Phosphoinositides and the Fate of Legionella in Phagocytes

Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires' disease. The environmental bacterium replicates in free-living amoebae as well as in lung macrophages in a distinct compartment, the Legionella-containing vacuole (LCV). The LCV communicates with a number of cellular vesicle trafficking pathways and is formed by a plethora of secreted bacterial effector proteins, which target host cell proteins and lipids. Phosphoinositide (PI) lipids are pivotal determinants of organelle identity, membrane dynamics and vesicle trafficking. Accordingly, eukaryotic cells tightly regulate the production, turnover, interconversion, and localization of PI lipids. L. pneumophila modulates the PI pattern in infected cells for its own benefit by (i) recruiting PI-decorated vesicles, (ii) producing effectors acting as PI interactors, phosphatases, kinases or phospholipases, and (iii) subverting host PI metabolizing enzymes. The PI conversion from PtdIns(3)P to PtdIns(4)P represents a decisive step during LCV maturation. In this review, we summarize recent progress on elucidating the strategies, by which L. pneumophila subverts host PI lipids to promote LCV formation and intracellular replication.

FIGURE 2 | Subcellular distribution of phosphoinositides. The subcellular distribution of phosphoinositide lipids is primarily arranged around the cellular dichotomy of the secretory and endocytic vesicle trafficking pathways. In the secretory pathway, PtdIns(4)P is synthesized in the endoplasmic reticulum (ER) and Golgi apparatus, localizes to secretory vesicles, and finally accumulates at the plasma membrane, where it is converted to PtdIns(4,5)P 2 and, transiently, to PtdIns(3,4,5)P 3 . In the endocytic pathway, PtdIns(3)P decorates early endosomes and the tubular endosomal network (TEN), and is converted to PtdIns(3,5)P 2 on multivesicular bodies (MVB), late endosomes and lysosomes (LYS). multivesicular bodies, which like late endosomes and lysosomes are also decorated with PtdIns(3,5)P 2 . PtdIns(4)P is the hallmark PI lipid of the secretory pathway and predominantly localizes to the Golgi apparatus and secretory vesicles (53,54,56,62). This PI lipid is formed from PtdIns on the ER and together with PtdIns(3)P also regulates phagosome-lysosome fusion (63).
On certain compartments and along some vesicle trafficking pathways, distinct PIs are functionally coupled, i.e., the product of a given PI-metabolizing enzyme is the substrate of a subsequent modification. This occurs, e.g., in the endocytic pathway, where PtdIns(3)P is phosphorylated to yield PtdIns(3,5)P 2 , as well as in the secretory pathway, where PtdIns(4)P serves as the precursor of PtdIns(4,5)P 2 at the plasma membrane. In turn, PtdIns(4,5)P 2 is phosphorylated by class I PI3K to transiently yield PtdIns(3,4,5)P 3 during phagocytosis.

EUKARYOTIC PI KINASES IMPLICATED IN UPTAKE AND ENDOCYTOSIS OF L. PNEUMOPHILA
PtdIns(3,4,5)P 3 and PtdIns(3)P are produced by class I or class III PI3Ks and are major regulators of phagocytosis or the endocytic pathway, respectively. Using the haploid social soil amoeba Dictyostelium discoideum, genetic and pharmacological disruption of class I PI3Ks indicated that these kinases are largely dispensable for uptake of wild-type L. pneumophila, but required for uptake of an icm/dot mutant strain (51,64). Moreover, using D. discoideum producing a fluorescent probe for PtdIns (3,4,5)P 3 , live-cell microscopy revealed that this PI lipid accumulated at bacterial entry sites and was cleared within approximately 40 s after uptake, regardless of whether the amoebae were infected with wild-type or icm/dot mutant L. pneumophila. In parallel, plasma membrane PtdIns(4,5)P 2 disappeared from the uptake sites (65). Similar to amoebae, the uptake of L. pneumophila wild-type, but not the icm/dot mutant strain by replication-permissive human U937 macrophage-like cells was not affected by the class I PI3K inhibitor wortmannin (66,67). In contrast, wortmannin or LY294002 inhibited the uptake of wild-type as well as icm/dot mutant L. pneumophila by non-permissive murine J774A.1 macrophages (64,66,67). The Icm/Dot T4SS controls the uptake of L. pneumophila by phagocytes (68,69); however, no effectors implicated in the process have been identified. These results suggest that during uptake of L. pneumophila class I PI3Ks are activated and the pathogen evades/inhibits downstream processes in an Icm/Dot-dependent manner to form the replication-permissive compartment.
Dictyostelium discoideum mutant strains were also used to examine the role of endosomal PI kinases, PI phosphatases and phospholipases for intracellular growth of L. pneumophila. Wildtype L. pneumophila replicated more efficiently in D. discoideum lacking two or five class I PI3Ks (51,64) or in amoebae lacking PIKfyve (70), a PI 5-kinase, which is recruited through its FYVE domain to early endosomes, where it phosphorylates PtdIns(3)P to yield PtdIns(3,5)P 2 . While it is not clear how lower levels of PtdIns(3,4,5)P 3 promote the intracellular replication of L. pneumophila, the reduction of PtdIns(3,5)P 2 impairs the bactericidal endocytic pathway, which restricts bacterial killing and thus benefits the pathogen (70). The disruption of D. discoideum PTEN (phosphatase and tensin homolog), a PI phosphatase antagonizing PI3Ks, reduces the uptake of L. pneumophila but does not affect intracellular growth (64). Finally, the inhibition of D. discoideum PLC (Phospholipase C), a hydrolase cleaving PI(4,5)P 2 to yield DAG and inositol 1,4,5phosphate (IP 3 ), also abolishes the uptake of L. pneumophila, but again has no effect on bacterial replication (64).

PHOSPHOINOSITIDE CONVERSION ON THE LEGIONELLA-CONTAINING VACUOLE
PtdIns(3)P accumulates on LCVs within 1 min after uptake, regardless of whether the vacuole contains wild-type or icm/dot mutant L. pneumophila (71). However, while phagosomes containing icm/dot mutant bacteria remain decorated with PtdIns(3)P, more than 80% of wild-type LCVs gradually lose this PI within 2 h. Concomitantly, major membrane rearrangements take place with PtdIns(3)P-positive membranes being segregated from the LCV and compacted at the cell center. PtdIns(4)P, on the other hand, transiently localizes to early phagosomes harboring wild-type or icm/dot mutant L. pneumophila, but is cleared within minutes after uptake. During the following 2 h, PtdIns(4)P steadily accumulates only on wild-type LCVs, which for at least 8 h maintain a discrete PtdIns(4)P identity spatially separated from the calnexin-positive ER. PtdIns(4)P decorates the LCV for a prolonged time (18 h p. i. and beyond) up to when the bacteria exit from the pathogen vacuole and the infected cell (71). Taken together, within 2 h post-infection, the LCV undergoes a PI conversion, replacing the endosomal PtdIns(3)P with the secretory PtdIns(4)P (Figure 3). Importantly, the LCV PI conversion occurs prior to and independently from ER recruitment, and the two compartments appear to remain separate throughout the intracellular life of L. pneumophila.
Mechanistically, the PI conversion on the LCV possibly proceeds along several, mutually non-exclusive pathways: (i) the LCV might communicate and selectively retain PI-decorated vesicles, (ii) L. pneumophila might produce (Icm/Dot-secreted) effectors acting directly as PI interactors, phosphatases or kinases, and/or (iii) the pathogen might subvert host PI metabolizing enzymes (Figure 3). Indeed, using D. discoideum producing fluorescent PtdIns(3)P and PtdIns(4)P probes in tandem, we recently showed by high-resolution real-time confocal laser scanning microscopy that nascent LCVs continuously capture and accumulate PtdIns(4)P-positive vesicles derived from the trans-Golgi network (72). The sustained association of the PtdIns(4)P-positive vesicles, but not the LCV-vesicle interactions per se, require a functional T4SS. Thus, L. pneumophila exploits the cellular dynamics of vesicle-bound PtdIns(4)P for LCV formation. At different stages of infection L. pneumophila effectors might modulate the host PI pattern in different ways (73).
The L. pneumophila Icm/Dot substrate SidC and its paralogue SdcA localize to the LCV membrane (115) and almost exclusively bind to PtdIns(4)P [(51); Figure 4 and Table 1]. The 105 kDa effector proteins harbor a unique 20 kDa C-terminal domain termed P4C [PtdIns(4)P-binding domain of SidC], which does not show similarity to any eukaryotic PI-binding motif and was used as a PtdIns(4)P probe in eukaryotic cells (116,136). SidC and the P4C domain are conserved in Legionella longbeachae, where the 111 kDa effector represents the major PtdIns(4)P binding protein (75). The SidC orthologs of L. pneumophila and L. longbeachae bind PtdIns(4)P with a low dissociation constant (K d ) of ca. 240 or 70 nM, respectively. The crystal structure of SidC revealed a unique PtdIns(4)P-binding domain essential for targeting the effector to the pathogen vacuole (137).
LCVs harboring an L. pneumophila sidC-sdcA mutant strain recruit the ER slower and to a smaller extent; yet, the formation of the spatially separated PtdIns(4)P-positive limiting LCV membrane is not affected (28,51,65,116). The interaction with the ER is catalyzed by a 70 kDa N-terminal fragment of SidC (116). The crystal structure of the N-terminal fragment revealed a novel fold (117,121), comprising a catalytic Cys-His-Asp triad, which is essential for SidC to promote the polyubiquitination of protein substrates on the LCV (118). Indeed, SidC and SdcA act as E3 ubiquitin ligases, which show a broad and nonoverlapping specificity for ubiquitin-conjugating E2 enzymes (118,119). Hence, the L. pneumophila effector SidC links and subverts two different eukaryotic pathways, phosphoinositide and ubiquitination signaling. In L. pneumophila-infected phagocytes, SidC decorates the LCV selectively, uniformly and in copious amounts (51,116). We exploited this feature to isolate LCVs from homogenates of infected host cells by establishing a two-step procedure comprising immuno-affinity enrichment with an anti-SidC antibody, followed by Histodenz density gradient centrifugation (138,139). Using this protocol, intact LCVs were isolated from D. discoideum amoeba (28,140), murine RAW 264.7 macrophage-like cells (24,27) and bone marrow-derived primary macrophages (141). The isolated LCVs were utilized for biochemical fusion experiments (28) and proteomics analysis (24,27,140,141), which identified small GTPases and their effectors (Rab family, Rap1, Ran, RanBP1), large GTPases, components of the endosomal and late secretory trafficking pathways, as well as protein or lipid kinases and phosphatases. LCV localization of some of these proteins was confirmed by fluorescence microscopy using D. discoideum strains producing the corresponding GFP-fusion proteins (24, 26-28, 140, 142).
The Icm/Dot substrate SidM (alias DrrA) localizes to the LCV membrane early during L. pneumophila infection (92) and is the major PtdIns(4)P-binding protein, as it was exclusively identified as such in a non-biased pulldown approach [(84); Figure 4 and Table 1]. In lysates of L. pneumophila sidM, no other PI-binding protein (not even SidC) was identified. The 73 kDa effector protein harbors the 12 kDa C-terminal domain P4M [PtdIns(4)P-binding domain of SidM], which does not show similarity to any eukaryotic PI-binding motif or the P4C domain of SidC, but is shared with two other effectors, Lpg1101 (alias Lem4) and Lpg2603 (alias Lem28) [(102); Table 1]. The P4M domain has been ectopically produced and used as a PtdIns(4)P probe in eukaryotic cells (143) and Drosophila photoreceptor cells (144). The crystal structure of SidM and biochemical analysis revealed a unique PtdIns(4)P-binding domain and a very high binding affinity (K d = 4-18 nM) (40,74).
SidM, i.e., its central domain, exerts GEF activity toward Rab1-GDI complexes, thus leading to GTP loading and Rab1 activation on LCV membranes (38,39,92,(124)(125)(126)(127). Moreover, the N-terminal domain of SidM catalyzes the covalent attachment of AMP to Rab1, a reaction termed AMPylation (128), which renders Rab1(GTP) inaccessible to GAPs and causes the constitutive activation of the small GTPase on LCVs (93). The AMPylation reaction is reversible, and the L. pneumophila effector protein SidD can remove the AMP residue from Rab1 by a deAMPylation reaction (145)(146)(147). The removal of the covalent modification allows the GAP LepB to inactivate Rab1 (92,94). Through activation of Rab1, SidM catalyzes the non-canonical pairing of plasma membrane t-SNARE syntaxin proteins (present on the LCV membrane) with the ER-localized v-SNARE protein Sec22b (148,149). Thus, the SidM-catalyzed activation of Rab1 seems to promote the tethering and fusion of the LCV with ER-derived vesicles, which has been described many years ago (150,151). In summary, the L. pneumophila effector SidM links and subverts two different eukaryotic pathways, phosphoinositide and small GTPase signaling.
The Icm/Dot substrate RidL specifically binds PtdIns(3)P and localizes to the LCV, juxtaposed to where the polar Icm/Dot T4SS connects to the pathogen vacuole membrane [(44); Figure 4 and Table 1]. RidL binds the Vps29 subunit of the retromer coat complex, inhibits retrograde trafficking and thereby promotes intracellular bacterial replication (19,20). Structural studies revealed that a hydrophobic β-hairpin in the N-terminal domain of RidL interacts with Vps29, thus displacing the Rab7 GAP TBC1D5 [a regulator of retrograde trafficking; (43,45,46)].
The Icm/Dot substrate RavZ targets autophagosomes and binds PtdIns(3)P on high-curvature membranes trough a C-terminal domain [(49); Figure 4 and Table 1]. RavZ inhibits autophagy by deconjugating Atg8/LC3 from phosphatidylethanolamine (PE) (48). In contrast to the eukaryotic deconjugating factor Atg4, the cysteine protease RavZ irreversible decouples Atg8 from PE by hydrolyzing the amide bond between the C-terminal glycine and an adjacent aromatic amino acid in Atg8.
The Icm/Dot substrates SetA (110,120) and LtpM (85) localize to LCVs and endosomes through C-terminal PtdIns(3)P-binding domains (Figure 4 and Table 1). The N-terminal domains of these effectors show similarities with glycosyl transferases, and indeed, the purified enzymes were found to exhibit glycohydrolase and glycosyltransferase activity in vitro, using UDP-glucose as a sugar donor. Intriguingly, PtdIns(3)P activates the glycosyltransferase activity of LtpM (85).
The Icm/Dot substrates LtpD (109) and RavD (114) also localize to the LCV through C-terminal PtdIns(3)Pbinding domains ( Table 1). LtpD might bind to the inositol monophosphatase IMPA1, which has indeed been detected on isolated LCVs (140). LpnE is a 41 kDa L. pneumophila virulence factor that binds to PtdIns(3)P and the eukaryotic PI  Figure 4 and Table 1]. The Sel1 repeat-containing LpnE is secreted independently of the Icm/Dot T4SS or the Lsp T2SS and promotes uptake of L. pneumophila by phagocytes and intracellular replication (157,158). Finally, a recent bioinformatics-based screen identified three novel PtdIns(3)P-binding domains, which are present in at least 14 known Icm/Dot substrates, including LepB and RavZ (95).
The crystal structure of the N-terminal catalytic domain in complex with its substrate PtdIns(3,4)P 2 revealed a positively charged groove in the catalytic center, similar to other PI phosphatases harboring the "CX 5 R" motif (123). The 102 kDa effector SidF harbors two predicted C-terminal transmembrane motifs, which anchor the protein to the LCV membrane. SidF specifically hydrolyses in vitro PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 typically occurring on early phagosomes, and it likely contributes to the production of PtdIns(4)P on LCVs, since vacuoles harboring L. pneumophila sidF accumulate lower amounts of the PtdIns(4)P-binding effector SidC. Yet, the sidF mutant strain is not impaired for intracellular growth.
The Icm/Dot substrate SidP acts as a PI 3-phosphatase in vitro and converts PtdIns(3,5)P 2 to PtdIns(5)P as well as PtdIns(3)P to PtdIns (Figure 4 and Table 1). However, its PI-phosphatase activity was not assessed in L. pneumophilainfected cells, and a sidP mutant strain is not impaired for intracellular growth (129). The crystal structure of SidP from L. longbeachae revealed three distinct domains: a large N-terminal catalytic domain, an appendage domain inserted into the catalytic domain, and a C-terminal α-helical domain. Based largely on biochemical studies, SidF and SidP were postulated to produce PtdIns(4)P and hydrolyze PtdIns(3)P on LCVs, thus contributing to the PI conversion on the pathogen vacuole.
The Icm/Dot substrates VipD, PlcC, and LpdA are lipases, which possess broad range activity against phospholipids including mono-phosphorylated PIs (Figure 4 and Table 1). VipD was identified as an Icm/Dot substrate that impairs membrane trafficking in yeast (130,131). The effector hydrolyzes PE as well as phosphatidylcholine (PC) (132) and, intriguingly, binds Rab5 as well as Rab22 and acts as a Rab5-activated phospholipase A 1 (133)(134)(135). Accordingly, VipD removes PtdIns(3)P from endosomal membranes and thus might promote the evasion of the endocytic pathway by LCVs (133,134). Analogously, the Icm/Dot substrate PlcC (alias CegC1) is a metallophospholipase C, which hydrolyzes a broad spectrum of lipids including PC, phosphatidylglycerol (PG), and PtdIns (111). The effector can degrade PtdIns(3)P and likely destabilizes target membranes. Finally, the Icm/Dot substrate LpdA is a phospholipase D that binds to membranes through C-terminal prenylation and hydrolyzes PG, PtdIns and PtdIns(3)P as well as PtdIns(4)P yielding phosphatidic acid (PA) (83). While LpdA does not seem to affect the cellular PI pattern, the phospholipase triggers Golgi fragmentation.

SUBVERSION OF HOST PHOSPHOINOSITIDE KINASES AND PHOSPHATASES BY L. PNEUMOPHILA
In addition to directly modulating PI lipids, L. pneumophila effectors also subvert the host cell PI pattern indirectly by targeting eukaryotic PI phosphatases and kinases (Figure 4). The PtdIns(3)P-binding virulence factor LpnE binds mammalian OCRL (Oculocerebrorenal syndrome of Lowe) and its Dictyostelium homolog Dd5P4 (D. discoideum 5-phosphatase 4) via their N-terminal domains (156). The interaction of LpnE with OCRL was recently confirmed by size exclusion chromatography and supported by the crystal structure of the bacterial protein (161). OCRL and Dd5P4 are PI 5-phosphatases, which hydrolyse PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 to yield PtdIns(4)P and PtdIns(3,4)P 2 , respectively (162, 163). Dd5P4 is likely catalytically active on LCVs and increases the PtdIns(4)P available for binding by effectors such as SidC or SidM (156). Consequently, LpnE might increase the concentration of PtdIns(4)P on LCVs by recruiting OCRL/Dd5P4, and thereby promote PI conversion. L. pneumophila grows more efficiently in D. discoideum lacking Dd5P4, and thus, the pleiotropic PI 5-phosphatase restricts intracellular bacterial growth. Mechanistic details of this process are not known, but Dd5P4 modulates the recruitment of calnexin, Rab1 and retromer components to LCVs, which might account for growth restriction (156,164).
The Icm/Dot substrates RalF and SidM possibly contribute indirectly to the modulation of the LCV PI pattern through the recruitment and activation of small host GTPases. RalF is an Arf1 GEF and activates the small GTPase on the LCV [ (22,112); Figure 4 and Table 1]. RalF harbors a C-terminal globular "capping" domain, which regulates GEF activity by auto-inhibition (77). Activated Arf1 recruits PI 4-kinase IIIβ (PI4KIIIβ) to the trans Golgi network (165), and hence, RalF might indirectly increase the PtdIns(4)P concentration on LCVs. Indeed, the depletion by RNA interference of PI4KIIIβ, but not PI4KIIIα or PI4KIIα decreases the amount of the PtdIns(4)P-binding effector SidC on LCVs, suggesting that in absence of PI4KIIIβ the level of PtdIns(4)P is reduced (84). Analogously, SidM recruits and activates Rab1 on LCVs (see above). Activated Rab1 (166) as well as Arf1 (167) recruit OCRL to endosomal membranes. Accordingly, SidM might not only bind to PtdIns(4)P, but also indirectly contribute to an increase of this PI on LCV membranes.
The Icm/Dot substrates LpdA and LecE localize to LCVs and might also indirectly modulate the LCV PI pattern by promoting DAG biosynthesis [(99); Table 1]. LpdA is a phospholipase D, which hydrolyzes PC to yield PA (see above). LecE enhances the activity of the eukaryotic PA phosphatase Pah1, which dephosphorylates PA yielding DAG. The second messenger DAG recruits protein kinase D (PKD) and its activator protein kinase C (PKC) to membranes. Activated PKD then interacts with PI4KIIIβ, thereby possibly also contributing to an increase in PtdIns(4)P on LCVs (99).

CONCLUSIONS AND OUTLOOK
Legionella pneumophila replicates intracellularly in phagocytes within an LCV, a complex compartment tightly associated with the ER. The nascent LCV undergoes a PI conversion from PtdIns(3)P to PtdIns(4)P, and thereby is rerouted from the bactericidal endocytic to the replication-permissive secretory pathway. To modulate the PI pattern in infected cells, L. pneumophila (i) recruits PI-decorated vesicles, (ii) produces effectors acting as PI interactors, phosphatases, kinases or phospholipases, or (iii) subverts host PI-metabolizing enzymes. To this end, at least 21 T4SS-translocated effector proteins have been shown to target the host PI metabolism ( Table 1). Intriguingly, a number of these effectors harbor 2-3 different functional domains and link PI signaling to other pivotal cellular pathways, e.g., SidC (PI interactor, ubiquitin ligase), SidM (PI interactor, Rab1 GEF, Rab1 AMPylase), LepB (PI interactor, PI 4-kinase, Rab1 GAP), SetA and LtpM (PI interactor, glycosyltransferase), and VipD (Rab5 interactor, phospholipase). LCV formation and the contribution of PI lipids to this process are incompletely understood. Among the more than 300 T4SS-translocated effector proteins of L. pneumophila only about 50 have been thoroughly investigated. Future studies will focus on the structural, molecular and cellular characterization of novel effectors implicated in host cell PI pattern subversion, as well as on the spatiotemporal regulation of effector translocation and function.

AUTHOR CONTRIBUTIONS
ALS and HH wrote the manuscript.

FUNDING
Research in the laboratory of HH was supported by the Swiss National Science Foundation (SNF; 31003A_153200, 31003A_175557), the University of Zürich, the Novartis Foundation for Medical-Biological Research, and the OPO foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.