Subversion of Cell-Autonomous Immunity and Cell Migration by Legionella pneumophila Effectors

Bacteria trigger host defense and inflammatory processes, such as cytokine production, pyroptosis, and the chemotactic migration of immune cells toward the source of infection. However, a number of pathogens interfere with these immune functions by producing specific so-called “effector” proteins, which are delivered to host cells via dedicated secretion systems. Air-borne Legionella pneumophila bacteria trigger an acute and potential fatal inflammation in the lung termed Legionnaires’ disease. The opportunistic pathogen L. pneumophila is a natural parasite of free-living amoebae, but also replicates in alveolar macrophages and accidentally infects humans. The bacteria employ the intracellular multiplication/defective for organelle trafficking (Icm/Dot) type IV secretion system and as many as 300 different effector proteins to govern host–cell interactions and establish in phagocytes an intracellular replication niche, the Legionella-containing vacuole. Some Icm/Dot-translocated effector proteins target cell-autonomous immunity or cell migration, i.e., they interfere with (i) endocytic, secretory, or retrograde vesicle trafficking pathways, (ii) organelle or cell motility, (iii) the inflammasome and programed cell death, or (iv) the transcription factor NF-κB. Here, we review recent mechanistic insights into the subversion of cellular immune functions by L. pneumophila.

Pathogenic bacteria target eukaryotic cells either as adversaries in the case of potentially bactericidal cells of the metazoan immune system and/or as a rich source of nutrients (8,9). Accordingly, pathogens developed means to counteract the arsenal of the humoral and cellular components of the innate and acquired immune system and to survive and replicate within eukaryotic cells, including phagocytes (10). To this end, a number of pathogens produce specific so-called "effector" proteins, which are delivered via dedicated secretion systems into host cells, where they interfere with immune functions and cell migration (11).
Legionella pneumophila employs the intracellular multiplication/defective for organelle trafficking (Icm/Dot) type IV secretion system (T4SS) and as many as 300 different effector proteins to govern host-cell interactions. The role and molecular mode of action of effectors involved in pathogen vacuole formation has recently been reviewed in detail (12). Here, we will review mechanistic insights into the subversion of cell-autonomous immunity and cell migration by L. pneumophila.

Pathogenesis of Legionella pneumophila
Legionella pneumophila is an opportunistic pathogen and causes a severe pneumonia termed Legionnaires' disease. The Gramnegative genus Legionella comprises more than 55 species with several serogroups; yet, at least 85% of human infections are caused by L. pneumophila (13). Evolutionary adaptation allows L. pneumophila to persist in a variety of extra-and intracellular niches. The aerobic bacterium can not only replicate in biofilms but also resists degradation by free-living protozoa and replicates within, e.g., Acanthamoeba, Hartmannella, and Tetrahymena species, as well as in Dictyostelium discoideum, although the latter amoeba is likely not a natural host (14,15). L. pneumophila is ubiquitously found in natural and technical water systems, including cooling towers, whirlpools, and showers.
Upon inhalation of contaminated aerosols, L. pneumophila resists degradation and replicates within alveolar macrophages, which is a precondition for the onset of disease (16). The acquisition of the pathogen from environmental sources is the only infection route; transmission between humans has never been observed. Since L. pneumophila probably mainly evolved as a parasite of free-living protozoa, the human host represents a dead-end for this "accidental" pathogen. Thus, L. pneumophila likely has not been exposed to a rigorous evolutionary selection to avoid recognition by mammalian PRRs, and accordingly, the bacteria trigger the activation of all PRR families (17).
Most humans and mice are able to clear a Legionella infection, and therefore, the development of a suitable small animal model was crucial. Initial studies using guinea pigs exposed to L. pneumophila-containing aerosols revealed a high susceptibility of these animals, which developed an illness reminiscent of typical human Legionnaires' disease (18). While most inbred mouse strains are resistant to L. pneumophila infection and disease progression, the A/J mouse strain was found to be susceptible and to present with acute pneumonia that resembled human disease (19). These results correspond to in vitro infections of peritoneal mouse macrophages, which indicated that cells from A/J mice were much more permissive for intracellular replication of L. pneumophila than macrophages from other mouse strains, such as C57BL/6 and BALB/c (20,21). Macrophages from C57BL/6 and BALB/c mice restrict L. pneumophila by the activation of a programed cell death pathway as an ultimate line of defense against the intracellular pathogen (see below). Accordingly, mice lacking components of this pathway fail to restrict L. pneumophila and faithfully mimic Legionella pathology (17,22).

Formation of the intracellular Replication Niche
In permissive macrophages as well as in protozoa, L. pneumophila employs a complex and apparently evolutionarily conserved mechanism to establish its replication-permissive membranebound niche, the Legionella-containing vacuole (LCV) (Figure 1). LCV formation is governed by the Icm/Dot T4SS, which translocates approximately 300 different effector proteins into the host cell (12,(33)(34)(35)(36)(37). Since L. pneumophila-host interactions are defined to a large extent by the Icm/Dot apparatus, the T4SS represents a major virulence factor of L. pneumophila.

inhibition of Retrograde Trafficking and Autophagy
Retrograde trafficking in eukaryotes comprises the transport from early or late endosomes through the trans-Golgi network (TGN) to the ER (84). The cation-independent mannose 6-phosphate receptor (CI-MPR) is a well-documented substrate of the retrograde transport pathway. CI-MPR binds mannose present on hydrolases and transports the cargo destined for the endocytic pathway from the TGN to the endosomal system. After releasing the cargo in the lysosomal lumen, the receptor is recycled back through the retrograde pathway to the TGN (85). Proper recycling requires a protein complex termed the retromer, composed of a cargo recognition subcomplex (Vps26, Vps29, and Vps35) and a membrane-deforming subcomplex, which consists of a dimer of sorting nexins (SNXs) (86). The cargo recognition complex is recruited to the membrane by the activated small GTPases Rab5 and Rab7 (87).
Recent findings indicate that L. pneumophila communicates with the retrograde trafficking pathway. The small GTPases Rab5 and Rab7 (88)(89)(90)(91), as well as the retromer subunits Vps26, Vps29, and Vps35 (42,92) localize to the LCV. Interestingly, the Icm/ Dot T4SS-translocated substrate RidL functions as a bacterial interactor of the eukaryotic retromer complex (42). RidL, which localizes on LCVs in D. discoideum cells and macrophages, binds to the Vps29 subunit of the cargo recognition subcomplex of the retromer. Subunits of the retromer cargo recognition complex localize to the LCV in an Icm/Dot-dependent but RidLindependent manner. RidL competes with SNX1 and SNX2 in its capacity for membrane binding specifically through PtdIns(3)P, thus possibly triggering the removal of SNXs. By altering the retrograde trafficking cascade, RidL might promote the formation of a non-lysosomal replicative vacuole and intracellular replication of L. pneumophila in protozoan and metazoan phagocytes (42). Earlier work already indicated that the retrograde pathway might control intracellular replication of L. pneumophila. The PtdIns(4,5)P2 5-phosphatase OCRL1 and its D. discoideum homolog Dd5P4 are implicated in retrograde trafficking (93,94) and localize to LCVs (60). Depletion of OCRL by RNA interference (42) or deletion of Dd5P4 (60) increased intracellular growth of L. pneumophila, indicating that the PI 5-phosphatase OCRL/Dd5P4 indeed restricts pathogen replication.
Autophagy is a major cell-autonomous defense mechanism used by infected cells against intracellular bacteria. The process of macroautophagy is responsible for the degradation of cytoplasmic constituents, such as bacteria or damaged organelles, which are engulfed by autophagosomes and subsequently fuse with lysosomes (95). In the course of microautophagy, the constituents are directly delivered to the lysosomes. Over 30 autophagy-related genes (Atg) have been discovered during the last years (96). An Atg protein essential for autophagy is the microtubule-associated protein light chain 3 (LC3, alias Atg8), which is conjugated to phosphatidylethanolamine and localized on autophagosomal membranes (97). Measurement of LC3/Atg8 during L. pneu mophila infection revealed an Icm/Dot-dependent inhibition of autophagy by the bacteria. Intriguingly, the bacterial protein RavZ was identified as an effector required for autophagy inhibition, yet further effectors could be involved, since L. pneumophila lacking ravZ still evaded the autophagy cascade (98). RavZ is a cysteine protease, which irreversibly deconjugates phosphatidylethanolamine from LC3/Atg8, thus reducing its membrane accumulation and activity. While most autophagy factors are not necessary for L. pneumophila replication in D. discoideum (99), amoebae lacking Atg9 do not internalize the pathogen as efficient as wild-type cells, yet allow more effective intracellular growth (100). In summary, these findings suggest that L. pneumophila inhibits retrograde vesicle trafficking as well as autophagy to promote intracellular growth.

Modulation of Organelle Motility and Cell Migration
The Icm/Dot T4SS is crucially involved in the formation of the LCV, and many L. pneumophila effectors selectively decorate the pathogen vacuole membrane. Intact LCVs can be purified by a straight-forward two-step protocol involving immuno-affinity enrichment using an antibody against the L. pneumophila effector SidC selectively decorating the pathogen vacuole and a secondary antibody coupled to magnetic beads, followed by a conventional density centrifugation step. Proteomics analysis of purified preparations of LCVs from infected D. discoideum amoebae (91) and RAW 264.7 macrophages (90) revealed the presence of 670 and 1150 host proteins, respectively, including 13 small GTPases of the Rab family, as well as the small GTPase Ran and its effector Ran binding protein 1 (RanBP1).
Ran is a member of the Ras superfamily of small GTPases and is fundamental in numerous cellular processes, such as nuclear pore translocation (101), or mitotic spindle assembly and postmitotic nuclear envelope formation (102,103). Ran GTPase also plays an important role in cytoplasmic processes involving noncentrosomal microtubules, e.g., endocytic receptor trafficking and retrograde signaling along microtubules in nerve axons (104). Ran can be activated by a nuclear (or in mitotic cells: chromatin bound) Ran GEF termed regulator of chromosome condensation 1 (RCC1) (105). Ran(GTP) is inactivated by the cytoplasmic Ran GTPase-activating protein 1 (RanGAP1) together with RanBP1, which harbors a Ran(GTP)-binding domain (104).
Intriguingly, L. pneumophila produces an Icm/Dottranslocated effector termed LegG1 that harbors eukaryotic-like RCC1 domains (106,107). The corresponding gene is conserved among the L. pneumophila genomes sequenced to date, including the strains Philadelphia-1, Paris, Lens, Corby, Alcoy, and Lorraine. LegG1 (Lpg1976/PieG) is encoded in the plasticity island of effectors (Pie) gene cluster (108). The C-terminal CAAX tetrapeptide motif of LegG1 is lipidated by the host prenylation machinery (109), thus promoting the membrane localization of the effector to small vesicular structures upon ectopic expression in eukaryotic cells (108).
Recent work revealed that LegG1 is indeed an L. pneumophila virulence factor, which promotes intracellular bacterial replication but is dispensable for uptake (110) (Figure 1). A ΔlegG1 mutant strain is not less cytotoxic than the parental strain, but outcompeted by wild-type L. pneumophila upon co-infection of A. castellanii amoebae. Moreover, Ran, RanBP1, and LegG1 accumulate in an Icm/Dot-dependent manner on the LCV, and the bacterial effector localizes to the cytosolic face of the LCV in L. pneumophila-infected phagocytes. L. pneumophila wildtype but not the ΔlegG1 mutant strain activates Ran on LCVs and in cell lysates; yet, while LegG1 promotes the accumulation of RanBP1 on LCVs, the effector is dispensable for the recruitment of the small GTPase. Several experimental approaches indicated that in infected phagocytes L. pneumophila triggers the polymerization of microtubules in a LegG1-dependent manner (110) (Figure 1). "Microbial microinjection" of LegG1 by Yersinia enterocolitica confirmed the positive effect of LegG1 on microtubule stabilization. Here, Y. enterocolitica harboring a T3SS, but lacking endogenous effectors, translocated LegG1 fused to a type III secretion signal into HeLa cells. Furthermore, while LCVs harboring wild-type L. pneumophila vividly move along microtubules in infected D. discoideum, the pathogen vacuole harboring ΔlegG1 mutant bacteria is stalled (110). In summary, the discovery and characterization of the L. pneumophila Ran activator LegG1 revealed an unexpected role of the small GTPase Ran in the formation of pathogen vacuoles.
Microtubule polarization and dynamics represent pivotal determinants of eukaryotic cell migration (111) (Figure 2). Given the prominent role of Ran and LegG1 on the dynamics of the microtubule cytoskeleton, we investigated the effect of L. pneumophila and LegG1 on host cell motility (112). Studies using D. discoideum amoebae or immune cells, such as RAW 264.7 macrophage-like cells or primary PMN, in different migration assays (under-agarose and Boyden chamber assays), revealed an Icm/Dot-dependent inhibition of migration. Phagocytes infected with wild-type L. pneumophila or Legionella longbeachae showed a substantially reduced migration when compared to cells lacking a functional Icm/Dot T4SS. Uptake and cytotoxicity assays demonstrated that the observed effect is not due to a defect in infection.
Based on these findings, the modulation of cell migration by LegG1 was assessed in D. discoideum as well as in RAW 264.7 macrophages or PMN (112) (Figure 1). Interestingly, the ΔlegG1 mutant strain hyper-inhibited the directed migration of phagocytes in the under-agarose assay, even to a larger extent than wild-type L. pneumophila. Overproduction of LegG1 in the ΔlegG1 mutant background re-established the migration range to an extent comparable to cells infected with a ΔicmT mutant strain. Single cell tracking revealed that the forward migration and the velocity of cells infected with wild-type or ΔlegG1 L. pneumophila was impaired. Similarly, upon "microbial microinjection" by Y. enterocolitica, LegG1 was sufficient to stimulate migration of epithelial cells in scratch assays. Moreover, using RNA interference a role of Ran in the LegG1dependent migration inhibition was demonstrated. Upon depletion of Ran, cells infected with the strain overproducing LegG1 were not able to migrate and to close scratch wounds over time (112). Taken together, the Ran activator LegG1 promotes cell motility by modulating microtubule dynamics and thus antagonizes Icm/Dot-dependent inhibition of cell migration. LegG1 might reverse the otherwise deleterious impact of other L. pneumophila effectors on the host cytoskeleton, and thereby Subversion of cellular immune functions by Legionella Frontiers in Immunology | www.frontiersin.org sustain vesicle trafficking and organelle motility required for the establishment and maintenance of LCVs.
In addition to PAMPs, such as flagellin, a number of specific Icm/Dot substrates are implicated in the regulation of programed host cell death. The L. pneumophila PI 3-phosphatase SidF inactivates the pro-apoptotic factors BNIP3 and Bcl-rambo by an unknown mechanism, and thereby counteracts cell death induction (128). Furthermore, the effector SdhA plays a role in maintaining the LCV membrane integrity and also contributes to the prevention of cell death (129,130). Cell death induction in absence of sdhA is suppressed by a secreted bacterial phospholipase A through an unknown mechanism. L. pneumophila lacking sdhA resides in the cytoplasm and triggers caspase-1 activation and IL-1β secretion, as well as macrophage pyroptosis through the DNA-sensing AIM2 inflammasome (131). The Icm/Dot substrate SdhA is also a key suppressor of the type I IFN (IFN-α/β) response to L. pneumophila through nucleic acid-sensing PRRs. Accordingly, RNA from L. pneumophila lacking sdhA triggers the RIG-I-dependent production of type I IFNs (132).
Legionella pneumophila also promotes programed cell death in an Icm/Dot-dependent manner. The phospholipase VipD destabilizes mitochondrial membranes by means of its phospholipase A activity, thus potentially contributing to the release of cytochrome c into the cytosol. This triggers pro-apoptotic caspase-3 activation and subsequently promotes host cell death (133). Analogously, the Icm/Dot-translocated phospholipase PlcC hydrolyzes several lipids, including phosphatidylcholine, phosphatidylglycerol, and phosphatidylinositol, which might destabilize target membranes and cause cell toxicity (134). Stimulation of apoptosis by an intracellular pathogen seems counterintuitive, but might reflect a tight spatial and temporal control of LCV maturation, followed by the release of the bacteria from the pathogen compartment and the host cell at the end of an infection cycle. Thus, the elaborate coordination of anti-and pro-apoptotic factors optimally supports intracellular bacterial proliferation.
Finally, caspases not only regulate cell death during L. pneu mophila infection but also control vesicle trafficking pathways and thus contribute to the formation of LCVs. Dependent on the Nlrc4 inflammasome and Naip5, L. pneumophila activates caspase-7 downstream of caspase-1, and consequently, the pathogen is delivered to lysosomes (117,135). In turn, upon deletion of the caspases, L. pneumophila forms an ER-derived replicative compartment. Similarly (but independently of caspase-1), active caspase-11 restricts the replication of L. pneumophila by promoting the fusion of the pathogen compartment with lysosomes (136).

Activation of the Transcription Factor NF-κB
The transcription factor NF-κB is a master regulator of the mammalian innate immune response and controls the production of anti-apoptotic and pro-survival factors as well as inflammatory mediators (137). Thus, the activation of NF-κB (which is not conserved in amoebae) represents another, more indirect way to prevent host cell death. NF-κB is composed of five Rel family proteins: RelA, RelB, c-Rel, and the precursors p100 and p105, which are processed to their mature forms, p52 and p50. The transcription factor is maintained in its inactive form in the cytoplasm by a family of inhibitors termed IκBs (IκBa, IκBb, IκBe, and Bcl-3).
Bacterial PAMPs, including LPS or flagellin, initiate the NF-κB cascade through TLR2 or TLR5 and the adaptor MyD88. L. pneumophila triggers the production of pro-inflammatory cytokines, such as IL-1β, and the activation of the inflammasome platform through these receptors (138)(139)(140)(141). The deletion of single TLRs does not dramatically alter L. pneumophila infection; yet, mice lacking MyD88 fail to produce cytokines, such as NK cell-derived IFN-γ, and are highly susceptible to L. pneumophila infection (142)(143)(144)29). Moreover, mice lacking the IL-1 receptor are impaired for PMN recruitment and bacterial clearance (145). Therefore, IL-1R/MyD88-dependant signaling is critical for host resistance to L. pneumophila infection.
The NF-κB pathway can be activated not only by bacterial PAMPs via a TLR-dependent pathway but also by distinct bacterial effectors (146). The Icm/Dot T4SS and its substrates LnaB and LegK1 strongly induce NF-κB (147-149). While the molecular mechanism of the novel effector LnaB is not understood, LegK1 is a Ser/Thr kinase that phosphorylates the NF-κB inhibitor IκBα, leading to a robust NF-κB activation by triggering the release and nuclear translocation of the transcription factor (150). Since L. pneumophila lacking legK1 is not impaired for intracellular replication, other effectors might also modulate the NF-κB response. Among the five L. pneumophila Icm/Dot substrates that show protein kinase activity in vitro (LegK1-LegK5), LegK2 is a virulence factor, which promotes intracellular replication and efficient recruitment of the ER to LCVs (151).
Translation inhibition by the Icm/Dot substrates Lgt1-3, SidI, and SidL also specifically decreases the production of IκB, even though the effectors are overall cytotoxic (152,153). To shut down translation, the UDP-glucosyltransferases Lgt1-3 modify the elongation factor eEF1A (154,155), and SidI inactivates eEF1A/eEF1Bγ by an unknown mechanism (153). Finally, L. pneumophila also modulates host transcription more directly. The Icm/Dot substrate RomA/LegAS4 promotes intracellular replication as a SET domain-containing histone methyltransferase that modifies (immune) gene expression (156,157). In summary, L. pneumophila modulates protein production and turnover through the activation of NF-κB and by altering the epigenetic pattern, as well as through the inhibition of translation.

Conclusion and Outlook
The ubiquitous environmental bacterium L. pneumophila triggers an acute and potential fatal pneumonia termed Legionnaires' disease. The opportunistic pathogen employs the Icm/Dot T4SS and as many as 300 different effector proteins to govern interactions with phagocytes and form an intracellular replication niche, the LCV. Some Icm/Dot-translocated effector proteins interfere with (i) endocytic, secretory, or retrograde vesicle trafficking pathways, (ii) organelle or cell motility, (iii) the inflammasome and programed cell death, or (iv) the transcription factor NF-κB. Future studies will address the molecular mechanisms of action of the many Icm/Dot substrates, which remain uncharacterized to date. The further analysis of L. pneumophila effector proteins will likely continue to provide novel insights into the elaborate pathogen-host interactions between a highly adapted opportunistic pathogen and its phagocytic host cells.