Formation of the Legionella Replicative Compartment at the Crossroads of Retrograde Trafficking
Kevin Bärlocher
Amanda Welin
Hubert Hilbi- Institute of Medical Microbiology, University of Zürich, Zurich, Switzerland
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.
Components of the Retrograde Trafficking Pathway
Eukaryotic cells employ complex endomembrane systems to process internalized material and transport newly synthesized proteins from the endoplasmic reticulum (ER) via the Golgi apparatus to target organelles and membranes (Johannes and Wunder, 2011). The sub-compartments of these endocytic and secretory (anterograde) pathways, respectively, communicate through vesicular trafficking. Endosomes represent sorting hubs, where the endocytic, secretory and lysosomal pathways meet and diverge.
Retrograde transport from endosomes back to the ER maintains organelle integrity, prevents lysosomal degradation and recycles cargo receptors of the secretory transport machinery (Johannes and Popoff, 2008; Seaman, 2012; Burd and Cullen, 2014; Lu and Hong, 2014; Progida and Bakke, 2016). Dependent on the cargo and various endogenous factors, retrograde transport can occur from different endosomal donor compartments such as early, late and recycling endosomes (EE, LE, RE) as well as the tubular endosomal network (TEN) (Bonifacino and Rojas, 2006; Figure 1). Each membrane is defined by the presence of specific mono- or polyphosphorylated phosphoinositide (PI) lipids and small GTPases of the Rab family, which together determine the spatio-temporal recruitment of specific transport machineries (Di Paolo and De Camilli, 2006; Wandinger-Ness and Zerial, 2014).
Figure 1. Positioning of the Legionella-containing vacuole in the retrograde route. Tubular/vacuolar early endosomes (EE; green) represent a sorting hub, where endosomal, recycling and retrograde trafficking pathways emerge to deliver cargo to late and recycling endosomes, the plasma membrane or the trans-Golgi network (TGN). The pathways employ distinct transport machineries (indicated with gray arrows and boxes), including small Rab GTPases, the PI 5-phosphatase OCRL, the retromer and sorting nexins (SNXs). Some of these retrograde trafficking components as well as retrograde cargos (MPR, sortilin) are also found on the LCV (indicated in red), suggesting that the pathogen vacuole is participating in these pathways. Possible retrograde transport routes involving the LCV are shown with red arrows. Inhibition of retrograde trafficking by RidL targeting the retromer might occur in cis, considering the LCV as (A) acceptor or (B) donor compartment for retrograde transport vesicles, or (C) in trans with the endosome as donor compartment. A possible role in LCV formation of late secretory (anterograde) trafficking emanating from the TGN is shown in blue. For details see text.
The PI 5-phosphatase OCRL (Oculocerebrorenal syndrome of Lowe) is required for retrograde trafficking from EEs to the trans-Golgi network (TGN) and for receptor recycling between endosomes and the plasma membrane (PM) (Noakes et al., 2011; Vicinanza et al., 2011; Sharma et al., 2015). OCRL and its Dictyostelium homolog Dd5P4 (D. discoideum 5-phosphatase 4) hydrolyze PtdIns(4,5)P2 and PtdIns(3,4,5)P3 to yield PtdIns(4)P and PtdIns(3,4)P2 (Zhang et al., 1995; Loovers et al., 2007). OCRL binds to clathrin-coated pits (Ungewickell et al., 2004; Mao et al., 2009) as well as to a range of Rab GTPases including Rab5 on EEs (Hyvola et al., 2006; Fukuda et al., 2008; Hou et al., 2011).
Cargo shuttled via the retrograde endosome-to-TGN pathway comprises many different membrane and soluble proteins as well as lipids. Membrane protein cargos include Vps10 domain family cargo receptors such as cation-dependent or cation-independent mannose 6-phosphate receptor (CD- or CI-MPR) and sortilin/Vps10 (Seaman et al., 1997; Seaman, 2007). Soluble protein cargos include well-studied bacterial AB toxins, such as Shiga toxin (STxB) and cholera toxin (CTxB), which bind to the cell surface glycosphingolipids Gb3 (Lingwood, 1993) or GM1 (Kuziemko et al., 1996), respectively, and hijack the retrograde trafficking pathway to reach the host cell cytosol.
A key component of retrograde trafficking is the retromer complex consisting of a Vps26-Vps29-Vps35 heterotrimer (herein referred to as “retromer”) and different combinations of sorting nexin (SNX) proteins, which harbor a PI-binding PX (phox homology) domain (Cullen and Korswagen, 2011; Teasdale and Collins, 2012). Additional factors required for retromer recruitment are retrograde cargo receptors and the small GTPase Rab7a, a regulator of late endosome dynamics, in its activated, GTP-bound form (Rojas et al., 2008; Lucas et al., 2016). The binding of Rab7a to the membrane is negatively regulated by the GTPase activating protein (GAP) TBC1D5, which itself binds to Vps29 (Seaman et al., 2009; Harrison et al., 2014; Jia et al., 2016).
The retromer interacts with the membrane-associated heterodimer of SNX-BAR subfamily members SNX1/2 and SNX5/6, which harbor an additional dimerization and membrane curvature sensing BAR (Bin/Amphiphysin/Rvs) domain (Rojas et al., 2007; Wassmer et al., 2007; Frost et al., 2009). Together with further accessory proteins including the WASH complex (Gomez and Billadeau, 2009; Harbour et al., 2010), EHD1/dynamin large GTPases (Gokool et al., 2007) and p150glued/microtubule motor dynein (Wassmer et al., 2009), the retromer-SNX-BAR complex gives rise to tubule formation, elongation, scission, and transport of vesicles along microtubules to receiver compartments (Carlton et al., 2004; van Weering et al., 2012).
SNXs also include the SNX-PX subfamily member SNX3, which comprises of only a PX domain, binds to Vps35-Vps26 and is necessary for retromer recruitment to the endosomal membrane and the recognition of some cargo (Chen et al., 2013; Harrison et al., 2014; Lucas et al., 2016). The SNX-FERM subfamily member SNX27 interacts with cargo, Vps26, SNX1-SNX2, and the WASH complex (Temkin et al., 2011; Steinberg et al., 2013; Gallon et al., 2014). The SNX27-retromer complex is necessary for Rab4-dependent endosome-to-PM recycling of different cargos including many transporters such as glucose transporter 1 (GLUT1) (Steinberg et al., 2013).
After un-coating of the transport carrier, different components such as tethering factors and SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) proteins are necessary for selective delivery to target membranes (Progida and Bakke, 2016). Thus, eukaryotic cells employ many distinct types of cargo-specific retrograde trafficking machinery to retrieve proteins and lipids from endosomes for recycling and to maintain organelle integrity.
Formation and Maturation of the Legionella-Containing Vacuole
The opportunistic pathogen L. pneumophila is the clinically most relevant Legionella species causing the severe pneumonia Legionnaires' disease (Bangsborg, 1997; Newton et al., 2010). Legionella spp. are ubiquitous Gram-negative environmental bacteria that colonize both natural and technical water systems, periodically causing disease outbreaks tracing back to cooling towers, whirlpools and showers. In the environment, L. pneumophila survives in both extra- and intracellular niches, i.e., the bacteria colonize biofilms, but also parasitize free-living protozoa (Hilbi et al., 2011). Natural hosts for L. pneumophila include Acanthamoeba, Hartmannella, and Tetrahymena species, and the bacterium also thrives within Dictyostelium discoideum, a commonly employed protozoan experimental host (Hoffmann et al., 2014b).
Through an evolutionarily conserved mechanism, L. pneumophila avoids killing by protozoan and mammalian phagocytes alike, allowing replication within a distinct intracellular compartment termed the Legionella-containing vacuole (LCV) (Isberg et al., 2009; Hubber and Roy, 2010; Hilbi and Haas, 2012; Sherwood and Roy, 2013; Finsel and Hilbi, 2015). Accidental infection of humans occurs through the inhalation of contaminated aerosols, although recently, a probable person-to-person transmission was reported for the first time (Correia et al., 2016). If the alveolar macrophages fail to eliminate the invading bacteria through cell-autonomous processes, intracellular replication of the bacteria will ensue, possibly culminating in a severe pneumonia (Simon and Hilbi, 2015).
The formation of the LCV is a complex process governed by the Icm/Dot type IV secretion system (T4SS). This T4SS injects more than 300 different “effector” proteins into the host cell, dictating every step from uptake to interference with various vesicle trafficking pathways and autophagy, interaction with the ER, and finally escape from the host cell (Finsel and Hilbi, 2015; Ensminger, 2016). To this end, the Icm/Dot-translocated proteins subvert numerous host cell targets, including PI lipids, which are used by several effectors as membrane anchors, and small GTPases (Haneburger and Hilbi, 2013; Rothmeier et al., 2013; Finsel and Hilbi, 2015). LCVs avoid luminal acidification and fusion with lysosomes, but continuously and extensively communicate with multiple vesicle trafficking routes (Horwitz, 1983; Urwyler et al., 2009a; Xu et al., 2010; Zhao et al., 2017).
Interaction with the endocytic pathway is indicated by the presence of the small GTPases Rab5a, Rab7a, and Rab21 on the LCV membrane (Urwyler et al., 2009b; Hoffmann et al., 2014a). Moreover, LCVs acquire PtdIns(3)P within 1 min of bacterial uptake, and gradually lose this PI lipid within 2 h (Weber et al., 2014). In the endocytic pathway, PtdIns(3)P is a crucial regulator, necessary for recruitment of early endosomal antigen 1 (EEA1) and for downstream events leading to fusion of the phagosome with bactericidal lysosomes (Stenmark, 2009).
A hallmark of LCV maturation is the intimate interaction of the pathogen vacuole with the ER (Swanson and Isberg, 1995; Lu and Clarke, 2005; Robinson and Roy, 2006), as a consequence of Rab1-dependent recruitment of secretory vesicles at ER exit sites (Kagan and Roy, 2002; Arasaki and Roy, 2010; Arasaki et al., 2012). PtdIns(4)P is a major regulator of secretory vesicle trafficking through the Golgi apparatus (Jean and Kiger, 2012), required for late steps of endocytosis (Jeschke et al., 2015) and present on LCVs (Weber et al., 2006, 2014). In fact, PtdIns(4)P transiently localizes to LCVs independently of the Icm/Dot T4SS immediately following bacterial uptake, but is then rapidly cleared. Over the following 2 h, PtdIns(4)P again accumulates on LCVs in an Icm/Dot-dependent manner, preceding attachment of the ER (Weber et al., 2014). In addition to Rab1, several other small GTPases involved in the secretory pathway are present on the LCV (Urwyler et al., 2009b; Hoffmann et al., 2014a). Of these, Rab8a, Rab10, and Rab32, all implicated in Golgi to endosome trafficking, promote intracellular replication of L. pneumophila (Hoffmann et al., 2014a). Thus, L. pneumophila exploits the secretory trafficking pathway to promote formation of the replication-permissive LCV.
Restriction of Intracellular Replication of L. pneumophila by Retrograde Trafficking
A number of intracellular bacterial pathogens subvert retrograde trafficking (Personnic et al., 2016). These include the obligate intracellular pathogen Chlamydia trachomatis (Aeberhard et al., 2015; Mirrashidi et al., 2015), facultative intracellular Salmonella enterica serovar Typhimurium (McGourty et al., 2012), and Coxiella burnetii (McDonough et al., 2013).
An early study using D. discoideum already implicated a role for retrograde trafficking during intracellular replication of L. pneumophila (Weber et al., 2009). This study revealed that the PI 5-phophatase OCRL/Dd5P4 restricts intracellular growth of L. pneumophila, while promoting the accumulation of ER on LCVs as well as the transition from “tight” to “spacious” pathogen vacuoles (Weber et al., 2009). In a more recent study, depletion of individual components of the retrograde machinery, such as the retromer subunits Vps26a/Vps26b or Vps29, or the retrograde cargo CI-MPR, led to increased intracellular replication of L. pneumophila (Finsel et al., 2013). Among the small Rab GTPases implicated in retrograde trafficking, Rab5a but not Rab7a, Rab9a or Rab11a restrict intracellular replication (Hoffmann et al., 2014a). Furthermore, in D. discoideum the TBC1D5 homolog promotes intracellular growth of L. pneumophila (Bärlocher et al., 2017). Since mammalian TBC1D5 functions as a Rab7 GAP and negatively regulates Rab7a, which is implicated in retrograde trafficking, these findings corroborate a function of the retrograde pathway in restricting L. pneumophila replication.
Interaction of LCVs with the Retrograde Trafficking Pathway
Proteomics analysis of isolated LCVs from infected protozoan and mammalian host cells revealed components of the retrograde trafficking pathway including small GTPases (Shevchuk et al., 2009; Urwyler et al., 2009b; Hoffmann et al., 2014a; Herweg et al., 2015; Schmölders et al., 2017). Whereas the late endosomal marker Rab9 is present on L. pneumophila wild-type as well as ΔicmT mutant-containing vacuoles, Rab4, Rab5, Rab7, and Rab11 are recruited to LCVs in an Icm/Dot-dependent manner (Finsel et al., 2013; Hoffmann et al., 2014a; Bärlocher et al., 2017). Rab4 and Rab11 associate predominantly with EE/RE or with RE/TGN, and are involved in EE/RE-to-PM or RE-to-PM/TGN trafficking, respectively (Zerial and McBride, 2001; Miserey-Lenkei et al., 2007; Figure 1).
With respect to retromer components, Vps26, Vps29, and Vps35 as well as SNX1, SNX2, SNX5, and TBC1D5 can be detected on LCVs (Finsel et al., 2013; Bärlocher et al., 2017). Moreover, retrograde cargos including Vps10, MPR, and Vamp4 are present on the pathogen vacuole (Finsel et al., 2013; Hoffmann et al., 2014a; Figure 1). The CI-MPR is shuttled between endosomes and the TGN in retromer-, SNX1/2- as well as Rab7b- and Rab9-dependent pathways (Lombardi et al., 1993; Arighi et al., 2004; Rojas et al., 2007; Progida et al., 2010, 2012). The SNARE Vamp4 is a retrograde cargo predominantly found at the TGN, which cycles to the cell surface and back via EE/RE (Tran et al., 2007).
The L. pneumophila Icm/Dot substrate RidL (Retromer interactor decorating LCVs) was identified as a binding partner of the retromer complex, and the effector protein promotes intracellular bacterial replication (Finsel et al., 2013). Upon infection of D. discoideum or macrophages, RidL decorates the LCV membrane and accumulates preferentially at the bacterial poles. Pulldown and protein-lipid overlay experiments revealed that RidL interacts specifically with the retromer Vps29 subunit and the PI lipid PtdIns(3)P, respectively. Ectopically produced RidL inhibits retrograde trafficking of fluorescently labeled STxB in HeLa cells, as evidenced by reduced Golgi localization of the toxin. Furthermore, in macrophages infected with wild-type L. pneumophila, retrograde trafficking of fluorescently labeled CTxB is impaired in a RidL-dependent manner. In presence of RidL, CTxB colocalizes with an EE/RE marker (transferrin receptor), but not with markers for lysosomes (dextran) or the Golgi apparatus (GM130). In absence of RidL, however, the toxin colocalizes with both EE/RE and Golgi markers, but not with the lysosome marker, consistent with a RidL-dependent inhibition of retrograde trafficking at endosome exit sites (Finsel et al., 2013).
A recent study revealed that the 29 kDa N-terminal domain of RidL (RidL2-281) adopts a new “foot-like” fold comprising a protruding hydrophobic β-hairpin at its “heel,” which interacts with Vps29 (Bärlocher et al., 2017). In HeLa cells, the fragment RidL9-258 co-localizes with the retromer complex and displaces the Rab7 GAP TBC1D5 from the retromer. Similarly, RidL translocated by L. pneumophila reduces the amount of TBC1D5 on LCVs during infection of D. discoideum. Thus, displacement of TBC1D5 from the Vps29 retromer subunit by the hydrophobic β-hairpin of RidL might contribute to effector function.
The LCV as an Acceptor Compartment in the Retrograde Trafficking Pathway
Identification of the site of action of RidL and the position of the LCV in the retrograde trafficking route are pivotal for an understanding of effector function (Figure 1). Further insights into the role of RidL were obtained by studying the effects of ridL deletion on the levels of retrograde trafficking components on LCVs (Finsel et al., 2013). Deletion of ridL does not affect the localization of the retromer subunits to the LCV, but the retrograde cargos Vps10 (a D. discoideum homolog of mammalian sortilin) and CI-MPR decorate a larger portion of LCVs. Moreover, the late endosomal/lysosomal marker LAMP1 increasingly localizes to LCVs in the absence of RidL. A larger portion of LCVs also stain positive for SNX1 and SNX2 in the absence of RidL, possibly because RidL competes with the SNXs for PtdIns(3)P binding. Additionally, the level of the retrograde trafficking regulator and Rab7 GAP TBC1D5 is increased on LCVs in absence of RidL (Bärlocher et al., 2017). Collectively, these findings suggest that RidL and its inhibition of retrograde trafficking results in decreased levels of retrograde cargos and some components of the retrograde machinery on LCVs.
Regarding the position of the LCV in the retrograde trafficking route different scenarios are conceivable. The LCV might represent a donor compartment in the retrograde trafficking route. Blockage of the pathway at the LCV by RidL would prevent vesicles from budding, elongation or scission, resulting in increased levels of retrograde components on the LCV in the presence of RidL (Figure 1). However, this is the opposite of what was observed, and therefore, it is unlikely that the LCV is a donor compartment in the retrograde pathway.
Alternatively, the LCV might be an acceptor compartment of retrograde trafficking vesicles (Figure 1). Blockage of incoming vesicles by RidL would prevent tethering, un-coating, attachment or fusion, leading to decreased levels of retrograde cargo, as observed (Finsel et al., 2013). Under these circumstances, the components of the retrograde transport machinery on the LCV would likely not be directly affected, unless incomplete vesicle un-coating or “kiss-and-run” interactions take place (Trahey and Hay, 2010). Furthermore, the presence of Rab4 (EE) and Rab11 (RE) on the LCV is also in agreement with the pathogen vacuole being a compartment accepting incoming retrograde trafficking from endosomes.
Interestingly, the retrograde cargos STxB and CTxB do not accumulate on LCVs, regardless of whether RidL is present or not (Finsel et al., 2013), and thus, the pathogen vacuole seems to serve as an acceptor compartment in some retrograde routes, e.g., the CI-MPR trafficking pathway, but not in others. At this point, we cannot rule out that the LCV is an acceptor compartment in the secretory pathway. Inhibition of retrograde trafficking by RidL might also reduce anterograde delivery of cargos such as MPR or Vps10/sortilin to the LCV by affecting the whole transport cycle (Riederer et al., 1994; Arighi et al., 2004). However, given the accumulation of retrograde machinery on LCVs such an indirect scenario seems less plausible.
Conclusions and Perspectives
The retrograde vesicle trafficking pathway appears to represent a pivotal component of cell-autonomous immunity, since several bacterial pathogens produce effector proteins subverting the pathway. L. pneumophila is unique in translocating an effector, RidL, which directly targets the eukaryotic retromer complex (Finsel et al., 2013). RidL binds the Vps29 retromer subunit, inhibits retrograde vesicle trafficking and promotes intracellular bacterial replication. The 29 kDa N-terminal domain of RidL adopts a novel fold including a hydrophobic β-hairpin, which binds Vps29, thereby displacing the Rab7 GAP TBC1D5 and contributing to subversion of retrograde trafficking in L. pneumophila-infected cells (Bärlocher et al., 2017).
The exact mode of action of RidL remains to be determined. While its 29 kDa N-terminal fragment is sufficient to displace TBC1D5, the C-terminal 102 kDa fragment of RidL likely harbors additional (perhaps catalytic) activities. Indeed, several L. pneumophila Icm/Dot substrates are large (>80 kDa) and represent multi-domain proteins with distinct (catalytic) activities (Itzen and Goody, 2011; Sherwood and Roy, 2013; Finsel and Hilbi, 2015). Thus, in addition to anchoring to Vps29 and displacing TBC1D5, RidL likely has other functions contributing to the inhibition of retrograde trafficking.
Moreover, the site of RidL action and the position of the LCV in the retrograde trafficking route are ill-defined at present. Cargos of the retrograde trafficking route (e.g., CI-MPR, sortilin) as well as the retromer subunits and the bacterial effector localize to LCVs. Yet, the retrograde toxin cargos STxB or CTxB cannot be detected on the LCV membrane, although their trafficking is affected by RidL. Due to its presence on the pathogen vacuole, RidL seems to act in cis (on the LCV) but might also function in trans (in a distance from the pathogen vacuole). Most available data is in agreement with the pathogen vacuole being an acceptor compartment in the retrograde trafficking pathway, such that RidL blocks the productive interaction of the LCV with incoming retrograde vesicles in cis, rather than inhibits the formation of retrograde transport vesicles at endosome exit sites in trans (Figure 1). Further studies will address the molecular function of RidL and the position of the LCV along the retrograde route.
Author Contributions
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgments
Research in the laboratory of HH was supported by the Swiss National Science Foundation (SNF; 31003A_153200), the University of Zürich, the Novartis Foundation for Medical-Biological Research, the OPO foundation, and the German Ministry of Education and Research (BMBF) in the context of the EU Infect-ERA initiative (project EUGENPATH). AW was supported by a grant from the Swedish Research Council (2014-396). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Abbreviations
Icm/Dot, intracellular multiplication/defective organelle trafficking; LCV, Legionella-containing vacuole; OCRL, Oculocerebrorenal syndrome of Lowe; PI, phosphoinositide; PtdIns, phosphatidylinositol; SNARE, soluble N-ethylmaleimide-sensitive factor attachment receptor; SNX, sorting nexin; T4SS, type IV secretion system; TGN, trans-Golgi network.
References
Aeberhard, L., Banhart, S., Fischer, M., Jehmlich, N., Rose, L., Koch, S., et al. (2015). The proteome of the isolated Chlamydia trachomatis containing vacuole reveals a complex trafficking platform enriched for retromer components. PLoS Pathog. 11:e1004883. doi: 10.1371/journal.ppat.1004883
Arasaki, K., and Roy, C. R. (2010). Legionella pneumophila promotes functional interactions between plasma membrane syntaxins and Sec22b. Traffic 11, 587–600. doi: 10.1111/j.1600-0854.2010.01050.x
Arasaki, K., Toomre, D. K., and Roy, C. R. (2012). The Legionella pneumophila effector DrrA is sufficient to stimulate SNARE-dependent membrane fusion. Cell Host Microbe 11, 46–57. doi: 10.1016/j.chom.2011.11.009
Arighi, C. N., Hartnell, L. M., Aguilar, R. C., Haft, C. R., and Bonifacino, J. S. (2004). Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor. J. Cell Biol. 165, 123–133. doi: 10.1083/jcb.200312055
Bangsborg, J. M. (1997). Antigenic and genetic characterization of Legionella proteins: contributions to taxonomy, diagnosis and pathogenesis. APMIS Suppl. 70, 1–53.
Bärlocher, K., Hutter, C. A. J., Swart, A. L., Steiner, B., Welin, A., Hohl, M., et al. (2017). Structural insights into Legionella RidL-Vps29 retromer subunit interaction reveal displacement of the regulator TBC1D5. Nat. Commun. 8:1543. doi: 10.1038/s41467-017-01512-5
Bonifacino, J. S., and Rojas, R. (2006). Retrograde transport from endosomes to the trans-Golgi network. Nat. Rev. Mol. Cell Biol. 7, 568–579. doi: 10.1038/nrm1985
Burd, C., and Cullen, P. J. (2014). Retromer: a master conductor of endosome sorting. Cold Spring Harb. Perspect. Biol. 6:a016774. doi: 10.1101/cshperspect.a016774
Carlton, J., Bujny, M., Peter, B. J., Oorschot, V. M., Rutherford, A., Mellor, H., et al. (2004). Sorting nexin-1 mediates tubular endosome-to-TGN transport through coincidence sensing of high-curvature membranes and 3-phosphoinositides. Curr. Biol. 14, 1791–1800. doi: 10.1016/j.cub.2004.09.077
Chen, C., Garcia-Santos, D., Ishikawa, Y., Seguin, A., Li, L., Fegan, K. H., et al. (2013). SNX3 regulates recycling of the transferrin receptor and iron assimilation. Cell Metab. 17, 343–352. doi: 10.1016/j.cmet.2013.01.013
Correia, A. M., Ferreira, J. S., Borges, V., Nunes, A., Gomes, B., Capucho, R., et al. (2016). Probable person-to-person transmission of Legionnaires' disease. N. Engl. J. Med. 374, 497–498. doi: 10.1056/NEJMc1505356
Cullen, P. J., and Korswagen, H. C. (2011). Sorting nexins provide diversity for retromer-dependent trafficking events. Nat. Cell Biol. 14, 29–37. doi: 10.1038/ncb2374
Di Paolo, G., and De Camilli, P. (2006). Phosphoinositides in cell regulation and membrane dynamics. Nature 443, 651–657. doi: 10.1038/nature05185
Ensminger, A. W. (2016). Legionella pneumophila, armed to the hilt: justifying the largest arsenal of effectors in the bacterial world. Curr. Opin. Microbiol. 29, 74–80. doi: 10.1016/j.mib.2015.11.002
Finsel, I., and Hilbi, H. (2015). Formation of a pathogen vacuole according to Legionella pneumophila: how to kill one bird with many stones. Cell. Microbiol. 17, 935–950. doi: 10.1111/cmi.12450
Finsel, I., Ragaz, C., Hoffmann, C., Harrison, C. F., Weber, S., Van Rahden, V. A., et al. (2013). The Legionella effector RidL inhibits retrograde trafficking to promote intracellular replication. Cell Host Microbe 14, 38–50. doi: 10.1016/j.chom.2013.06.001
Frost, A., Unger, V. M., and De Camilli, P. (2009). The BAR domain superfamily: membrane-molding macromolecules. Cell 137, 191–196. doi: 10.1016/j.cell.2009.04.010
Fukuda, M., Kanno, E., Ishibashi, K., and Itoh, T. (2008). Large scale screening for novel Rab effectors reveals unexpected broad Rab binding specificity. Mol. Cell. Proteomics 7, 1031–1042. doi: 10.1074/mcp.M700569-MCP200
Gallon, M., Clairfeuille, T., Steinberg, F., Mas, C., Ghai, R., Sessions, R. B., et al. (2014). A unique PDZ domain and arrestin-like fold interaction reveals mechanistic details of endocytic recycling by SNX27-retromer. Proc. Natl. Acad. Sci. U.S.A. 111, E3604–E3613. doi: 10.1073/pnas.1410552111
Gokool, S., Tattersall, D., and Seaman, M. N. (2007). EHD1 interacts with retromer to stabilize SNX1 tubules and facilitate endosome-to-Golgi retrieval. Traffic 8, 1873–1886. doi: 10.1111/j.1600-0854.2007.00652.x
Gomez, T. S., and Billadeau, D. D. (2009). A FAM21-containing WASH complex regulates retromer-dependent sorting. Dev. Cell 17, 699–711. doi: 10.1016/j.devcel.2009.09.009
Haneburger, I., and Hilbi, H. (2013). Phosphoinositide lipids and the Legionella pathogen vacuole. Curr. Top. Microbiol. Immunol. 376, 155–173. doi: 10.1007/82_2013_341
Harbour, M. E., Breusegem, S. Y., Antrobus, R., Freeman, C., Reid, E., and Seaman, M. N. (2010). The cargo-selective retromer complex is a recruiting hub for protein complexes that regulate endosomal tubule dynamics. J. Cell Sci. 123, 3703–3717. doi: 10.1242/jcs.071472
Harrison, M. S., Hung, C. S., Liu, T. T., Christiano, R., Walther, T. C., and Burd, C. G. (2014). A mechanism for retromer endosomal coat complex assembly with cargo. Proc. Natl. Acad. Sci. U.S.A. 111, 267–272. doi: 10.1073/pnas.1316482111
Herweg, J. A., Hansmeier, N., Otto, A., Geffken, A. C., Subbarayal, P., Prusty, B. K., et al. (2015). Purification and proteomics of pathogen-modified vacuoles and membranes. Front. Cell. Infect. Microbiol. 5:48. doi: 10.3389/fcimb.2015.00048
Hilbi, H., and Haas, A. (2012). Secretive bacterial pathogens and the secretory pathway. Traffic 13, 1187–1197. doi: 10.1111/j.1600-0854.2012.01344.x
Hilbi, H., Hoffmann, C., and Harrison, C. F. (2011). Legionella spp. outdoors: colonization, communication and persistence. Environ. Microbiol. Rep. 3, 286–296. doi: 10.1111/j.1758-2229.2011.00247.x
Hoffmann, C., Finsel, I., Otto, A., Pfaffinger, G., Rothmeier, E., Hecker, M., et al. (2014a). Functional analysis of novel Rab GTPases identified in the proteome of purified Legionella-containing vacuoles from macrophages. Cell. Microbiol. 16, 1034–1052. doi: 10.1111/cmi.12256
Hoffmann, C., Harrison, C. F., and Hilbi, H. (2014b). The natural alternative: protozoa as cellular models for Legionella infection. Cell. Microbiol. 16, 15–26. doi: 10.1111/cmi.12235
Horwitz, M. A. (1983). The Legionnaires' disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J. Exp. Med. 158, 2108–2126. doi: 10.1084/jem.158.6.2108
Hou, X., Hagemann, N., Schoebel, S., Blankenfeldt, W., Goody, R. S., Erdmann, K. S., et al. (2011). A structural basis for Lowe syndrome caused by mutations in the Rab-binding domain of OCRL1. EMBO J. 30, 1659–1670. doi: 10.1038/emboj.2011.60
Hubber, A., and Roy, C. R. (2010). Modulation of host cell function by Legionella pneumophila type IV effectors. Annu. Rev. Cell Dev. Biol. 26, 261–283. doi: 10.1146/annurev-cellbio-100109-104034
Hyvola, N., Diao, A., McKenzie, E., Skippen, A., Cockcroft, S., and Lowe, M. (2006). Membrane targeting and activation of the Lowe syndrome protein OCRL1 by Rab GTPases. EMBO J. 25, 3750–3761. doi: 10.1038/sj.emboj.7601274
Isberg, R. R., O'connor, T. J., and Heidtman, M. (2009). The Legionella pneumophila replication vacuole: making a cosy niche inside host cells. Nat. Rev. Microbiol. 7, 13–24. doi: 10.1038/nrmicro1967
Itzen, A., and Goody, R. S. (2011). Covalent coercion by Legionella pneumophila. Cell Host Microbe 10, 89–91. doi: 10.1016/j.chom.2011.08.002
Jean, S., and Kiger, A. A. (2012). Coordination between Rab GTPase and phosphoinositide regulation and functions. Nat. Rev. Mol. Cell Biol. 13, 463–470. doi: 10.1038/nrm3379
Jeschke, A., Zehethofer, N., Lindner, B., Krupp, J., Schwudke, D., Haneburger, I., et al. (2015). Phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate regulate phagolysosome biogenesis. Proc. Natl. Acad. Sci. U.S.A. 112, 4636–4641. doi: 10.1073/pnas.1423456112
Jia, D., Zhang, J. S., Li, F., Wang, J., Deng, Z., White, M. A., et al. (2016). Structural and mechanistic insights into regulation of the retromer coat by TBC1d5. Nat. Commun. 7:13305. doi: 10.1038/ncomms13305
Johannes, L., and Popoff, V. (2008). Tracing the retrograde route in protein trafficking. Cell 135, 1175–1187. doi: 10.1016/j.cell.2008.12.009
Johannes, L., and Wunder, C. (2011). Retrograde transport: two (or more) roads diverged in an endosomal tree? Traffic 12, 956–962. doi: 10.1111/j.1600-0854.2011.01200.x
Kagan, J. C., and Roy, C. R. (2002). Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites. Nat. Cell Biol. 4, 945–954. doi: 10.1038/ncb883
Kuziemko, G. M., Stroh, M., and Stevens, R. C. (1996). Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance. Biochemistry 35, 6375–6384. doi: 10.1021/bi952314i
Lombardi, D., Soldati, T., Riederer, M. A., Goda, Y., Zerial, M., and Pfeffer, S. R. (1993). Rab9 functions in transport between late endosomes and the trans-Golgi network. EMBO J. 12, 677–682.
Loovers, H. M., Kortholt, A., De Groote, H., Whitty, L., Nussbaum, R. L., and Van Haastert, P. J. (2007). Regulation of phagocytosis in Dictyostelium by the inositol 5-phosphatase OCRL homolog Dd5P4. Traffic 8, 618–628. doi: 10.1111/j.1600-0854.2007.00546.x
Lu, H., and Clarke, M. (2005). Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae. Cell. Microbiol. 7, 995–1007. doi: 10.1111/j.1462-5822.2005.00528.x
Lu, L., and Hong, W. (2014). From endosomes to the trans-Golgi network. Semin. Cell Dev. Biol. 31, 30–39. doi: 10.1016/j.semcdb.2014.04.024
Lucas, M., Gershlick, D. C., Vidaurrazaga, A., Rojas, A. L., Bonifacino, J. S., and Hierro, A. (2016). Structural mechanism for cargo recognition by the retromer complex. Cell 167, 1623–1635. doi: 10.1016/j.cell.2016.10.056
Mao, Y., Balkin, D. M., Zoncu, R., Erdmann, K. S., Tomasini, L., Hu, F., et al. (2009). A PH domain within OCRL bridges clathrin-mediated membrane trafficking to phosphoinositide metabolism. EMBO J. 28, 1831–1842. doi: 10.1038/emboj.2009.155
McDonough, J. A., Newton, H. J., Klum, S., Swiss, R., Agaisse, H., and Roy, C. R. (2013). Host pathways important for Coxiella burnetii infection revealed by genome-wide RNA interference screening. MBio 4:e00606-12. doi: 10.1128/mBio.00606-12
McGourty, K., Thurston, T. L., Matthews, S. A., Pinaud, L., Mota, L. J., and Holden, D. W. (2012). Salmonella inhibits retrograde trafficking of mannose-6-phosphate receptors and lysosome function. Science 338, 963–967. doi: 10.1126/science.1227037
Mirrashidi, K. M., Elwell, C. A., Verschueren, E., Johnson, J. R., Frando, A., Von Dollen, J., et al. (2015). Global mapping of the Inc-human interactome reveals that retromer restricts Chlamydia infection. Cell Host Microbe 18, 109–121. doi: 10.1016/j.chom.2015.06.004
Miserey-Lenkei, S., Waharte, F., Boulet, A., Cuif, M. H., Tenza, D., El Marjou, A., et al. (2007). Rab6-interacting protein 1 links Rab6 and Rab11 function. Traffic 8, 1385–1403. doi: 10.1111/j.1600-0854.2007.00612.x
Newton, H. J., Ang, D. K., Van Driel, I. R., and Hartland, E. L. (2010). Molecular pathogenesis of infections caused by Legionella pneumophila. Clin. Microbiol. Rev. 23, 274–298. doi: 10.1128/CMR.00052-09
Noakes, C. J., Lee, G., and Lowe, M. (2011). The PH domain proteins IPIP27A and B link OCRL1 to receptor recycling in the endocytic pathway. Mol. Biol. Cell 22, 606–623. doi: 10.1091/mbc.E10-08-0730
Personnic, N., Bärlocher, K., Finsel, I., and Hilbi, H. (2016). Subversion of retrograde trafficking by translocated pathogen effectors. Trends Microbiol. 24, 450–462. doi: 10.1016/j.tim.2016.02.003
Progida, C., and Bakke, O. (2016). Bidirectional traffic between the Golgi and the endosomes - machineries and regulation. J. Cell Sci. 129, 3971–3982. doi: 10.1242/jcs.185702
Progida, C., Cogli, L., Piro, F., De Luca, A., Bakke, O., and Bucci, C. (2010). Rab7b controls trafficking from endosomes to the TGN. J. Cell Sci. 123, 1480–1491. doi: 10.1242/jcs.051474
Progida, C., Nielsen, M. S., Koster, G., Bucci, C., and Bakke, O. (2012). Dynamics of Rab7b-dependent transport of sorting receptors. Traffic 13, 1273–1285. doi: 10.1111/j.1600-0854.2012.01388.x
Riederer, M. A., Soldati, T., Shapiro, A. D., Lin, J., and Pfeffer, S. R. (1994). Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network. J. Cell. Biol. 125, 573–582.
Robinson, C. G., and Roy, C. R. (2006). Attachment and fusion of endoplasmic reticulum with vacuoles containing Legionella pneumophila. Cell. Microbiol. 8, 793–805. doi: 10.1111/j.1462-5822.2005.00666.x
Rojas, R., Kametaka, S., Haft, C. R., and Bonifacino, J. S. (2007). Interchangeable but essential functions of SNX1 and SNX2 in the association of retromer with endosomes and the trafficking of mannose 6-phosphate receptors. Mol. Cell. Biol. 27, 1112–1124. doi: 10.1128/MCB.00156-06
Rojas, R., Van Vlijmen, T., Mardones, G. A., Prabhu, Y., Rojas, A. L., Mohammed, S., et al. (2008). Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7. J. Cell Biol. 183, 513–526. doi: 10.1083/jcb.200804048
Rothmeier, E., Pfaffinger, G., Hoffmann, C., Harrison, C. F., Grabmayr, H., Repnik, U., et al. (2013). Activation of Ran GTPase by a Legionella effector promotes microtubule polymerization, pathogen vacuole motility and infection. PLoS Pathog. 9:e1003598. doi: 10.1371/journal.ppat.1003598
Schmölders, J., Manske, C., Otto, A., Hoffmann, C., Steiner, B., Welin, A., et al. (2017). Comparative proteomics of purified pathogen vacuoles correlates intracellular replication of Legionella pneumophila with the small GTPase Ras-related protein 1 (Rap1). Mol. Cell. Proteomics 16, 622–641. doi: 10.1074/mcp.M116.063453
Seaman, M. N. (2007). Identification of a novel conserved sorting motif required for retromer-mediated endosome-to-TGN retrieval. J. Cell Sci. 120, 2378–2389. doi: 10.1242/jcs.009654
Seaman, M. N. (2012). The retromer complex - endosomal protein recycling and beyond. J. Cell Sci. 125, 4693–4702. doi: 10.1242/jcs.103440
Seaman, M. N., Harbour, M. E., Tattersall, D., Read, E., and Bright, N. (2009). Membrane recruitment of the cargo-selective retromer subcomplex is catalysed by the small GTPase Rab7 and inhibited by the Rab-GAP TBC1D5. J. Cell Sci. 122, 2371–2382. doi: 10.1242/jcs.048686
Seaman, M. N., Marcusson, E. G., Cereghino, J. L., and Emr, S. D. (1997). Endosome to Golgi retrieval of the vacuolar protein sorting receptor, Vps10p, requires the function of the VPS29, VPS30, and VPS35 gene products. J. Cell Biol. 137, 79–92. doi: 10.1083/jcb.137.1.79
Sharma, S., Skowronek, A., and Erdmann, K. S. (2015). The role of the Lowe syndrome protein OCRL in the endocytic pathway. Biol. Chem. 396, 1293–1300. doi: 10.1515/hsz-2015-0180
Sherwood, R. K., and Roy, C. R. (2013). A Rab-centric perspective of bacterial pathogen-occupied vacuoles. Cell Host Microbe 14, 256–268. doi: 10.1016/j.chom.2013.08.010
Shevchuk, O., Batzilla, C., Hägele, S., Kusch, H., Engelmann, S., Hecker, M., et al. (2009). Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium. Int. J. Med. Microbiol. 299, 489–508. doi: 10.1016/j.ijmm.2009.03.006
Simon, S., and Hilbi, H. (2015). Subversion of cell-autonomous immunity and cell migration by Legionella pneumophila effectors. Front. Immunol. 6:447. doi: 10.3389/fimmu.2015.00447
Steinberg, F., Gallon, M., Winfield, M., Thomas, E. C., Bell, A. J., Heesom, K. J., et al. (2013). A global analysis of SNX27-retromer assembly and cargo specificity reveals a function in glucose and metal ion transport. Nat. Cell Biol. 15, 461–471. doi: 10.1038/ncb2721
Stenmark, H. (2009). Rab GTPases as coordinators of vesicle traffic. Nat. Rev. Mol. Cell Biol. 10, 513–525. doi: 10.1038/nrm2728
Swanson, M. S., and Isberg, R. R. (1995). Association of Legionella pneumophila with the macrophage endoplasmic reticulum. Infect. Immun. 63, 3609–3620.
Teasdale, R. D., and Collins, B. M. (2012). Insights into the PX (phox-homology) domain and SNX (sorting nexin) protein families: structures, functions and roles in disease. Biochem. J. 441, 39–59. doi: 10.1042/BJ20111226
Temkin, P., Lauffer, B., Jager, S., Cimermancic, P., Krogan, N. J., and Von Zastrow, M. (2011). SNX27 mediates retromer tubule entry and endosome-to-plasma membrane trafficking of signalling receptors. Nat. Cell Biol. 13, 715–721. doi: 10.1038/ncb2252
Trahey, M., and Hay, J. C. (2010). Transport vesicle uncoating: it's later than you think. F1000 Biol. Rep. 2:47. doi: 10.3410/B2-47
Tran, T. H., Zeng, Q., and Hong, W. (2007). VAMP4 cycles from the cell surface to the trans-Golgi network via sorting and recycling endosomes. J. Cell Sci. 120, 1028–1041. doi: 10.1242/jcs.03387
Ungewickell, A., Ward, M. E., Ungewickell, E., and Majerus, P. W. (2004). The inositol polyphosphate 5-phosphatase Ocrl associates with endosomes that are partially coated with clathrin. Proc. Natl. Acad. Sci. U.S.A. 101, 13501–13506. doi: 10.1073/pnas.0405664101
Urwyler, S., Brombacher, E., and Hilbi, H. (2009a). Endosomal and secretory markers of the Legionella-containing vacuole. Commun. Integr. Biol. 2, 107–109. doi: 10.4161/cib.7713
Urwyler, S., Nyfeler, Y., Ragaz, C., Lee, H., Mueller, L. N., Aebersold, R., et al. (2009b). Proteome analysis of Legionella vacuoles purified by magnetic immunoseparation reveals secretory and endosomal GTPases. Traffic 10, 76–87. doi: 10.1111/j.1600-0854.2008.00851.x
van Weering, J. R., Sessions, R. B., Traer, C. J., Kloer, D. P., Bhatia, V. K., Stamou, D., et al. (2012). Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules. EMBO J. 31, 4466–4480. doi: 10.1038/emboj.2012.283
Vicinanza, M., Di Campli, A., Polishchuk, E., Santoro, M., Di Tullio, G., Godi, A., et al. (2011). OCRL controls trafficking through early endosomes via PtdIns4,5P(2)-dependent regulation of endosomal actin. EMBO J. 30, 4970–4985. doi: 10.1038/emboj.2011.354
Wandinger-Ness, A., and Zerial, M. (2014). Rab proteins and the compartmentalization of the endosomal system. Cold Spring Harb. Perspect. Biol. 6:a022616. doi: 10.1101/cshperspect.a022616
Wassmer, T., Attar, N., Bujny, M. V., Oakley, J., Traer, C. J., and Cullen, P. J. (2007). A loss-of-function screen reveals SNX5 and SNX6 as potential components of the mammalian retromer. J. Cell Sci. 120, 45–54. doi: 10.1242/jcs.03302
Wassmer, T., Attar, N., Harterink, M., Van Weering, J. R., Traer, C. J., Oakley, J., et al. (2009). The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network. Dev. Cell 17, 110–122. doi: 10.1016/j.devcel.2009.04.016
Weber, S. S., Ragaz, C., and Hilbi, H. (2009). The inositol polyphosphate 5-phosphatase OCRL1 restricts intracellular growth of Legionella, localizes to the replicative vacuole and binds to the bacterial effector LpnE. Cell. Microbiol. 11, 442–460. doi: 10.1111/j.1462-5822.2008.01266.x
Weber, S. S., Ragaz, C., Reus, K., Nyfeler, Y., and Hilbi, H. (2006). Legionella pneumophila exploits PI(4)P to anchor secreted effector proteins to the replicative vacuole. PLoS Pathog. 2:e46. doi: 10.1371/journal.ppat.0020046
Weber, S., Wagner, M., and Hilbi, H. (2014). Live-cell imaging of phosphoinositide dynamics and membrane architecture during Legionella infection. MBio 5:e00839-13. doi: 10.1128/mBio.00839-13
Xu, L., Shen, X., Bryan, A., Banga, S., Swanson, M. S., and Luo, Z. Q. (2010). Inhibition of host vacuolar H+-ATPase activity by a Legionella pneumophila effector. PLoS Pathog. 6:e1000822. doi: 10.1371/journal.ppat.1000822
Zerial, M., and McBride, H. (2001). Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2, 107–117. doi: 10.1038/35052055
Zhang, X., Jefferson, A. B., Auethavekiat, V., and Majerus, P. W. (1995). The protein deficient in Lowe syndrome is a phosphatidylinositol-4,5-bisphosphate 5-phosphatase. Proc. Natl. Acad. Sci. U.S.A. 92, 4853–4856. doi: 10.1073/pnas.92.11.4853
Keywords: Dictyostelium discoideum, effector protein, host-pathogen interaction, pathogen vacuole, retrograde transport, retromer, sorting nexin, type IV secretion
Citation: Bärlocher K, Welin A and Hilbi H (2017) Formation of the Legionella Replicative Compartment at the Crossroads of Retrograde Trafficking. Front. Cell. Infect. Microbiol. 7:482. doi: 10.3389/fcimb.2017.00482
Received: 30 August 2017; Accepted: 06 November 2017;
Published: 24 November 2017.
Edited by:
Elizabeth L. Hartland, Hudson Institute of Medical Research, AustraliaReviewed by:
James Samuel, Texas A&M University, United StatesIsabelle Vergne, Centre National de la Recherche Scientifique (CNRS), France
Copyright © 2017 Bärlocher, Welin and Hilbi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Hubert Hilbi, hilbi@imm.uzh.ch
†These authors have contributed equally to this work.