membrane contact sites (MCS)proteins possibly implicated in lipid exchange. Using duallyfluorescence-labeled Dictyostelium discoideum amoeba, we revealthat VAMP-associated protein (Vap) and the PtdIns(4)P 4-phosphatase Sac1 localize to the ER, and Vap also localizes to the LCV membrane. Furthermore, Vap as well as Sac1 promote intracellular replication of L.pneumophila and LCV remodeling. Oxysterol binding proteins (OSBPs) preferentially localize to the ER (OSBP8) or the LCV membrane (OSBP11), respectively, and restrict (OSBP8) or promote (OSBP11) bacterial replication and LCV expansion. The sterol probes GFP-D4H* and filipin indicate that sterols are rapidly depleted from LCVs, while PtdIns(4)P accumulates. In addition to Sac1, the PtdIns(4)P-subverting L.pneumophila effector proteins LepB and SidC also support LCV remodeling. Taken together, the Legionella- and host cell-driven PtdIns(4)P gradient at LCV-ER MCSs promotes Vap-, OSBP- and Sac1-dependent pathogen vacuole maturation.

Imaging Flow Cytometry of Legionella-Containing Vacuoles in Intact and Homogenized Wild-Type and Mutant Dictyostelium.

The causative agent of asevere pneumonia termed"Legionnaires' disease", Legionella pneumophila, replicates within protozoan and mammalian phagocytes in a specialized intracellular compartment called the Legionella-containing vacuole (LCV). This compartment does not fuse with bactericidal lysosomes but communicates extensively with several cellular vesicle trafficking pathways and eventually associates tightly with the endoplasmic reticulum. In order to comprehend in detail the complex process of LCV formation, the identification and kinetic analysis of cellular trafficking pathway markers on the pathogen vacuole are crucial. This chapter describes imaging flow cytometry (IFC)-based methods for the objective, quantitative and high-throughput analysis of different fluorescently tagged proteins or probes on the LCV. To this end, we use the haploid amoeba Dictyostelium discoideum as an infection model for L. pneumophila, to analyze either fixed intact infected host cells or LCVs from homogenized amoebae. Parental strains and isogenic mutant amoebae are compared in order to determine the contribution of a specific host factor to LCV formation. The amoebae simultaneously produce two different fluorescently tagged probes enabling tandem quantification of two LCV markers in intact amoebae or the identification of LCVs using one probe and quantification of the other probe in host cell homogenates. The IFC approach allows rapid generation of statistically robust data from thousands of pathogen vacuoles and can be applied to other infection models.

Mechanism of Lys6 poly-ubiquitin specificity by the L. pneumophila deubiquitinase LotA

The versatility of ubiquitination to control vast domains of eukaryotic biology is due, in part, to diversification through differently linked poly-ubiquitin chains. Deciphering signaling roles for some chain types, including those linked via K6, has been stymied by a lack of specificity among the implicated regulatory proteins. Forged through strong evolutionary pressures, pathogenic bacteria have evolved intricate mechanisms to regulate host ubiquitin during infection. Herein, we identify and characterize a deubiquitinase domain of the secreted effector LotA from Legionella pneumophila that specifically regulates K6-linked poly-ubiquitin. We demonstrate the utility of LotA for studying K6 poly-ubiquitin signals. We identify the structural basis of LotA activation and poly-ubiquitin specificity and describe an essential "adaptive" ubiquitin-binding domain. Without LotA activity during infection, the Legionella-containing vacuole becomes decorated with K6 poly-ubiquitin as well as the AAA ATPase VCP/p97/Cdc48. We propose that LotA's deubiquitinase activity guards Legionella-containing vacuole components from ubiquitin-dependent extraction.

Atypical Legionella GTPase effector hijacks host vesicular transport factor p115 to regulate host lipid droplet.

The intracellular bacterial pathogen Legionella pneumophila uses hundreds of effector proteins to manipulate multiple processes of the host cells to establish a replicative niche known as Legionella-containing vacuole (LCV). Biogenesis of the LCV has been known to depend on host small guanosine triphosphatases (GTPases), but whether bacterial effector GTPases are also involved remains unknown. Here, we show that an ankyrin repeat containing effector LegA15 localizes directly in host lipid droplets (LDs), leading to Golgi apparatus fragmentation of the host cells by hijacking the host vesicular transport factor p115. LegA15 is a GTPase with a unique catalytic mechanism, unlike any eukaryotic small GTPases. Moreover, the effector LegA15 co-opts p115 to modulate homeostasis of the host LDs in its GTPase-dependent manner. Together, our data reveal that an atypical GTPase effector regulates the host LDs through impeding the vesicle secretion system of the host cells for intracellular life cycle of Legionella.