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ScienceDirect Legionella pneumophila: homeward bound away from the phagosome Akriti Prashar1,2 and Mauricio R Terebiznik1,2 The intracellular pathogen Legionella pneumophila (Lp) survives and replicates inside a specialized vacuolar compartment that evades canonical phagosomal maturation. Through the action of a large number of effectors translocated into the host cytosol via the Dot/Icm type IV secretion system, Lp subverts host cell pathways to convert its nascent phagosome into an ER-derived compartment, the Legionella containing vacuole (LCV), which serves as bacterial replication niche. Addresses 1 Biological Sciences, University of Toronto at Scarborough, 1265 Military Trail, Scarborough, Ontario, Canada M1C 1A4 2 Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Ontario, Canada M5S 3G5 Corresponding author: Terebiznik, Mauricio R ([email protected])

Current Opinion in Microbiology 2015, 23:86–93 This review comes from a themed issue on Host–microbe interactions: bacteria Edited by David Holden and Dana Philpott

http://dx.doi.org/10.1016/j.mib.2014.11.008 1369-5274/# 2014 Elsevier Ltd. All rights reserved.

nascent phagosomes from the phagocytic maturation pathway to instead favor their fusion with ER-derived secretory vesicles (ESVs) and membranes. This process results in the formation the Legionella containing vacuole (LCV), the intracellular replicative niche for Lp. The LCVs acquire ER and ESVs markers, associate with mitochondria and polyubiquitinated proteins, and later interact with ER membranes and become studded with ribosomes. Although the LCVs lack endosomal markers for most of intracellular lifecycle of Lp, the presence of late endosomal proteins and hydrolytic proteases can be detected on the LCV 18 hours after Lp infection, which is followed by the escape of bacteria to the cytoplasm where they undergo a few rounds of replication before escaping the wasted host cell. Through this highly orchestrated process Lp can survive and replicate in human macrophages and protozoa. Interestingly, macrophages from most inbred strains of mice are resistant to Lp replication. Naip5/Birc1e NOD-like receptors detect cytosolic flagellin and activate the NLRC4 inflammasome complex leading to pyroptosis, thus preventing bacterial replication [1]. A hypomorphic allele for Naip5 makes A/J mice strain permissive for Lp growth [1]. Here we will discuss recent findings that have furthered our understanding of how LCV biogenesis occurs, with a particular emphasis on the role of T4SS translocated effectors in this process.

The way from a phagosome to the LCV Introduction Legionella pneumophila (Lp) is the causative agent of Legionnaires disease, a severe form of pneumonia. Lp normally thrives in fresh water environments as freeliving forms, in biofilms and as the intracellular parasite in protozoa. Human infection occurs when aerosolized droplets of contaminated water are inhaled and reach the alveolar mucosa. In the lungs Lp invades and replicates in macrophages, which are considered the primary target of Lp although, evidence indicates that Lp can also invade and replicate in epithelial cells. Amoeba and macrophages engulf bacteria by phagocytosis, leading to the formation of plasma membrane derived nascent phagosomes that mature by fusing with endosomal compartments to acquire hydrolytic and antimicrobial properties that kill and digest bacteria. However, Lp delivers effectors into the host cytosol through Lp Dot/ Icm type IV secretion system (T4SS) that segregate its Current Opinion in Microbiology 2015, 23:86–93

Lp requires a functional T4SS to deliver effectors into the host cell, which are necessary to escape phagolysosomal killing and to sustain its intracellular replication in the LCV. Among the 275 Lp effectors, some of them have recently been identified to interfere with the different stages of phagocytic maturation pathway. Escaping the phagosomal maturation pathway

The effectors VipA, VipD and VipF that target the host endocytic pathway were identified in a yeast screening for Lp genes causing membrane trafficking defects [2]. VipA acts as an actin nucleator that localizes to the early endosomes [3]. By altering actin dynamics and vesicle trafficking, VipA could play a role in modulating the endocytic pathway. VipD can disrupt the endocytic pathway and block phagosomal maturation in two different ways; by binding to active Rab5 and Rab22 on endosomes through its C-terminal domain and preventing their function in endosomal trafficking, and by eliminating PI(3)P from endosomes utilizing the phospholipase A1 activity www.sciencedirect.com

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from its N-terminal region [4,5]. VipF is a N-acetyltransferase domain but whether it plays any role during Lp infection remains unknown [2]. Phagosomal maturation requires the acidification of the compartment by vacuolar ATPase proton pumps (VATPase). Despite containing V-ATPases, LCV acidification is delayed for several hours after infection [6], with the effector SidK playing a role in this process [7]. Through its N-terminal domain, SidK interacts with the catalytic subunit of V-ATPase, leading to inhibition of ATP hydrolysis required for organelle acidification [7]. Macrophages expressing SidK show impaired endosomal acidification and degradative capacity [7]. During maturation, phagosomes move along microtubules (MTs) toward the perinuclear zone where they fuse with lysosomes. Lp also takes advantage of MTs for the initial trafficking of the LCVs. Lp effector LegG1 co-opts host Ran GTPase leading to enhanced MT stabilization. This in turn supports LCV motility, however no MT motors have been associated with this phenomenon [8]. LegG1, RanGTPase and its effector RanBP1 all localize at the LCV and LegG1 promotes Lp replication in protozoa and macrophages [8]. It has been proposed that by positioning the LCVs in close proximity to intracellular compartments or by promoting the trafficking of intracellular vesicles that associate with the LCVs, LegG1 and Ran dependent MT stabilization could contribute to LCV biogenesis [8]. Effector independent ways to modulate endocytic maturation pathway

Lp outer membrane vesicles (OMVs) play a role in inhibiting phagosome–lysosome fusion. Indeed, phagosomes containing latex beads coated with these LPS rich OMVs fail to acquire lysosomal markers [9]. Similarly, Lp outer membrane chaperone HtpB contributes to the inhibition of phagosome–lysosome fusion [10]. KupA is an Lp K+ transporter that is required for Lp replication in eukaryotic cells [11]. Inhibiting vacuolar acidification by bafilomycin rescues this defect, suggesting that functional K+ transporters play a role in LCV formation. Indeed in the absence of kupA, fewer LCVs acquire polyubiquitinated proteins indicating a defect in its biogenesis [11]. However, the precise mechanism responsible for this phenomenon remains to be elucidated. Lp exhibits length polymorphism and filamentous forms of Lp are present in biofilms as well as in patients [12,13]. Filaments can invade both macrophages and epithelial cells [13,14] and this morphology can contribute to Lp survival in macrophages [14]. Macrophages engulf filamentous Lp gradually from their short axis, into tubular phagocytic cups (TPC) [14]. Although, the TPCs undergo www.sciencedirect.com

phagosomal maturation, they are leaky to the extracellular milieu and cannot acidify or retain hydrolases to develop hydrolytic properties until they seal to form a phagosome [14]. Therefore, bacterial length causes a delay in the formation of the nascent phagosomes and thus, filamentous Lp spend a longer time in a harmless compartment from where it can translocate effectors to favor the formation of the LCV more efficiently than their shorter counterparts that are delivered to phagolysosomes more frequently [14].

LCV biogenesis Hijacking host early secretory pathway

Lp effectors hijack modulators of the host early secretory pathway to convert the nascent phagosomes into an LCV. This modification occurs in two phases; an early association of the LCV with ER secretory vesicles (ESVs), characterized by the presence of small GTPases Sar1 and Rab1 on the LCV, followed by Arf1 GTPase dependent fusion with ER membranes [15]. Rab1, the main regulator of ER-Golgi trafficking pathway, is rapidly sequestered to Lp containing phagosomes. Recent advances have identified the role of a network of Lp effectors that control Rab1 recruitment and activity at the LCV to co-opt ESVs and mediate their fusion with the LCV (Figure 1) [16]. Lp effectors SidM/DrrA and LidA are responsible for sequestering Rab1 on the LCV [17,18]. SidM/DrrA is a multifunctional effector that targets various host cell molecules. Its central domain possesses GEF (guanosine exchange factor) activity that activates Rab1 [18]. In addition, SidM/DrrA can also act as a Rab1 GDF (guanosine diphosphate dissociation inhibitor displacement factor) that extracts Rab1 from GDI its (guanosine diphosphate dissociation inhibitor) in the host cytoplasm [19]. The N-terminal domain of SidM/DrrA possesses enzymatic activity that catalyzes the AMPylation of the switch domain of Rab1, making it resistant to GAPs and locking it in its GTP bound form [20,21]. Indeed without AMPylation, Rab1 associated with the LCVs is more susceptible to inactivation by GAPs and is rapidly removed from the LCVs. Therefore, SidM/DrrA GEF activity allows Lp to efficiently sequester and activate Rab1 on the LCV surface while its AMPylase activity retains Rab1 on the LCV [22]. The effector AnkX contains a FIC domain that modulates Rab1 activity by phosphorylcholinating (PC) the switch II region of Rab1 [23,24]. This post-translational modification makes Rab1 resistant to endogenous GEFs or GAP. Therefore, it is speculated that PCylation of Rab1 sequesters it from host trafficking pathway to serve in the LCV biogenesis. Lp effectors also inactivate Rab1 on the LCV. This is initiated by the de-AMPylation and de-PCylation of Rab1 by the effectors SidD and Lem3 respectively [25–27]. The activities of these effectors render Rab1 susceptible to the GAP activity of Lp effector LepB, and host GAPs Current Opinion in Microbiology 2015, 23:86–93

88 Host–microbe interactions: bacteria

Figure 1

Cytoplasm GDI

ESVs GDI

Sec22b GDI

CDP choline CMP

Pi

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Rab1 GDP ATP

GTP SidM/DrrA

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LCV membrane LCV lumen Current Opinion in Microbiology

Legionella pneumophila effectors control Rab1 activation cycle. The effector SidM/DrrA anchors to PI(4)P in the LCV membrane and acts as a GEF. Active Rab1 (GTP bound), which leaves its GDI gets inserted into the LCV membrane. SidM/DrrA also AMPylates Rab1, and AnkX phosphocholinates Rab1, both making Rab1 resistant to GAP activity. Rab1 promotes the docking and fusion of ER-derived secretory vesicles (ESVs) with the LCV, mediated by tetherin proteins like sec22b. Lp effectors SidD and Lem3 de-adenylylate and de-phosphocholinate modified Rab1 respectively, making it susceptible to inactivation by the effector LepB and endogenous GAPs (not shown here) releasing Rab1 from the LCV.

and GDIs. Interestingly, in vitro analysis has shown that LepB can also act as GAP for Rabs 3a, 8a, 10, 13 and 35, however, the role of these Rabs during Lp replication is unknown [28]. The ectopic activation of Rab1 by SidM/DrrA and AnkX has been associated with cytotoxic effects due to disruption of Golgi apparatus and secretory trafficking in yeast and mammalian cells [17]. Thus, Rab1 inactivation by Lp effectors not only contributes to LCV maturation, it also preserves host cell viability during infection. The effector LidA binds to both GDP and GTP–Rab1 with high affinity [29,30]. Interestingly, it also binds with Rab1Amp and Rab1-PC and facilitates the recruitment of ESVs to the LCV by protecting Rab1 from de-AMPylation and de-PCylation by SidD and Lem3 respectively [20,30]. SNARE proteins mediate membrane fusion events in eukaryotic cells. Rab1 controls the formation of SNARE complexes between v-SNARE Sec22b, present on ESVs and the t-SNARES syntaxin5, membrin and rBET1 to mediate the fusion of these vesicles with the Golgi complex [31]. The arrival of Rab1 at the LCV prompts the recruitment of Sec22b containing ESVs to the LCVs, however the LCV lacks all of the aforementioned Golgi Current Opinion in Microbiology 2015, 23:86–93

t-SNAREs. Instead, plasma membrane t-SNAREs syntaxins 2,3 and 4 become a part of the LCV during bacterial uptake [31]. Therefore, by activating Rab1 on the LCVs, SidM/DrrA stimulates the non-canonical fusion between Sec22b ESVs and plasma membrane syntaxins required for the fusion of ESVs with the LCV [32]. RalF mediates ESV and ER membrane fusion to the LCV by recruiting and activating Arf1, a host GTPase that regulates vesicle trafficking from the ER to Golgi. RalF N-terminal contains a Sec 7 homology region conserved among known Arf1 GEFs [33] and a C-terminal domain that can block the Sec7 domain to control its access to Arf1 [34]. The capping domain has membrane-sensor activity through which it likely localizes RalF to the LCV membranes [35]. RalF C-terminal domain is suggested to ensure its correct targeting to the LCV membrane, which can then efficiently regulate the spatial activation of Arf1, ultimately leading to LCV-ER membrane fusion [35].

Modulation of the host ubiquitin pathway

Protein ubiquitination regulates protein trafficking, sorting and degradation in eukaryotic cells [36]. Lp subverts host cell ubiquitination machinery to exert www.sciencedirect.com

Biogenesis of Legionella vacuole Prashar and Terebiznik 89

spacio-temporal control over its effectors and for proteasomal degradation of host proteins [36]. Several Lp effectors contain F-box and U-box eukaryotic motifs present in E3 ligases, which attach ubiquitin monomers to target proteins [36]. The effector LubX shows functional and structural homology to the U-Box containing E3 ligases [37]. LubX exerts spatiotemporal control over the effector SidH by marking it for proteasomal degradation [37]. Thus, LubX might modulate the function of other effectors. This action could be critical to reduce the cytotoxic effects of Lp effectors for the host cells as LubX mutant strains showed a hyper-lethality phenotype in the Drosophila melanogaster host [37]. LubX ubiquitinates host cell cycle kinase Clk1 but whether this leads to proteasomal degradation of Clk1 is unknown and its consequence in Lp replication is are unknown [36]. Lp philadelphia effectors, AnkB (LegAU13), LegU1 and LicA contain the F-box domains found in eukaryotic proteins that form the SCF (Skp-Cullin-F-box) complex family of ubiquitin ligases [36]. AnkB has two additional domains with homology to eukaryotic proteins. It contains two ankyrin repeats usually involved in proteinprotein interactions, through which it likely binds targets proteins to be ubiquitinated and a C-terminal CAAX motif that gets farnesylated in the host cell [38]. Farnesylated AnkB can anchor in the LCV membrane, facilitating the SCF complex dependent polyubiquitination of proteins on the LCV, which contributes to Lp replication [38,39]. Among the possible roles for protein ubiquitination for Lp replication, it has been suggested that AnkB mediated ubiquitination and degradation of proteins by proteasome supplies amino acids to sustain Lp replication inside the LCV [40]. A different role is played by AnkB ortholog in Lp strain Paris, Lpp2082. This effector inhibits the ubiquitination of cytoskeletal signaling protein ParvinB, by potentially competing with the endogenous E3 ligase [41]. Lp replication is reduced in cells lacking ParvinB, likely suggesting a link between AnkB–PavinB interaction and LCV formation [41]. The effector LegU1 forms a complex with SCF and ubiquitinates host proteins BAT3, a host chaperone that regulates ER stress and apoptosis [42]. LicA has been speculated to form a non-canonical SCF complex but this has not been experimentally demonstrated [42]. The N-terminal domain of the effector SidC has E3 ligase activity, and in Lp infected Dictyostelium discoideum is important for the recruitment of ER proteins and ubiquitinated proteins to the LCV [43]. SidC can indirectly monoubiquitinate Rab1. However, how this occurs remains unknown [43,44]. Modulation of the Host Autophagy Pathway

The conserved housekeeping pathway, autophagy, targets intracellular pathogens for their destruction in www.sciencedirect.com

autophagolysosomes [45]. On the other hand, several intracellular pathogens take over autophagy trafficking for the biogenesis of their intracellular compartment [45]. On the basis of the presence of markers of the autophagic pathway on the LCV, it was shown that Lp infection triggers autophagy in the host cell [15,46,47]. Recently, an Lp protein LegA9 has been suggested to be important for inducing autophagy as a greater number of LegA9 mutants avoid autophagy and are able to replicate intracellularly in mouse macrophages and in vivo in mice [46]. The effector RavZ has been shown to disrupt autophagosome formation in human embryonic kidney cells [47]. Conjugation of cytosolic autophagy protein Atg8, also known as LC3, with phosphatidylethanolamine (PE) on autophagosomal membranes is an essential step required for autophagosome formation [45]. RavZ acts as a protease and specifically targets PE-conjugated Atg8, causing its deconjugation from PE. Deconjugated Atg8 lacks the C-terminal glycine needed for reconjugation by Atg7 and Atg3 and consequently, autophagosome formation is inhibited [47].

Hijacking host phosphoinositides for LCV biogenesis Phosphatidyl phosphoinositides (PIPs) are eukaryotic membrane lipids that regulate critical cellular processes including membrane trafficking, therefore, they are common targets for intracellular bacteria. Lp express several effectors that subvert host PIPs by anchoring to them or by directly modifying them for LCV biogenesis (Figure 2) [48]. PIPs as anchors for Lp effectors

The PIPs, PI(3)P and PI(4)P are present on the LCV membrane and serve as anchors for several Lp effectors [48]. SidC and its paralog SdcA bind to PI(4)P on the LCV. Anchoring to PI(4)P occurs through its C-terminal domain, referred to as P4C, while the N-terminal domain is responsible for recruiting ER vesicles to the LCV [49]. Crystal structure of the effector SidM/DrrA shows that it also binds to PI(4)P through a C-terminal region, P4M, which is unrelated to the SidC P4C region [50,51]. Recently, sequence analysis for Lp proteins containing a conserved PI(4)P binding domain present in SidM/ DrrA, led to the identification of Lpg1101 and Lpg2603, both of which are T4SS substrates and bind to PI(4)P [52]. However, their role in Lp replication has not been described yet. PI(3)P binding has been demonstrated for the effectors LidA [50], SetA [53], LpnE [54], LtpD [55] and RidL [56]. LidA uses its central domain to binds to PI(3)P [30] and also binds several host Rabs including Rab1, Rab6 and Rab8 [29,30,57,58], likely recruiting them to the LCV. It also binds to PI(4)P to a lesser extent [50]. SetA was identified in a yeast screen to find Lp effectors that Current Opinion in Microbiology 2015, 23:86–93

90 Host–microbe interactions: bacteria

Figure 2

(a) Cytosol SidM/DrrA

LidA

SidC

LpnE

LidA

LCV membrane (b) Cytosol SidP

SidF

SidF

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PI(4,5)P2

Current Opinion in Microbiology

Lp effectors and host PIs in LCV biogenesis. (a) Lp effectors bind to PI(3)P and PI(4)P to anchor to the LCV membrane to faciliate their interaction with the host endocytic and secretory trafficking pathway. (b) Lp effectors can act as PI phosphatases to modify the PI composition of the LCV membrane. SidF and SidP act as D3 phosphatases. (c) Host cell PI metabolizing enzymes can be used to alter the LCV PI composition. PI(4)P, which is critical for effector binding and accumulates in the LCV, is produced from PI(4,5)P3 by ORCL1 mediated dephosphoryation. ORCL1 is recruited to the LCV by the effector LpnE. PI(4)P can also produced by the action of PI4KIII kinase. Sac1 phosphatase arriving at the LCV through ER-derived vesicles dephosphoryates PI(4)P, causing the detachment of PI(4)P binding Lp effectors from the LCV.

interfered with host vesicular trafficking [59]. The Cterminal domain of SetA binds to PI(3)P, localizing it to the LCV and the N-terminal has similarity to glycosyltransferases that can covalently modify host proteins [53]. The effector LpnE which contributes to the virulence of Lp by promoting bacterial entry into human macrophagelike cells also binds to PI(3)P [54]. In the absence of LpnE, bacterial virulence in the A/J mice model of Legionellosis is compromised [60,61]. LtpD is an Lp effector that localizes to endosomal compartments and plays an important role in bacterial replication in THP-1 macrophages, Galleria larvae and in vivo in mice [55]. Recently identified effector RidL also binds to PI(3)P and competes with sorting nexins for binding to VPS29 subunit of the retromer complex [56]. This inhibits host Current Opinion in Microbiology 2015, 23:86–93

retrograde trafficking, promoting Lp replication in RAW macrophages, and D. discoideum and Acanthamoeba castellanii [56]. Manipulating host lipid metabolizing enzymes

Lp effector SidF, possesses a CX5R motif characteristic of PI phosphatases [62]. SidF directly hydrolyses D3 phosphate of early endosomal PIPs, PI(3,4)P2 and PI(3,4,5)P3 to PI(4)P [62], transforming the phagosomes into compartments better suited for the anchoring of Lp effectors with a PI(4)P binding domain. SidP also acts as a PI-3phosphatase hydrolyzing PI(3)P and PI(3,5)P2 [63]. This effector was identified through bioinformatics screening for Lp effectors with PI-metabolizing activity. Similar to SidF, it contains the CX5R motif but has no additional www.sciencedirect.com

Biogenesis of Legionella vacuole Prashar and Terebiznik 91

sequence homology to other known PI-phosphatases [63]. It is speculated that by removing PI(3)P, SidP and SidF could restrict the fusion of early endosomes with the LCV [63]. The effector LpnE contains a Sel-1 repeat motif known to mediate protein-protein interactions [61]. Via an Nterminal domain, LpnE binds mammalian PI5-phosphatase OCRL1 and its Dictostelium homologue D5dP4, which convert PI(4,5)P2 and PI(3,4,5)P3 to PI(3,4)P2 and PI(4)P respectively [54]. Therefore, LpnE also might facilitate the anchoring of PI(4)P binding effectors on the LCV. The activity of host PI4KIII enzymes can also contribute to increasing PI(4)P levels on the LCV [50,52]. Although their direct recruitment on the LCV has not been shown, these kinases could arrive at the LCV on ESVs through SidM/DrrA mediated trafficking or by the activity of Lp effectors LpdA and LecE, both of which, likely cause an increase in DAG on the LCV and lead to the recruitment of protein kinase D and PI4KIIIb [48,64]. By altering the lipid profile of the LCV and increasing PI(4)P levels, lipid modifying Lp effectors could facilitate effector binding to the LCV and membrane trafficking events needed for LCV formation. Supporting this, removal of PI(4)P by phosphate Sac1 present on the ER vesicles causes effector dissociation from the LCV [52].

Maintenance of LCV stability During later stages of infection, Lp escape from the LCV into the host cytoplasm where they undergo a few rounds of replication before host cell lysis [65]. However, Lp mutants defective in certain effectors cause cell death by prematurely escaping the LCV, suggesting that Lp actively maintains the LCV. The effectors LidA and WipB were recently identified as contributors to maintaining LCV stability [66]. Replication of DLidA/ DWipB strains in bone marrow derived macrophages was inhibited and this defect was associated with the loss of LCV integrity [66]. Lp effector SdhA also contributes to the maintenance of LCV stability [67,68] by regulating Lp phospholipase PlaA [67]. In the absence of SdhA the LCV are less stable and more susceptible to PlaA activity [67], causing increased cytosolic access of the bacteria and the activation of cytosolic immune responses, including caspase activation and pyroptosis [67,68].

Conclusions and perspectives The vast repertoire of Lp effectors that block phagosomal maturation and form the LCV in the protozoa target highly conserved pathways and thus are equally effective for LCV formation in human macrophages and epithelial cells. The complex network of effectors that hijack the www.sciencedirect.com

activation cycle of Rab1, best exemplify how well Lp is adapted to control its hosts. The function of several Lp effectors has been characterized and much has been advanced in our understanding of their contribution in regulating the early secretory pathway for LCV biogenesis. However, only in recent years studies have begun to establish the role of Lp effectors in modulating host protein sorting and degradation, endocytic and autophagy pathways and much still needs to be done to understand how Lp interacts with these pathways to complete the intracellular roadmap of this bacteria. This is a challenging task as known Lp effectors have highly redundant functions and the role of vast number of them remains unknown.

Acknowledgements This work was supported by the NSERC Discovery and Ontario Lung Association-Pfizer grants to MRT.

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20. Muller MP, Peters H, Blumer J, Blankenfeldt W, Goody RS, Itzen A: The Legionella effector protein DrrA AMPylates the membrane traffic regulator Rab1b. Science 2010, 329:946-949. 21. Goody RS, Muller MP, Schoebel S, Oesterlin LK, Blumer J, Peters H, Blankenfeldt W, Itzen A: The versatile Legionella effector protein DrrA. Commun Integr Biol 2011, 4:72-74. 22. Hardiman CA, Roy CR: AMPylation is critical for Rab1 localization to vacuoles containing Legionella pneumophila. MBio 2014, 5 e01035-13. 23. Mukherjee S, Liu X, Arasaki K, McDonough J, Galan JE, Roy CR: Modulation of Rab GTPase function by a protein phosphocholine transferase. Nature 2011, 477:103-106. 24. Campanacci V, Mukherjee S, Roy CR, Cherfils J: Structure of the Legionella effector AnkX reveals the mechanism of phosphocholine transfer by the FIC domain. EMBO J 2013, 32:1469-1477. 25. Neunuebel MR, Chen Y, Gaspar AH, Backlund PS Jr, Yergey A, Machner MP: De-AMPylation of the small GTPase Rab1 by the pathogen Legionella pneumophila. Science 2011, 333:453-456. 26. Tan Y, Luo ZQ: Legionella pneumophila SidD is a deAMPylase that modifies Rab1. Nature 2011, 475:506-509. 27. Tan Y, Arnold RJ, Luo ZQ: Legionella pneumophila regulates the small GTPase Rab1 activity by reversible phosphorylcholination. Proc Natl Acad Sci U S A 2011, 108:21212-21217.

40. Price CT, Al-Quadan T, Santic M, Rosenshine I, Abu Kwaik Y: Host proteasomal degradation generates amino acids essential for intracellular bacterial growth. Science 2011, 334:1553-1557. 41. Lomma M, Dervins-Ravault D, Rolando M, Nora T, Newton HJ, Sansom FM, Sahr T, Gomez-Valero L, Jules M, Hartland EL et al.: The Legionella pneumophila F-box protein Lpp2082 (AnkB) modulates ubiquitination of the host protein parvin B and promotes intracellular replication. Cell Microbiol 2010, 12:12721291. 42. Ensminger AW, Isberg RR: E3 ubiquitin ligase activity and targeting of BAT3 by multiple Legionella pneumophila translocated substrates. Infect Immun 2010, 78:3905-3919. 43. Hsu F, Luo X, Qiu J, Teng YB, Jin J, Smolka MB, Luo ZQ, Mao Y: The Legionella effector SidC defines a unique family of ubiquitin ligases important for bacterial phagosomal remodeling. Proc Natl Acad Sci U S A 2014, 111:10538-10543. 44. Horenkamp FA, Mukherjee S, Alix E, Schauder CM, Hubber AM, Roy CR, Reinisch KM: Legionella pneumophila subversion of host vesicular transport by SidC effector proteins. Traffic 2014, 15:488-499. 45. Huang J, Brumell JH: Bacteria-autophagy interplay: a battle for survival. Nat Rev Microbiol 2014, 12:101-114.  This excellent review highlights the antimicrobial role of autophagy and the ways in which pathogens modulate autophagy for their suvival.

28. Mihai Gazdag E, Streller A, Haneburger I, Hilbi H, Vetter IR, Goody RS, Itzen A: Mechanism of Rab1b deactivation by the Legionella pneumophila GAP LepB. EMBO Rep 2013, 14: 199-205.

46. Khweek AA, Caution K, Akhter A, Abdulrahman BA, Tazi M, Hassan H, Majumdar N, Doran A, Guirado E, Schlesinger LS et al.: A bacterial protein promotes the recognition of the Legionella pneumophila vacuole by autophagy. Eur J Immunol 2013, 43:1333-1344.

29. Cheng W, Yin K, Lu D, Li B, Zhu D, Chen Y, Zhang H, Xu S, Chai J, Gu L: Structural insights into a unique Legionella pneumophila

47. Choy A, Dancourt J, Mugo B, O‘Connor TJ, Isberg RR, Melia TJ,  Roy CR: The Legionella effector RavZ inhibits host autophagy

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Biogenesis of Legionella vacuole Prashar and Terebiznik 93

through irreversible Atg8 deconjugation. Science 2012, 338:1072-1076. This study demonstrates that Legionella effector RavZ can inhibit host cell autophagy.

58. Schoebel S, Cichy AL, Goody RS, Itzen A: Protein LidA from Legionella is a Rab GTPase supereffector. Proc Natl Acad Sci U S A 2011, 108:17945-17950.

48. Haneburger I, Hilbi H: Phosphoinositide lipids and the Legionella pathogen vacuole. Curr Top Microbiol Immunol 2013, 376:155-173.

59. Heidtman M, Chen EJ, Moy MY, Isberg RR: Large-scale identification of Legionella pneumophila Dot/Icm substrates that modulate host cell vesicle trafficking pathways. Cell Microbiol 2009, 11:230-248.

49. Ragaz C, Pietsch H, Urwyler S, Tiaden A, Weber SS, Hilbi H: The Legionella pneumophila phosphatidylinositol-4 phosphatebinding type IV substrate SidC recruits endoplasmic reticulum vesicles to a replication-permissive vacuole. Cell Microbiol 2008, 10:2416-2433.

60. Newton HJ, Sansom FM, Bennett-Wood V, Hartland EL: Identification of Legionella pneumophila-specific genes by genomic subtractive hybridization with Legionella micdadei and identification of lpnE, a gene required for efficient host cell entry. Infect Immun 2006, 74:1683-1691.

50. Brombacher E, Urwyler S, Ragaz C, Weber SS, Kami K, Overduin M, Hilbi H: Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila. J Biol Chem 2009, 284:4846-4856.

61. Newton HJ, Sansom FM, Dao J, McAlister AD, Sloan J, Cianciotto NP, Hartland EL: Sel1 repeat protein LpnE is a Legionella pneumophila virulence determinant that influences vacuolar trafficking. Infect Immun 2007, 75:5575-5585.

51. Del Campo CM, Mishra AK, Wang YH, Roy CR, Janmey PA, Lambright DG: Structural basis for PI(4)P-specific membrane recruitment of the Legionella pneumophila effector DrrA/ SidM. Structure 2014, 22:397-408. 52. Hubber A, Arasaki K, Nakatsu F, Hardiman C, Lambright D, De  Camilli P, Nagai H, Roy CR: The machinery at endoplasmic reticulum-plasma membrane contact sites contributes to spatial regulation of multiple Legionella effector proteins. PLoS Pathog 2014, 10:e1004222. This study showed that host cell PI(4)P metabolizing enzymes can regulate the anchoring of PI(4)P binding Legionella effectors for LCV biogenesis. 53. Jank T, Bohmer KE, Tzivelekidis T, Schwan C, Belyi Y, Aktories K: Domain organization of Legionella effector SetA. Cell Microbiol 2012, 14:852-868. 54. Weber SS, Ragaz C, Hilbi H: The inositol polyphosphate 5phosphatase OCRL1 restricts intracellular growth of Legionella, localizes to the replicative vacuole and binds to the bacterial effector LpnE. Cell Microbiol 2009, 11:442-460. 55. Harding CR, Mattheis C, Mousnier A, Oates CV, Hartland EL, Frankel G, Schroeder GN: LtpD is a novel Legionella pneumophila effector that binds phosphatidylinositol 3phosphate and inositol monophosphatase IMPA1. Infect Immun 2013, 81:4261-4270. 56. Finsel I, Ragaz C, Hoffmann C, Harrison CF, Weber S, van Rahden VA, Johannes L, Hilbi H: The Legionella effector RidL inhibits retrograde trafficking to promote intracellular replication. Cell Host Microbe 2013, 14:38-50. 57. Chen Y, Machner MP: Targeting of the small GTPase Rab6A0 by the Legionella pneumophila effector LidA. Infect Immun 2013, 81:2226-2235.

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62. Hsu F, Zhu W, Brennan L, Tao L, Luo ZQ, Mao Y: Structural basis for substrate recognition by a unique Legionella phosphoinositide phosphatase. Proc Natl Acad Sci U S A 2012, 109:13567-13572. 63. Toulabi L, Wu X, Cheng Y, Mao Y: Identification and structural characterization of a Legionella phosphoinositide phosphatase. J Biol Chem 2013, 288:24518-24527. 64. Viner R, Chetrit D, Ehrlich M, Segal G: Identification of two Legionella pneumophila effectors that manipulate host phospholipids biosynthesis. PLoS Pathog 2012, 8:e1002988. 65. Molmeret M, Bitar DM, Han L, Kwaik YA: Disruption of the phagosomal membrane and egress of Legionella pneumophila into the cytoplasm during the last stages of intracellular infection of macrophages and Acanthamoeba polyphaga. Infect Immun 2004, 72:4040-4051. 66. O‘Connor TJ, Boyd D, Dorer MS, Isberg RR: Aggravating genetic  interactions allow a solution to redundancy in a bacterial pathogen. Science 2012, 338:1440-1444. This study utilized a novel genetic screening technique of combining bacterial insertion mutants with RNAi targeting host pathways to identify Legionella effectors important for maintenance of the LCV. 67. Creasey EA, Isberg RR: The protein SdhA maintains the integrity of the Legionella-containing vacuole. Proc Natl Acad  Sci U S A 2012, 109:3481-3486. 68. Ge J, Gong YN, Xu Y, Shao F: Preventing bacterial DNA release  and absent in melanoma 2 inflammasome activation by a Legionella effector functioning in membrane trafficking. Proc Natl Acad Sci U S A 2012, 109:6193-6198. Together with Ref. [66], this study showed that Legionella effector SdhA maintains the integrity of the LCV.

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