Legionella-containing Vacuole Research Articles

A bacterial effector deubiquitinase specifically hydrolyses linear ubiquitin chains to inhibit host inflammatory signalling.

Linear ubiquitin (Ub) chains regulate many cellular processes, including NF-κB immune signalling. Pathogenic bacteria have evolved to secrete effector proteins that harbour deubiquitinase activity into host cells to disrupt host ubiquitination signalling. All previously identified effector deubiquitinases hydrolyse isopeptide-linked polyubiquitin (polyUb). It has been a long-standing question whether bacterial pathogens have evolved an effector deubiquitinase to directly cleave linear Ub chains. In this study, we performed extensive screening of bacterial pathogens and found that Legionella pneumophila-the causative agent of human Legionnaire's disease-encodes an effector protein, RavD, which harbours deubiquitinase activity exquisitely specific for linear Ub chains. RavD hydrolyses linear Ub chains but not any type of isopeptide-linked polyUb. The crystal structure of RavD with linear diubiquitin reveals that RavD adopts a papain-like fold with a Cys-His-Ser catalytic triad. The Ub-binding surface and specific interacting residues in RavD determine its specificity for Met1 linkages. RavD prevents the accumulation of linear Ub chains on Legionella-containing vacuoles established by the pathogen in host cells to inhibit the NF-κB pathway during infection. This study identified a unique linear Ub chain-specific effector deubiquitinase and indicates its potential application as a tool to dissect linear polyUb-mediated signalling in mammalian cells.

The Legionella effector RavD binds phosphatidylinositol-3-phosphate and helps suppress endolysosomal maturation of the Legionella-containing vacuole

Upon phagocytosis into macrophages, the intracellular bacterial pathogen Legionella pneumophila secretes effector proteins that manipulate host cell components, enabling it to evade lysosomal degradation. However, the bacterial proteins involved in this evasion are incompletely characterized. Here we show that the L. pneumophila effector protein RavD targets host membrane compartments and contributes to the molecular mechanism the pathogen uses to prevent encounters with lysosomes. Protein-lipid binding assays revealed that RavD selectively binds phosphatidylinositol-3-phosphate (PI(3)P) in vitro We further determined that a C-terminal RavD region mediates the interaction with PI(3)P and that this interaction requires Arg-292. In transiently transfected mammalian cells, mCherry-RavD colocalized with the early endosome marker EGFP-Rab5 as well as the PI(3)P biosensor EGFP-2×FYVE. However, treatment with the phosphoinositide 3-kinase inhibitor wortmannin did not disrupt localization of mCherry-RavD to endosomal compartments, suggesting that RavD's interaction with PI(3)P is not necessary to anchor RavD to endosomal membranes. Using superresolution and immunogold transmission EM, we observed that, upon translocation into macrophages, RavD was retained onto the Legionella-containing vacuole and was also present on small vesicles adjacent to the vacuole. We also report that despite no detectable effects on intracellular growth of L. pneumophila within macrophages or amebae, the lack of RavD significantly increased the number of vacuoles that accumulate the late endosome/lysosome marker LAMP-1 during macrophage infection. Together, our findings suggest that, although not required for intracellular replication of L. pneumophila, RavD is a part of the molecular mechanism that steers the Legionella-containing vacuole away from endolysosomal maturation pathways.

The Legionella effector LtpM is a new type of phosphoinositide-activated glucosyltransferase

Legionella pneumophila causes Legionnaires' disease, a severe form of pneumonia. L. pneumophila translocates more than 300 effectors into host cells via its Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) type IV secretion system to enable its replication in target cells. Here, we studied the effector LtpM, which is encoded in a recombination hot spot in L. pneumophila Paris. We show that a C-terminal phosphoinositol 3-phosphate (PI3P)-binding domain, also found in otherwise unrelated effectors, targets LtpM to the Legionella-containing vacuole and to early and late endosomes. LtpM expression in yeast caused cytotoxicity. Sequence comparison and structural homology modeling of the N-terminal domain of LtpM uncovered a remote similarity to the glycosyltransferase (GT) toxin PaTox from the bacterium Photorhabdus asymbiotica; however, instead of the canonical DxD motif of GT-A type glycosyltransferases, essential for enzyme activity and divalent cation coordination, we found that a DxN motif is present in LtpM. Using UDP-glucose as sugar donor, we show that purified LtpM nevertheless exhibits glucohydrolase and autoglucosylation activity in vitro and demonstrate that PI3P binding activates LtpM's glucosyltransferase activity toward protein substrates. Substitution of the aspartate or the asparagine in the DxN motif abolished the activity of LtpM. Moreover, whereas all glycosyltransferase toxins and effectors identified so far depend on the presence of divalent cations, LtpM is active in their absence. Proteins containing LtpM-like GT domains are encoded in the genomes of other L. pneumophila isolates and species, suggesting that LtpM is the first member of a novel family of glycosyltransferase effectors employed to subvert hosts.

Quantitative Imaging Flow Cytometry of Legionella-Containing Vacuoles in Dually Fluorescence-Labeled Dictyostelium.

Legionella pneumophila enters and replicates within protozoan and mammalian phagocytes by forming through a conserved mechanism a specialized intracellular compartment termed the Legionella-containing vacuole (LCV). This compartment avoids fusion with bactericidal lysosomes but communicates extensively with different cellular vesicle trafficking pathways and ultimately interacts closely with the endoplasmic reticulum. In order to delineate the process of pathogen vacuole formation and to better understand L. pneumophila virulence, an analysis of markers of the different trafficking pathways on the pathogen vacuole is crucial. Here, we describe a method for rapid, objective and quantitative analysis of different fluorescently tagged proteins or probes on the LCV. To this end, we employ an imaging flow cytometry approach and use the D. discoideum -L. pneumophila infection model. Imaging flow cytometry enables quantification of many different parameters by fluorescence microscopy of cells in flow, rapidly producing statistically robust data from thousands of cells. We also describe the generation of D. discoideum strains simultaneously producing two different fluorescently tagged probes that enable visualization of compartments and processes in parallel. The quantitative imaging flow technique can be corroborated and enhanced by laser scanning confocal microscopy.

Perturbation of Legionella Cell Infection by RNA Interference.

Legionella pneumophila is a facultative intracellular bacterium, which grows in amoebae as well as in macrophages and epithelial cells. Depletion of genes of interest by RNA interference (RNAi) has proven to be a robust and economic technique to study L. pneumophila-host cell interactions. Predesigned and often validated double-stranded (ds) RNA oligonucleotides that silence specific genes are commercially available. RNAi results in a reduced level of distinct proteins, which allows studying the specific role of host cell components involved in L. pneumophila infection. Here, we describe how to assess RNAi-mediated protein depletion efficiency and cytotoxic effects in human A549 lung epithelial cells and murine RAW 264.7 macrophages. Moreover, we demonstrate how RNAi can be used to screen for novel host cell proteins involved in the formation of the Legionella-containing vacuole and intracellular replication of the pathogen.

The Caenorhabditis elegans Model of Legionella Infection.

Caenorhabditis elegans can serve as a simple genetic host to study interactions between Legionellaceae and their hosts and to examine the contribution of specific gene products to virulence and immunity. C. elegans nematodes have several appealing attributes as a host organism; they are inexpensive, have robust genetic analysis tools, have a simple anatomy yet display a wide range of complex behaviors, and, as invertebrates, do not require animal ethics protocols. Use of C. elegans as a host model complements cell-based models, providing additional support and consistency of the experimental data obtained from multiple models. The C. elegans innate immune system functions similarly to that of the alveolar macrophage including the apoptosis [a.k.a. programmed cell death (PCD)] pathway located within the germline. The digestive tract of C. elegans is a primary interface between the innate immune system and bacterial pathogens. Thus, the C. elegans host model provides an alternative approach to investigate L. pneumophila immunopathogenesis, particularly in the view of the recent discovery of Legionella-containing vacuoles within the gonadal tissues of Legionella-colonized nematodes supporting the plausible evolutionary origin of the strategies employed by L. pneumophila to counteract macrophage cellular responses.

The structure of Legionella effector protein LpnE provides insights into its interaction with Oculocerebrorenal syndrome of Lowe (OCRL) protein

Legionella pneumophila is a freshwater bacterium that replicates in predatory amoeba and alveolar macrophage. The ability of L.pneumophila to thrive in eukaryotic host cells is conferred by the Legionella containing vacuole (LCV). Formation and intracellular trafficking of the LCV are governed by an arsenal of effector proteins, many of which are secreted by the Icm/Dot Type 4 Secretion System. One such effector, known as LpnE (L.pneumophila Entry), has been implicated in facilitating bacterial entry into host cells, LCV trafficking, and substrate translocation. LpnE belongs to a subfamily of tetratricopeptide repeat proteins known as Sel1-like repeats (SLRs). All eight of the predicted SLRs in LpnE are required to promote host cell invasion. Herein, we report that LpnE(1-375) localizes to cis-Golgi in HEK293 cells via its signal peptide (aa 1-22). We further verify the interaction of LpnE(73-375) and LpnE(22-375) with Oculocerebrorenal syndrome of Lowe protein (OCRL) residues 10-208, restricting the known interacting residues for both proteins. To further characterize the SLR region of LpnE, we solved the crystal structure of LpnE(73-375) to 1.75Å resolution. This construct comprises all SLRs, which are arranged in a superhelical fold. The α-helices forming the inner concave surface of the LpnE superhelix suggest a potential protein-protein interaction interface. DATABASE: Coordinates and structure factors were deposited in the Protein Data Bank with the accession number 6DEH.

Legionella remodels the plasma membrane-derived vacuole by utilizing exocyst components as tethers.

During the initial stage of infection, Legionella pneumophila secretes effectors that promote the fusion of endoplasmic reticulum (ER)-derived vesicles with the Legionella-containing vacuole (LCV). This fusion leads to a remodeling of the plasma membrane (PM)-derived LCV into a specialized ER-like compartment that supports bacterial replication. Although the effector DrrA has been shown to activate the small GTPase Rab1, it remains unclear how DrrA promotes the tethering of host vesicles with the LCV. Here, we show that Sec5, Sec15, and perhaps Sec6, which are subunits of the exocyst that functions in the tethering of exocytic vesicles with the PM, are required for DrrA-mediated, ER-derived vesicle recruitment to the PM-derived LCV. These exocyst components were found to interact specifically with a complex containing DrrA, and the loss of Sec5 or Sec15 significantly suppressed the recruitment of ER-derived vesicles to the LCV and inhibited intracellular replication of Legionella Importantly, Sec15 is recruited to the LCV, and Rab1 activation is necessary for this recruitment.

Legionella pneumophila effector Lem4 is a membrane-associated protein tyrosine phosphatase

Legionella pneumophila is a Gram-negative pathogenic bacterium that causes severe pneumonia in humans. It establishes a replicative niche called Legionella-containing vacuole (LCV) that allows bacteria to survive and replicate inside pulmonary macrophages. To hijack host cell defense systems, L. pneumophila injects over 300 effector proteins into the host cell cytosol. The Lem4 effector (lpg1101) consists of two domains: an N-terminal haloacid dehalogenase (HAD) domain with unknown function and a C-terminal phosphatidylinositol 4-phosphate-binding domain that anchors Lem4 to the membrane of early LCVs. Herein, we demonstrate that the HAD domain (Lem4-N) is structurally similar to mouse MDP-1 phosphatase and displays phosphotyrosine phosphatase activity. Substrate specificity of Lem4 was probed using a tyrosine phosphatase substrate set, which contained a selection of 360 phosphopeptides derived from human phosphorylation sites. This assay allowed us to identify a consensus pTyr-containing motif. Based on the localization of Lem4 to lysosomes and to some extent to plasma membrane when expressed in human cells, we hypothesize that this protein is involved in protein-protein interactions with an LCV or plasma membrane-associated tyrosine-phosphorylated host target.

Crucial Role of Legionella pneumophila TolC in the Inhibition of Cellular Trafficking in the Protistan Host Paramecium tetraurelia.

Legionella pneumophila is a facultative intracellular Gram-negative bacterium, which is a major causative agent of Legionnaires’ disease. In the environment, this bacterium survives in free-living protists such as amoebae and Tetrahymena. The association of L. pneumophila and protists leads to the replication and spread of this bacterium. Thus, from a public health perspective, their association can enhance the risk of L. pneumophila infection for humans. Paramecium spp. are candidates of natural hosts of L. pneumophila, but their detailed relationships remain unclear. In the present study, we used an environmental strain, L. pneumophila Ofk308 (Ofk308) and Paramecium tetraurelia st110-1a to reveal the relationship between L. pneumophila and Paramecium spp. Ofk308 was cytotoxic to P. tetraurelia in an infection-dependent manner. We focused on TolC, a component of the type I secretion system, which is a virulence factor of L. pneumophila toward protists and found that cytotoxicity was dependent on TolC but not on other T1SS components. Further, the number of bacteria in P. tetraurelia was not associated with cytotoxicity and TolC was not involved in the mechanism of resistance against the digestion of P. tetraurelia in Ofk308. We used a LysoTracker to evaluate the maturation process of P. tetraurelia phagosomes containing Ofk308. We found that there was no difference between Ofk308 and the tolC-deletion mutant. To assess the phagocytic activity of P. tetraurelia, Texas Red-conjugated dextran-uptake assays were performed. Ofk308 inhibited phagosome formation by P. tetraurelia through a TolC-dependent mechanism. Further, we evaluated the excretion of Legionella-containing vacuoles from P. tetraurelia. We found that P. tetraurelia failed to excrete undigested Ofk308 and that Ofk308 remained within cells through a TolC-dependent mechanism. Our results suggest that TolC is essential for L. pneumophila to remain within Paramecium cells and to show cytotoxicity. Because of the high mobility and high cell division rate of Paramecium spp., living with Paramecium spp. would be beneficial for L. pneumophila to expand its habitat. To control Legionaries’ disease, understanding the ecology of L. pneumophila in the environment is essential.

LotA, a Legionella deubiquitinase, has dual catalytic activity and contributes to intracellular growth

The intracellular bacterial pathogen, Legionella pneumophila, establishes the replicative niche as a result of the actions of a large array of effector proteins delivered via the Legionella Type 4 secretion system. Many effector proteins are expected to be involved in biogenesis and regulation of the Legionella-containing vacuole (LCV) that is highly decorated with ubiquitin. Here, we identified a Legionella deubiquitinase, designated LotA, by carrying out a genome analysis to find proteins resembling the eukaryotic ovarian tumour superfamily of cysteine proteases. LotA exhibits a dual ability to cleave ubiquitin chains that is dependent on 2 distinctive catalytic cysteine residues in the eukaryotic ovarian tumour domains. One cysteine dominantly contributes to the removal of ubiquitin from the LCVs by its polyubiquitin cleavage activity. The other specifically cleaves conjugated Lys6-linked ubiquitin. After delivered by the Type 4 secretion system, LotA localises on the LCVs via its PI(3)P-binding domain. The lipid-binding ability of LotA is crucial for ubiquitin removal from the vacuoles. We further analysed the functional interaction of the protein with the recently reported noncanonical ubiquitin ligases of L.pneumophila, revealing that the effector proteins are involved in coordinated regulation that contributes to bacterial growth in the host cells.

Legionella Effector RavD Binds Host Phosphoinositide‐3‐phosphate and Contributes to Lysosomal Avoidance

Subversion of phosphoinositide lipids is a key strategy that bacterial pathogens employ to survive within hosts. Due to the importance of phosphoinositides in phagocytosis, bacterial pathogens that infect innate immune cells manipulate these lipids to alter phagosome maturation with the purpose of converting the nascent phagosome into a replication‐permissive niche. The intracellular bacterial pathogen, Legionella pneumophila, the causative agent of a severe pneumonia known as Legionnaires' disease, infects alveolar macrophages where it proliferates within the safety of a vacuole. Upon phagocytosis, L. pneumophila hijacks the nascent phagosome and remodels the cytosolic façade of the phagosomal membrane by stimulating fusion with vesicles derived from the endoplasmic reticulum. This process relies on the function of bacterial effector proteins translocated directly into the host cell via a specialized secretion system. A number of L. pneumophila effector proteins have been shown to bind phosphoinositides either for the purpose of manipulating their metabolism or as a means of targeting specific host membrane compartments. Our preliminary data indicate that many more phosphoinositide‐binding effectors remain to be discovered. Here, we show that the L. pneumophila effector, RavD, is a novel phosphoinositide‐3‐phosphate (PI3P) binding‐protein. We establish that RavD is found on the Legionella‐containing vacuole (LCV) at early stages of infection and harbors its PI3P binding domain within the C terminal region of the protein. In the absence of the ravD gene, the LCV accumulates lysosomal markers at 2 hours post‐infection, consistent with the notion that RavD function is necessary to arrest phagosome maturation.Support or Funding InformationThis work was supported by the University of Delaware Research Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The large GTPase atlastin controls ER remodeling around a pathogen vacuole

ABSTRACTThe ubiquitous environmental bacterium Legionella pneumophila is the causative agent of Legionnaires' pneumonia and replicates in free-living protozoa and mammalian macrophages in a specific compartment, the Legionella-containing vacuole (LCV). LCVs communicate with the endosomal, retrograde and secretory vesicle trafficking pathway, and eventually tightly interact with the endoplasmic reticulum (ER). In Dictyostelium discoideum amoebae and macrophages, the ER tubule-resident large GTPase Sey1/atlastin3 (Atl3) accumulates on LCVs and promotes LCV expansion and intracellular replication of L. pneumophila. Fluorescence microscopy of D. discoideum infected with L. pneumophila indicated that Sey1 is involved in extensive ER remodeling around LCVs. An ultrastructural analysis confirmed these findings. Moreover, dominant negative Sey1_K154A compromises ER accumulation on LCVs and causes an aberrant ER morphology in uninfected D. discoideum as well as in amoebae infected with avirulent L. pneumophila that lack a functional type IV secretion system. Thus, the large, dynamin-like GTPase Sey1/Atl3 controls circumferential ER remodeling during LCV maturation.

Acanthamoeba and Dictyostelium as Cellular Models for Legionella Infection.

Environmental bacteria of the genus Legionella naturally parasitize free-living amoebae. Upon inhalation of bacteria-laden aerosols, the opportunistic pathogens grow intracellularly in alveolar macrophages and can cause a life-threatening pneumonia termed Legionnaires' disease. Intracellular replication in amoebae and macrophages takes place in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates literally hundreds of “effector” proteins into host cells, where they modulate crucial cellular processes for the pathogen's benefit. The mechanism of LCV formation appears to be evolutionarily conserved, and therefore, amoebae are not only ecologically significant niches for Legionella spp., but also useful cellular models for eukaryotic phagocytes. In particular, Acanthamoeba castellanii and Dictyostelium discoideum emerged over the last years as versatile and powerful models. Using genetic, biochemical and cell biological approaches, molecular interactions between amoebae and Legionella pneumophila have recently been investigated in detail with a focus on the role of phosphoinositide lipids, small and large GTPases, autophagy components and the retromer complex, as well as on bacterial effectors targeting these host factors.

Host-Pathogen Interactions via Ubiquitination Pathways

Ankyrin B (AnkB) is a bacterial protein that plays an essential role in the intracellular proliferation of Legionella pneumophila, the causative agent of Legionnaires’ disease. It collects proteins to target for degradation into free amino acids, generating a source of carbon and energy, and preventing a starvation response. AnkB contains two eukaryotic-like domains, the combination of which have never been found in the same eukaryotic protein. The N-terminal F-box domain allows mimicry of host F-box proteins for interaction with the host's ubiquitination pathway via Skp1 of the SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase complex. The C-terminal ankyrin repeats allow AnkB to selectively bind targets for degradation. Here, we report the crystal structure of full length AnkB in complex with host Skp1. We found that AnkB contains an enlarged the substrate-binding site with three ankyrin repeats rather than the two expected based on its sequence. Structural analyses and mutational studies have identified key residues responsible for decorating the Legionella-containing vacuole with ubiquitin and for the replication of the pathogen during infection.Our study provides the first structural insights into the structural mimicry that allows the bacterial virulence factor, AnkB, to associate with the host ubiquitination complex and select substrates.

Differential Effects of Iron, Zinc, and Copper on Dictyostelium discoideum Cell Growth and Resistance to Legionella pneumophila.

Iron, zinc, and copper play fundamental roles in eucaryotes and procaryotes, and their bioavailability regulates host-pathogen interactions. For intracellular pathogens, the source of metals is the cytoplasm of the host, which in turn manipulates intracellular metal traffic following pathogen recognition. It is established that iron is withheld from the pathogen-containing vacuole, whereas for copper and zinc the evidence is unclear. Most infection studies in mammals have concentrated on effects of metal deficiency/overloading at organismal level. Thus, zinc deficiency or supplementation correlate with high risk of respiratory tract infection or recovery from severe infection, respectively. Iron, zinc, and copper deficiency or overload affects lymphocyte proliferation/maturation, and thus the adaptive immune response. Whether they regulate innate immunity at macrophage level is open, except for iron. The early identification in a mouse mutant susceptible to mycobacterial infection of the iron transporter Nramp1 allowed dissecting Nramp1 role in phagocytes, from the social amoeba Dictyostelium to macrophages. Nramp1 regulates iron efflux from the phagosomes, thus starving pathogenic bacteria for iron. Similar studies for zinc or copper are scant, due to the large number of copper and zinc transporters. In Dictyostelium, zinc and copper transporters include 11 and 6 members, respectively. To assess the role of zinc or copper in Dictyostelium, cells were grown under conditions of metal depletion or excess and tested for resistance to Legionella pneumophila infection. Iron shortage or overload inhibited Dictyostelium cell growth within few generations. Surprisingly, zinc or copper depletion failed to affect growth. Zinc or copper overloading inhibited cell growth at, respectively, 50- or 500-fold the physiological concentration, suggesting very efficient control of their homeostasis, as confirmed by Inductively Coupled Plasma Mass Spectrometry quantification of cellular metals. Legionella infection was inhibited or enhanced in cells grown under iron shortage or overload, respectively, confirming a major role for iron in controlling resistance to pathogens. In contrast, zinc and copper depletion or excess during growth did not affect Legionella infection. Using Zinpyr-1 as fluorescent sensor, we show that zinc accumulates in endo-lysosomal vesicles, including phagosomes, and the contractile vacuole. Furthermore, we provide evidence for permeabilization of the Legionella-containing vacuole during bacterial proliferation.

Legionella effector AnkX interacts with host nuclear protein PLEKHN1

BackgroundThe intracellular bacterial pathogen Legionella pneumophila proliferates in human alveolar macrophages, resulting in a severe pneumonia termed Legionnaires’ disease. Throughout the course of infection, L. pneumophila remains enclosed in a specialized membrane compartment that evades fusion with lysosomes. The pathogen delivers over 300 effector proteins into the host cell, altering host pathways in a manner that sets the stage for efficient pathogen replication. The L. pneumophila effector protein AnkX targets host Rab GTPases and functions in preventing fusion of the Legionella-containing vacuole with lysosomes. However, the current understanding of AnkX’s interaction with host proteins and the means through which it exerts its cellular function is limited.ResultsHere, we investigated the protein interaction network of AnkX by using the nucleic acid programmable protein array (NAPPA), a high-density platform comprising 10,000 unique human ORFs. This approach facilitated the discovery of PLEKHN1 as a novel interaction partner of AnkX. We confirmed this interaction through multiple independent in vitro pull-down, co-immunoprecipitation, and cell-based assays. Structured illumination microscopy revealed that endogenous PLEKHN1 is found in the nucleus and on vesicular compartments, whereas ectopically produced AnkX co-localized with lipid rafts at the plasma membrane. In mammalian cells, HaloTag-AnkX co-localized with endogenous PLEKHN1 on vesicular compartments. A central fragment of AnkX (amino acids 491–809), containing eight ankyrin repeats, extensively co-localized with endogenous PLEKHN1, indicating that this region may harbor a new function. Further, we found that PLEKHN1 associated with multiple proteins involved in the inflammatory response.ConclusionsAltogether, our study provides evidence that in addition to Rab GTPases, the L. pneumophila effector AnkX targets nuclear host proteins and suggests that AnkX may have novel functions related to manipulating the inflammatory response.

Hijacking of the Host Ubiquitin Network by Legionella pneumophila.

Protein ubiquitination is critical for regulation of numerous eukaryotic cellular processes such as protein homeostasis, cell cycle progression, immune response, DNA repair, and vesicular trafficking. Ubiquitination often leads to the alteration of protein stability, subcellular localization, or interaction with other proteins. Given the importance of ubiquitination in the regulation of host immunity, it is not surprising that many infectious agents have evolved strategies to interfere with the ubiquitination network with sophisticated mechanisms such as functional mimicry. The facultative intracellular pathogen Legionella pneumophila is the causative agent of Legionnaires' disease. L. pneumophila is phagocytosed by macrophages and is able to replicate within a niche called Legionella-containing vacuole (LCV). The biogenesis of LCV is dependent upon the Dot/Icm type IV secretion system which delivers more than 330 effector proteins into host cytosol. The optimal intracellular replication of L. pneumophila requires the host ubiquitin-proteasome system. Furthermore, membranes of the bacterial phagosome are enriched with ubiquitinated proteins in a way that requires its Dot/Icm type IV secretion system, suggesting the involvement of effectors in the manipulation of the host ubiquitination machinery. Here we summarize recent advances in our understanding of mechanisms exploited by L. pneumophila effector proteins to hijack the host ubiquitination pathway.

Formation of the Legionella Replicative Compartment at the Crossroads of Retrograde Trafficking.

Retrograde trafficking from the endosomal system through the Golgi apparatus back to the endoplasmic reticulum is an essential pathway in eukaryotic cells, serving to maintain organelle identity and to recycle empty cargo receptors delivered by the secretory pathway. Intracellular replication of several bacterial pathogens, including Legionella pneumophila, is restricted by the retrograde trafficking pathway. L. pneumophila employs the Icm/Dot type IV secretion system (T4SS) to form the replication-permissive Legionella-containing vacuole (LCV), which is decorated with multiple components of the retrograde trafficking machinery as well as retrograde cargo receptors. The L. pneumophila effector protein RidL is secreted by the T4SS and interferes with retrograde trafficking. Here, we review recent evidence that the LCV interacts with the retrograde trafficking pathway, discuss the possible sites of action and function of RidL in the retrograde route, and put forth the hypothesis that the LCV is an acceptor compartment of retrograde transport vesicles.

Structural insights into Legionella RidL-Vps29 retromer subunit interaction reveal displacement of the regulator TBC1D5

Legionella pneumophila can cause Legionnaires’ disease and replicates intracellularly in a distinct Legionella-containing vacuole (LCV). LCV formation is a complex process that involves a plethora of type IV-secreted effector proteins. The effector RidL binds the Vps29 retromer subunit, blocks retrograde vesicle trafficking, and promotes intracellular bacterial replication. Here, we reveal that the 29-kDa N-terminal domain of RidL (RidL2–281) adopts a “foot-like” fold comprising a protruding β-hairpin at its “heel”. The deletion of the β-hairpin, the exchange to Glu of Ile170 in the β-hairpin, or Leu152 in Vps29 abolishes the interaction in eukaryotic cells and in vitro. RidL2–281 or RidL displace the Rab7 GTPase-activating protein (GAP) TBC1D5 from the retromer and LCVs, respectively, and TBC1D5 promotes the intracellular growth of L. pneumophila. Thus, the hydrophobic β-hairpin of RidL is critical for binding of the L. pneumophila effector to the Vps29 retromer subunit and displacement of the regulator TBC1D5.