Subversion of Retrograde Trafficking by Translocated Pathogen Effectors.

TIMI 1298 No. of Pages 13

Review

Subversion of Retrograde Trafficking by Translocated Pathogen Effectors Nicolas Personnic,1 Kevin Bärlocher,1 Ivo Finsel,2 and Hubert Hilbi1,2,* Intracellular bacterial pathogens subvert the endocytic bactericidal pathway to form specific replication-permissive compartments termed pathogen vacuoles or inclusions. To this end, the pathogens employ type III or type IV secretion systems, which translocate dozens, if not hundreds, of different effector proteins into their host cells, where they manipulate vesicle trafficking and signaling pathways in favor of the intruders. While the distinct cocktail of effectors defines the specific processes by which a pathogen vacuole is formed, the different pathogens commonly target certain vesicle trafficking routes, including the endocytic or secretory pathway. Recently, the retrograde transport pathway from endosomal compartments to the trans-Golgi network emerged as an important route affecting pathogen vacuole formation. Here, we review current insight into the host cell's retrograde trafficking pathway and how vacuolar pathogens of the genera Legionella, Coxiella, Salmonella, Chlamydia, and Simkania employ mechanistically distinct strategies to subvert this pathway, thus promoting intracellular survival and replication. Secreted Bacterial Effectors and Pathogen Compartments All intracellular bacterial pathogens colonize a unique niche. While only a few pathogens escape the bactericidal endocytic pathway by dissolving the phagosome, most pathogens create a distinct replication-permissive vacuolar compartment. To this end, the pathogens employ type III secretion systems (T3SSs) (see Glossary) or type IV secretion systems (T4SSs) to translocate a plethora of effector proteins into host cells, where they manipulate vesicle trafficking and signaling pathways, for example, by acting as a guanine nucleotide exchange factor (GEF) for small GTPases. Legionella species employ a T4SS and more than 300 different effector proteins to form a distinct Legionella-containing vacuole (LCV), which communicates extensively with the endoplasmic reticulum (ER) [1,2]. Coxiella burnetii uses a closely related T4SS to create an acidic lysosomal replication compartment [3], the Coxiella-containing vacuole (CCV). In contrast, Salmonella and Chlamydia spp. utilize a T3SS and dozens of effectors to create[17_TD$IF] a distinct [18_TD$IF]compartment termed Salmonella-containing [19_TD$IF]vacuole ([20_TD$IF]SCV) [4,5], or Chlamydia [21_TD$IF]inclusion ([2_TD$IF]CIN), respectively [6,7]. Another member of the Chlamydiales, the obligate intracellular bacterium Simkania negevensis, forms a complex compartment called the Simkania-containing vacuole (SnCV) [8]. Again, while the T3SSs used by Salmonella, Chlamydia, or Simkania spp. are highly homologous, the pathogen vacuoles are vastly different. Salmonella grows in a compartment that extensively communicates with late endosomes/lysosomes and tubular structures, Chlamydia spp. differentiate in a membrane-bound inclusion from infectious spore-like elementary bodies (EB) to replication-competent reticulate bodies (RB), and S. negevensis replicates in a vacuole characterized by numerous multilayered ER contact sites. It is well established that

Trends in Microbiology, Month Year, Vol. xx, No. yy

Trends Intracellular bacterial pathogens translocate various effector proteins into host cells, where they modulate membrane dynamics and vesicle trafficking in order to establish a replicative niche termed the pathogen-containing vacuole. The retrograde vesicle trafficking pathway promotes transport from early, late, and recycling endosomes to the trans-Golgi network (TGN) and back to the endoplasmic reticulum (ER). Key components of this pathway are the COPI and retromer coats, sorting nexin (SNX) proteins, and phosphoinositide (PI) lipids, as well as the PI 5-phosphatase OCRL and small GTPases of the Rab family (Rab5, Rab7, and Rab9). Bacterial effector proteins blocking retrograde transport are (i) Legionella pneumophila RidL, which binds to the retromer, (ii) Salmonella Typhimurium SifA, which interacts with SKIP/ kinesin, and (iii) Chlamydia trachomatis IncE, which binds to SNX5/6.

1 Institute of Medical Microbiology, Department of Medicine, University of Zürich, Gloriastrasse 30/32, 8006 Zürich, Switzerland 2 Max von Pettenkofer Institute, Ludwig-Maximilians University Munich, Pettenkoferstrasse 9a, 80336 Munich, Germany

*Correspondence: [email protected] (H. Hilbi).

http://dx.doi.org/10.1016/j.tim.2016.02.003 © 2016 Elsevier Ltd. All rights reserved.

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pathogen vacuoles communicate with the endosomal pathway [5], the ER and the anterograde (secretory) trafficking from the ER to the Golgi apparatus [9]. Recent proteomics and functional studies revealed that components of the retrograde trafficking pathway decorate pathogen compartments, restrict intracellular bacterial growth, and are targeted by translocated effector proteins [10–15]. The eukaryotic retrograde vesicle trafficking pathway promotes transport from the endosomal system to the trans-Golgi network (TGN) and further to the ER [16–19]. Here we review recent insight into the function and composition of specific [23_TD$IF]branches of the host cell's retrograde trafficking pathway. To this end, we first summarize available insight on retrograde transport from Rab5/Rab7- or Rab9-positive endosomes to the TGN, and within the Golgi stack back to the ER. Subsequently, we discuss how bacterial vacuolar pathogens employ mechanistically distinct strategies to subvert distinct branches and key factors of the retrograde trafficking pathway, thus promoting intracellular replication.

Bidirectional ER-Endosome Transport: Anterograde and Retrograde Trafficking The anterograde and retrograde vesicle trafficking pathways constitute a fundamental cellular transport cycle. Newly synthesized proteins, destined for secretion or residence in specific subcellular compartments, are produced in the ER where they undergo folding and posttranslational modifications before being packed into vesicles coated with the COPII coat complex [20]. Budding vesicles deliver their content and membrane to the nascent Golgi cisternae in the ER–Golgi Intermediate Compartment (ERGIC) and cis-Golgi interface of the Golgi stack [21]. During their anterograde (cis to trans) passage across the polarized Golgi stack, proteins undergo limited proteolysis and/or glycosylation in a stepwise fashion as they encounter the corresponding resident enzymes [22,23]. At the final stage of secretory trafficking, the TGN produces clathrin-coated transport carriers [24] to deliver matured proteins to either the plasma membrane or to intracellular compartments such as the endolysosomal system. The endosomal tubulo-vesicular system is a functional assembly of specialized subcompartments that communicate with each other, the plasma membrane, the lysosomal compartment, and the secretory pathway. Endosomes can be grouped into three major compartments: the early endosomes (EE), the recycling endosomes (RE), and the late endosomes (LE) [25]. In EE both secretory and endocytic routes converge, and therefore, these compartments are considered the main sorting hub that segregates cargo to be degraded via the LE-lysosome pathway from material to be recycled via either the RE or the Golgi pathway. LE also sort cargo and interact with the lysosomal compartment to deliver newly synthesized acidic hydrolases as well as material to be degraded. The various retrograde retrieval mechanisms from endosomes to the Golgi and the ER are required for a wide range of cellular processes. Retrograde routes return membrane-associated trafficking components for another round of cargo transport, and they maintain resident proteins in their correct compartment, thereby ensuring organelle identity and function [16–19], as well as lipid homeostasis [26]. The two major membrane-associated molecule classes defining the identity of any cellular compartment, subdomain, or vesicle trafficking route are phosphoinositide (PI) lipids and Rab small GTPases [27–30]. The seven species of monophosphorylated or polyphosphorylated phosphatidylinositol (PtdIns) and the 70 human Rab GTPases are spatio-temporally tightly controlled and serve as a recruiting platform for[24_TD$IF] endogenous effector proteins to assemble

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Glossary Adaptor: protein complex that sorts transmembrane protein cargos into nascent vesicles by binding directly to their cytosolic domains. Cargo: any membrane and soluble material directly or indirectly sorted by cargo adaptors into nascent vesicles. Coat: protein complex composed of numerous core and accessory proteins that binds to membrane, recognizes cargo proteins, bends the local membranes into a bud, and pinches the nascent bud off to form a vesicle. Effector: bacterial protein translocated by a secretion system into a host cell, where it subverts signal transduction and membrane trafficking pathways. GTPase activating protein (GAP): small GTPase inactivator that catalyzes hydrolysis of GTP by several orders of magnitude. Guanine nucleotide exchange factor (GEF): small GTPase activator that dissociates GDP from the guanine nucleotide-binding pocket, thereby allowing binding of GTP, which is present at higher intracellular concentrations than GDP. Pathogen vacuole: distinct membrane-bound compartment within a host cell, which serves as a microbial replicative niche. Phosphoinositide (PI) lipid: family of lipids present at the cytosolic leaflet of cellular membranes. PI lipids consist of a glycerol moiety with two fatty acid chains and an inositol headgroup, which can be mono- or poly-phosphorylated and codetermines the spatio-temporally controlled recruitment of specific endogenous effectors. Retromer: heterotrimeric complex of Vps26, Vps29, and Vps35, which, in association with SNXs, drives retrograde transport of specific cargoes, for example, from the endosomal system to the TGN. Small GTPase: member of an enzyme family (e.g., Arf, Rab, Ran, and Rho) that switches from an inactive, GDP-bound form to an active, GTP-bound form. Sorting nexin (SNX): protein containing a PI lipid-binding (usually PX) domain responsible for membrane attachment (and in some cases curvature induction). Tethering factor: (coiled-coil) protein that mediates the first (long-distance)


various functional organelle transport machineries at the correct membrane location and time, to sort specific cargoes, and to define transport directionality. The Golgi is enriched in PtdIns(4)P, and Rab6A is mainly present at the TGN, while Rab1 and Rab2 concentrate at the cis-side. EE are positive for PtdIns(3)P and specifically harbor Rab5, whereas the maturating pathway from EE to LE displays a shift from Rab5 to Rab7 [25]. RE are positive for PtdIns(4)P and PtdIns(4,5)P2 and contain Rab4 or Rab11, defining fast and slow recycling pathways, respectively [31]. Finally, LE accumulate PtdIns(3,5)P2 as well as PtdIns(3,4)P2, and they are positive for Rab9 [29,30]. Most retrograde routes from the endosomal network to the TGN emerge from Rab5/Rab7positive endosomes and from Rab9-positive LE.

interaction between a vesicle carrier and its target membrane. Translocation: protein transport across membranes; for example, transport by a dedicated bacterial type III or type IV secretion system across the two membranes of a Gram-negative bacterium and a host membrane. Transport vesicle: small intermediate carrier of 50–100 nm that buds and separates from a distinct donor organelle and actively ferries its contents to its destination compartment. Type III/IV secretion system (T3/ 4SS): bacterial secretion system related to flagella (type III) or conjugation systems (type IV) that spans the two membranes of Gramnegative bacteria and penetrates the host cell membrane, thus allowing the delivery of effector proteins. Vacuolar protein sorting (Vps): factor implicated in selection and accumulation of cargo destined for transport in a vesicle.

Retrograde Transport from the Endosomal Network to the TGN Retrograde Transport from Rab5/Rab7-Positive Endosomes to the TGN A key factor implicated in endosome–TGN retrograde trafficking is the retromer, formerly known as cargo-selective complex [32]. The retromer is an evolutionary conserved trimer of proteins termed vacuolar protein sorting (Vps) 26, Vps29 and Vps35 (Figure 1). Vps35 binds both Vps26 and Vps29. Vps29–Vps35 form a stable structure protected from polyubiquitinylation and subsequent proteasome degradation [33], whereas the Vps26 variants Vps26A and Vps26B define functionally distinct retromer complexes [34]. The assembly of the canonical retromer machinery requires association of Vps26–Vps29–Vps35 with heterodimers of membrane curvature-inducing proteins termed Bin-Amphiphysin-Rvs (BAR) domain containing sorting nexins (SNXs), that is, SNX1 or SNX2 with SNX5 or SNX6 [35,36]. Other SNXs might be involved in retrograde trafficking as the family comprises 49 members [28]. Retromer recruitment to the

Endosomal network

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(II A)

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+ Pi

Key: Clathrin

Rab7(GDP)

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p150Glued/dynein

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PtdIns(3)P PtdIns(3,5)P₂

EHD1/dynamin

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WASH complex

TBC1D5

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Figure 1. Recruitment of the Retromer, Endosome Tubulation, and Retrograde Vesicle Trafficking. (I) Cargo receptors accumulate through clathrin. (II) The retromer (Vps26-Vps29-Vps35) is recruited to the endosomal membrane by activated, GTP-bound Rab7 (IIA), which, in turn, is inactivated by the Vps29-bound GAP TBC1D5 (IIB). (III) Resident PtdIns(3)P binds to SNX1/2 followed by SNX5/6, and (IV) the heterodimer induces membrane curvature. (V) Further remodeling forms tubules that bind the WASH complex, which (VI) triggers actin polymerization pushing the nascent vesicle, while binding of SNX5/6 to the microtubule motor complex protein p150Glued/dynein pulls the tubule. Finally, scission factors, including EHD1 and dynamin, separate the cargo-loaded vesicle from endosomes, and the newly formed vesicle is uncoated prior to fusion with the destination compartment (adapted from [18]).


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endosome requires the interaction of Vps35–Vps26 with activated, GTP-bound Rab7A [37–39]. Rab5, in turn, recruits the class III PI 3-kinase Vps34, thereby locally enriching PtdIns(3)P, which binds Phox homology (PX) domain-containing SNXs. Other SNXs might anchor to PtdIns(4,5)P2 (SNX5) or PtdIns(4)P (SNX6), respectively. Noteworthy, retromer association to the membrane does not require SNX1/2 or SNX5/6 and vice versa. Several accessory proteins participate in retromer-mediated sorting, tubule elongation, and stabilization, scission and vesicle transport (Figure 1). These include the WASH actin nucleation complex, as well as the EHD1, EHD3, or p150Glued[2_TD$IF] subunits of the motor protein dynactin [35]. Moreover, the Rab7 GTPase activating protein (GAP) TBC1D5 binds Vps29 and promotes the release of the retromer [38]. Another Vps29 binding partner, the multidomain protein VARP, plays a role in the recycling of cargo from endosomes to the plasma membrane [40]. Finally, the membrane-curving coat protein clathrin is involved in retromer-mediated (as well as retromerindependent) retrograde trafficking by supporting cargo concentration before sorting [41]. A PI-metabolizing enzyme crucially involved in endosome–TGN retrograde trafficking is OCRL (oculocerebrorenal syndrome of Lowe). OCRL [42] and its Dictyostelium homologue Dd5P4 (Dictyostelium discoideum 5-phosphatase 4) [43], are type II PI 5-phosphatases that hydrolyse PtdIns(4,5)P2 as well as PtdIns(3,4,5)P3 to yield PtdIns(4)P and PtdIns(3,4)P2, respectively [44]. The loss of OCRL or its phosphatase activity impairs the retrograde transport of cargo, which accumulates in Rab5-positive EE [45]. OCRL binds clathrin through its N-terminal PH domain [46] and the small GTPases Rab5 via its ASH domain, thereby targeting the PI 5-phosphatase to EE [47] and providing a link to clathrin-mediated retrograde transport. Retrograde Transport from Rab9-positive LE to the TGN Rab9 is predominantly bound to LE membranes and is required for the retrieval to the TGN of cation-independent (CI[3_TD$IF]) and cation-dependent (CD[4_TD$IF]) mannose-6-phosphate receptors (MPRs) after they have delivered newly synthesized enzymes to the endosomal network [48,49]. The Rab9 effectors Tip47 and GCC185 localize to LE or Golgi membranes, respectively [50], and are involved in the recycling of MPRs from LE, along with the PI 5-kinase PIKfyve [51]. The current model proposes that Tip47 remains on Rab9-positive transport vesicles as a coat and is removed prior to arrival of the vesicle at the Golgi to allow fusion [52]. Strikingly, at least in [25_TD$IF]Caenorhabditis elegans, Rab9 interacts with Vps35 and acts sequentially with Rab5 and Rab7 for sorting the protein serpentin to LE and from LE to the TGN [53].

Retrograde Transport within the Golgi Stack and to the ER The retrograde transport of most cargo within the Golgi stack and from the cis-Golgi back to the ER involves the coat complex COPI [54,55]. The basic assembly unit of the COPI coat is the heteroheptameric coatomer complex [56]. Contrary to clathrin or COPII coat subunits, the coatomer COPI subunits are simultaneously recruited to the Golgi membranes during the vesiculation process in an atypical non-‘adaptor-cage’ assembly process upon both membrane and cargo binding [56,57]. COPI is recruited to membranes by the activated small GTPase ARF1 (ADP ribosylation factor 1) [58]. Activated ARF1 also recruits PtdIns 4-kinase IIIb [59] and OCRL [60], which likely increases local PtdIns(4)P concentrations. Rab6A, the main Rab GTPase present at the TGN, is also required in the COPI-dependent retrograde trafficking within the Golgi stack [61,62]. Rab6A binds to OCRL and stimulates its 5-phosphatase activity, thereby enriching the membrane in PtdIns(4)P [47].

Cargo Delivery along the Retrograde Transport Route Many cargoes depend on the retromer for their retrieval from the endosomal network to the Golgi, and require the COPI coat complex for Golgi to ER trafficking. These cargoes include the

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MPRs and sortilins [63], the plant toxin ricin [64], or bacterial toxins such as Shiga toxin or cholera toxin [65]. Sorting of transmembrane proteins is driven by a direct recognition of their cytosolic exposed lysine- and arginine-containing motifs by different COPI coatomer subunits [66]. Soluble proteins to be retrieved to the ER contain a typical C-terminal KDEL sequence, which is recognized by KDEL-receptor proteins that interact with COPI components [66]. Once correctly collected and transported, the cargo must be selectively delivered to the right destination organelle. The soluble N-ethylmaleinimide-sensitive factor attachment receptors (SNAREs) are a class of binding partners present at both the carrier membrane (v/R-SNARES) and the acceptor compartment (t/Q-SNARES), which promote membrane fusion [67]. The diversity of cellular SNAREs, their differential localization and Rab dependency, and their selective pairwise interactions, confer specificity to membrane traffic. However, due to their relatively small size, SNAREs can only interact if vesicle and recipient membranes are closely positioned. A process called tethering attaches the carrier to the organelle before SNARE complex assembly. Tethering factors form a membrane-bound docking platform, which recognizes specific features of the carrier membrane, such as SNAREs, tethers, coats, and small GTPases. The long coiledcoil tether proteins, for example, golgins [68], seize vesicles destined for the Golgi. Another class of tethers is the CATCHR (complex associated with tethering containing helical rod [69]). CATCHR complexes termed GARP, COG (conserved oligomeric Golgi), or NZM localize either to the TGN, intra-Golgi or the ER, respectively, where they participate in retromer- [36,70], clathrin- [71], and COPI-mediated retrograde trafficking [72,73]. Before fusion with the target membrane, carriers traveling along the retrograde routes must shed their coat and release their motor complex. The PI 5-phosphatase OCRL might promote this stage, since its product PtdIns(4)P triggers clathrin uncoating in endocytic vesicles [74], as well as the dissociation of motor proteins during retromer-associated retrograde trafficking [36,75]. In summary, retrograde trafficking is a highly regulated, multibranched and essential cellular transport route, which[26_TD$IF] as such is targeted by a number of vacuolar bacterial pathogens.

The Legionella Effector RidL Binds the Retromer Vps29 Subunit The environmental bacterium Legionella pneumophila is a natural parasite of amoebae [76]. Upon inhalation of bacteria-laden aerosols, the pathogen grows within lung macrophages, thereby triggering a severe pneumonia termed Legionnaires’ disease [77,78]. Employing a largely conserved mechanism, L. pneumophila replicates in protozoan and mammalian host cells in a distinct membrane-bound compartment, the LCV. LCVs avoid fusion with bactericidal lysosomes, but extensively communicate with the endosomal and secretory vesicle trafficking pathways of the host cell [1,9,79]. LCV formation implicates interactions with early secretory vesicles at ER exit sites and the activity of the small GTPases Arf1, Sar1, and Rab1 [80–82]. Upon movement along microtubules, the pathogen vacuole adheres to and finally fuses with the ER [83–85]. Formation of LCVs is a complex and robust process, which requires the bacterial Icm/Dot T4SS that secretes approximately 300 different effector proteins [2,79,86–88]. These proteins are injected into host cells, where they modulate pivotal components of eukaryotic signal transduction and membrane dynamics, such as the small GTPases Arf1 [89], Rab1 [90– 93] or Ran [94,95], the PI lipids PtdIns(3,4,5)P3 [96], PtdIns(3)P [97] or PtdIns(4)P [98–101], the vacuolar H+-ATPase [102], or the autophagy machinery [103]. Proteomics studies on intact LCVs from L. pneumophila-infected Dictyostelium amoebae or RAW 264.7 macrophages, purified by immunomagnetic separation using an antibody against the PtdIns(4)P-binding Icm/Dot substrate SidC [98], revealed the presence of many host factors implicated in vesicle trafficking, including small GTPases, PI-metabolizing enzymes and the


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Key Figure

Subversion of Retrograde Trafficking by Bacterial Vacuolar Pathogens

Endocyc pathway Early endosome Lysosome Late endosome CCV

Recycling endosome

Rab7

Rab7

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LCV Rab7 Vps35, 29, 26 SNX1, 2 OCRL

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? Rab6

TGN COPI COG complex

ER

COPI

IncE SNX5/6

CIN Rab6, 11 Vps35, 29 SNX1, 2, 4, 5, 6 OCRL COG complex

Golgi

Figure 2. Legionella, Coxiella, Salmonella, Chlamydia, and Simkania spp. employ mechanistically distinct strategies to subvert retrograde trafficking. The Legionella pneumophila effector RidL directly binds the Vps29 subunit of the retromer [15_TD$IF]complex, thus likely inhibiting retromer function. Coxiella burnetii forms the acidic CCV decorated with retrograde components. The Salmonella enterica serovar Typhimurium effector SifA binds to the host factor SKIP, thereby sequestering Rab9 and subverting Rab9-dependent retrograde trafficking and lysosome function. The Chlamydia trachomatis effector IncE binds to SNX5/6, which relocalizes these PI-binding and membrane-deforming proteins to the inclusion membrane. The Simkania negevensis-containing compartment accumulates components of the COPI complex, and inhibitors of retrograde trafficking impair intracellular growth. Abbreviations: CCV, Coxiella-containing vacuole; CIN, Chlamydia inclusion; LCV, Legionella-containing vacuole; SCV, Salmonella-containing vacuole; SnCV, Simkania-containing vacuole. Colour code: red, retrograde pathway; green, endocytic pathway; blue,[16_TD$IF] pathogen vacuoles and bacterial effector proteins; black, interactions of pathogen compartments with cell organelles or vesicles.

retromer [10,104,105] (Figure 2, Key Figure). While the TGN Rab GTPase Rab6A is absent from LCVs, the pathogen vacuole is enriched for Rab7 [10,104,106], which is required for the recruitment of retromer to membranes. Upon depletion of components of the retromer complex, L. pneumophila grows more efficiently in mammalian cells, suggesting that a functional retrograde pathway restricts intracellular replication [107]. Interestingly, the Icm/Dot substrate RidL was found to selectively bind the Vps29 retromer subunit as well as PtdIns(3)P (Figure 2), and the retromer accumulates to the same extent on LCVs in D. discoideum or macrophages infected with either wild-type or DridL L. pneumophila [107]. Yet, retrograde cargo receptors and SNXs preferentially localize to LCVs in the absence of RidL. L. pneumophila lacking ridL was impaired for intracellular growth in macrophages or

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amoebae, and outcompeted by the [28_TD$IF]parental strain in amoebae. The ectopic production of RidL inhibited retrograde trafficking of fluorescently labeled Shiga toxin subunit B (STxB) in epithelial cells, and dependent on ridL, L. pneumophila blocked retrograde transport of fluorescently labeled cholera toxin subunit B (CTxB) at endosome exit sites in infected macrophages. Together, these findings indicate that a single L. pneumophila effector, RidL, inhibits host retromer function and promotes bacterial replication. A role for retrograde trafficking for intracellular replication of L. pneumophila was already previously indicated. Deletion of the D. discoideum OCRL homologue Dd5P4 promoted intracellular growth of L. pneumophila [108], and depletion by RNA interference of OCRL had the same effect [107]. OCRL and Dd5P4 localize to LCVs via an N-terminal domain, and the enzymes appear to be catalytically active on LCVs, thus increasing the[29_TD$IF] amount of PtdIns(4)P available for binding of Icm/Dot substrates [108]. Taken together, a functional retrograde trafficking pathway restricts intracellular growth of L. pneumophila, which produces the T4SS-translocated effector RidL that binds retromer and blocks retrograde transport at endosome exit sites.

Retrograde Trafficking Promotes Vacuolar Coxiella Growth Coxiella burnetii is a facultative intracellular pathogen which causes the zoonosis Q fever [109]. The pathogen is aspirated through contaminated aerosols and causes an acute, flu-like disease which, in rare cases, can develop into chronic endocarditis. The intracellular replication niche, termed CCV, is an acidic large membrane compartment and characterized by the presence of late endosomal/lysosomal markers such as Rab7, LAMP-1, and LAMP-2, as well as the VATPase [3] (Figure 2). The CCV is clearly distinct from a classical phagolysosome, as the compartment is highly fusogenic and interacts in particular with endolysosomal vesicles. Moreover, the CCV membrane is enriched in cholesterol, and inhibitors of cholesterol metabolism reduce intracellular replication of C. burnetii [110]. To form CCVs, C. burnetii requires an Icm/Dot T4SS that is very similar to its L. pneumophila counterpart [111–115]. Indeed, a number of putative C. burnetii effector proteins have been shown to be translocated by L. pneumophila. However, the function of most of these effectors is currently unknown. To further characterize the CCV and identify functionally important host factors, a genome-wide screen was performed using gene silencing by [30_TD$IF]siRNA[7_TD$IF]. Newly identified, functionally relevant host factors included the retromer complex [15]. Interestingly, the depletion of the retromer subunits Vps29 or Vps35 (but not Vps26A or Vps26B), as well as depletion of retromer-associated SNXs (SNX2, -3, -5, and -6; but not SNX1), abolished intracellular replication of C. burnetii. Thus, the retromer (and in consequence retrograde trafficking) appears to promote the replication of the pathogen. These findings are in contrast to studies with L. pneumophila, where the depletion or deletion of components of the retrograde trafficking machinery resulted in increased growth. Perhaps, these differences reflect characteristics of the distinct pathogen vacuoles that are created by C. burnetii or L. pneumophila: while intracellular growth of the former appears to be promoted by certain cues and conditions of a retrograde proficient, acidic compartment, the growth of the latter in a neutral compartment is [31_TD$IF]enhanced upon inhibition of retrograde trafficking.

The Salmonella Effector SifA Binds to the Rab9 Interactor SKIP Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that causes a self-limiting gastroenteritis [116]. Pathogenesis of the bacteria is dependent on two T3SSs encoded on the Salmonella pathogenicity islands 1 (SPI-1) and 2 (SPI-2). The T3SSs translocate more than 40 effector proteins, which govern uptake by epithelial cells and the formation of the SCV connected to tubular structures collectively named Salmonella-induced tubules [4,5].


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Recently, S. Typhimurium was found to inhibit retrograde retrieval of CI- and CD-MPRs from endosomes [117]. This study was spurred by the paradox that, although SCVs are acidic and contain late endosomal markers (Rab7, LAMP1, and V-ATPase) [32_TD$IF]as well as lysosomal membrane glycoproteins, MPRs and their cargo hydrolases are excluded from the SCV (Figure 2). Upon infection with S. Typhimurium, the MPRs normally localizing in the TGN are dispersed throughout the cell, suggesting that the pathogen interferes with receptor trafficking [117]. Remarkably, retrograde trafficking of CTxB, which requires the SNARE syntaxin6, instead of syntaxin10 like the MPRs, was not affected by the bacteria. S. Typhimurium infection did not alter the localization of syntaxin10, but CD-MPR redistributed to structures containing Vps26, indicating that the pathogen intercepts MPR trafficking between the Vps26-dependent sorting and the syntaxin10-dependent fusion of the vesicle with the TGN. Moreover, these findings indicated a role for Rab9, which is also required for MPR recycling. The S. Typhimurium effector SifA directs the formation of Salmonella-induced filaments (SIF) along microtubules [118][3_TD$IF]. [34_TD$IF]On SCVs[35_TD$IF] SifA directly binds to SKIP (SifA- and kinesin-interacting protein), a host protein that down-regulates the recruitment of kinesin on the pathogen vacuole and thus controls vacuolar membrane dynamics [119]. Infection studies with S. Typhimurium lacking the effector SifA (and also SseJ), heterologous production of SifA, or depletion of SKIP, revealed that the effector protein and its interaction with SKIP are responsible for the redistribution of the MPR cargo receptors. With both SifA and SKIP present, the percentage of Rab9-MPR colocalization was [36_TD$IF]lower, suggesting that SifA–SKIP captures and sequesters Rab9 to SCVs [117] (Figure 2). In agreement with this notion, the depletion of syntaxin6 increased lysosome potency and decreased replication of S. Typhimurium, whereas the depletion [37_TD$IF]or absence of syntaxin10 reduced the lysosomal efficiency and enhanced bacterial growth [117]. Moreover, the findings are in agreement with an earlier study documenting that interference with[8_TD$IF] MPR[38_TD$IF] recycling leads to misrouting and secretion of newly synthesized lysosomal hydrolases [120]. Taken together, formation of the SifA–SKIP–Rab9 complex [39_TD$IF]seems to prevent the interaction of the small GTPase with the TGN tethering factor GCC185, thus inhibiting retrograde trafficking of MPRs and lysosome function. SNXs also play an important role during SCV maturation (Figure 2). SNX1 is recruited to the SCV and forms extensive long-ranging filaments, termed spacious vacuole-associated tubules (SVATs), shortly after bacterial uptake [121]. SVAT-formation coincided with a decrease in vacuolar size, suggesting membrane loss by tubulation. Depletion of SNX1 led to an overall delay of bacterial replication and the accumulation of CI-MPR on SCVs, indicating the removal of this receptor via SVATs. Furthermore, SNX3 accumulates on SCVs early after infection and is crucial for the induction of SNX3-positive tubules [122]. SNX3 possesses no membrane-deforming BARdomain, and thus, SNX1 and SNX2 are required for tubulation. Moreover, depletion of SNX3 impairs Rab7 and LAMP-1 recruitment to the SCV and alters SIF formation, indicating a role in the regulation of SCV maturation. Finally, in a recent proteomics approach analyzing purified Salmonella-modified membranes, the retromer subunit Vps26B and components of the COPI complex as well as the COPII complex were identified [11]. In summary, S. Typhimurium highjacks the function of SNXs and other endosomal retrograde components to establish its replicative niche.

The Chlamydia Effector IncE Binds the Sorting Nexins 5/6 The obligate intracellular pathogen Chlamydia trachomatis causes sexually transmitted [40_TD$IF]diseases that can cause sterility in women and ocular infections leading to blindness [123]. Chlamydia spp. adopt a biphasic life cycle comprising two morphologically distinct forms: the infectious, metabolically inert EB, and the noninfectious, replicative RB. The RB form replicates within a membrane-bound compartment termed CIN, which does not fuse with endosomes or lysosomes and instead captures TGN-derived secretory vesicles on their way to the plasma membrane [124,125].

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Inclusion formation also [41_TD$IF]involves the acquisition of sphingomyelin (produced in the cis-Golgi network from ER-derived ceramide) and other lipids. C. trachomatis infection of epithelial cells triggers the fragmentation of the Golgi apparatus, a process which is required for intracellular bacterial growth [126]. The fragmentation of the Golgi[10_TD$IF] is induced by the cleavage of the integral Golgi[1_TD$IF] protein golgin-84 by the chlamydial protease-like activity factor (CPAF) [127]. Golgi fragmentation further requires the retrograde and recycling small GTPases Rab6 and Rab11, respectively, which decorate inclusions (Figure 2), but not the endosomal[12_TD$IF] GTPases Rab5, Rab7, or Rab9 [128]. Finally, similar to LCVs, the Chlamydia inclusion membrane also [42_TD$IF]harbors PtdIns(4)P, Arf1, and the PI 5-phosphatase OCRL [129]. Depletion by RNA interference of the small GTPase[43_TD$IF] Arf1 or the PI phosphatase[4_TD$IF] OCRL inhibited inclusion formation and RB replication. To form the inclusion, C. trachomatis employs a T3SS and its substrates, the Inc (inclusion membrane) proteins [6,130–132]. Notably, the C. trachomatis SNARE domain protein IncA promotes homotypic fusion of inclusions [133,134]. IncD interacts with the ceramide transfer protein CERT and thus exploits the nonvesicular ER–TGN lipid transport machinery to acquire the sphingomyelin precursor ceramide [135,136]. In an approach mapping the Inc-human cell interactome by affinity purification/mass spectrometry, IncE was found to bind SNX[2_TD$IF]5/6, thus sequestering components of the retrograde transport machinery and enhancing inclusion tubulation [12]. Moreover, a proteomics study of C. trachomatis inclusions, isolated by immunomagnetic separation with an anti-IncA antibody, revealed the accumulation of retrograde components, such as Vps29 and Vps35, as well as SNX1, -2, -4, -5, and -6 and the CIMPR [13] (Figure 2). The depletion of retromer enhanced C. trachomatis progeny production, indicating that – similar to L. pneumophila –[45_TD$IF] the retromer[46_TD$IF] complex restricts infection [12,13]. C. trachomatis also hijacks the COG complex implicated in intra-Golgi retrograde trafficking, as [47_TD$IF]this complex[48_TD$IF] also accumulates on inclusions, and its depletion reduced progeny production [137]. In summary, CIN are decorated with various, functionally relevant components of the retrograde trafficking pathway upstream or downstream of the Golgi en route from endosomes to the ER.

Outstanding Questions What is the mechanism by which retrograde trafficking restricts bacterial pathogens? Are there additional Legionella, Salmonella, or Chlamydia effectors targeting retrograde transport? (How) do Coxiella and Simkania effectors modulate retrograde trafficking? Do pathogens subvert retrograde routes to stimulate other (beneficial) coregulated membrane trafficking pathways? Is lipid acquisition of bacterial pathogens affected by subverting retrograde routes? Do pathogens regulate cargo sorting via post-translational modifications (e.g., ubiquitination)? Can the (pharmacological) manipulation of retrograde trafficking be exploited for therapeutic purposes?

The Simkania Compartment Is Decorated with Retrograde Components Another member of the order Chlamydiales, the obligate intracellular bacterium Simkania negevensis, has been linked to infections of the upper respiratory tract [123,138]. S. negevensis replicates in amoebae, mammalian macrophages, or epithelial cells in a continuous membrane system that forms numerous contact sites with the ER [8]. Thus, the SnCV is clearly distinct from the single vacuolar structure adopted by Chlamydia inclusions. Proteomics analysis of enriched SnCVs/ER revealed that components of endosomal and exocytic transport, as well as COPII-dependent ER to Golgi transport, were depleted from the inclusion preparation, while proteins of the endosomal recycling pathway remained unchanged[49_TD$IF]. [50_TD$IF]Interestingly, components of COPI-dependent retrograde Golgi to ER transport were enriched [139] (Figure 2). The pharmacological analysis with compounds selectively targeting the retrograde pathway (Retro-1, Retro-2, and VP-182) confirmed a central role of retrograde transport for intracellular replication and lipid (ceramide) acquisition of S. negevensis. Taken together, S. negevensis appears to exploit components of the retrograde trafficking pathway to establish its complex, ER-derived pathogen vacuole.

Concluding Remarks The cellular anterograde and retrograde vesicle trafficking pathways constitute a fundamental transport cycle linking the production, processing, and delivery of proteins with their turnover and replenishment. The retrograde vesicle trafficking pathway promotes transport from early, late, and recycling endosomes to the TGN, as well as trafficking within the Golgi apparatus and


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further back to the ER. Key components of this pathway are the COPI and retromer coats and membrane[38_TD$IF] curvature-inducing SNX proteins, as well as the PI 5-phosphatase OCRL and small GTPases of the Rab family (Rab5, Rab7, Rab9). Vacuolar bacterial pathogens employ T3SSs or T4SSs to translocate dozens or hundreds of socalled effector proteins into host cells, where they manipulate signaling and vesicle trafficking pathways, including retrograde trafficking. Recent proteomics and functional studies revealed that components of the retrograde trafficking pathway decorate pathogen compartments, modulate intracellular bacterial replication, and are targeted by translocated effector proteins. Only few bacterial effectors [51_TD$IF]affecting retrograde transport have been characterized: L. pneumophila RidL binds to Vps29, S. Typhimurium SifA interacts with SKIP/kinesin and Chlamydia IncE binds to [52_TD$IF]SNX5/6, thus blocking retromer-, Rab9- or SNX-dependent retrograde trafficking, respectively. A number of other bacterial effector proteins likely also manipulate retrograde transport – perhaps, by employing novel molecular mechanisms (see Outstanding Questions). While the retrograde route restricts the growth of some vacuolar pathogens, the underlying mechanisms are mostly not known. Yet, the retrograde pathway appears to be a common and important target of bacterial pathogens. Further research will aim at elucidating the molecular strategies that intracellular pathogens employ to counteract growth restriction by the host's retrograde trafficking pathway. Acknowledgments Research in the authors’ laboratory was generously supported by the Swiss National Science Foundation (SNF; 31003A_[1_TD$IF]153200), the Deutsche Forschungsgemeinschaft (DFG; HI 1511/1-1, SPP 1580) and the German Ministry of Education and Research (BMBF) in the context of the EU Infect-ERA initiative (project EUGENPATH).

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Intervention of Phytohormone Pathways by Pathogen Effectors.

Host FIH-Mediated Asparaginyl Hydroxylation of Translocated Legionella pneumophila Effectors.

Filamentous plant pathogen effectors in action.

A translocated effector required for Bartonella dissemination from derma to blood safeguards migratory host cells from damage by co-translocated effectors.

Do pathogen effectors play peek-a-boo?

Non-coding RNAs in Host-Pathogen Interactions: Subversion of Mammalian Cell Functions by Protozoan Parasites.

Creating a customized intracellular niche: subversion of host cell signaling by Legionella type IV secretion system effectors.

Post-modern pathogens: surprising activities of translocated effectors from E. coli and Legionella.

Pathogen small RNAs: a new class of effectors for pathogen attacks.

The intersection of Golgi-ER retrograde and autophagic trafficking.

Global temporal dynamic landscape of pathogen-mediated subversion of Arabidopsis innate immunity.

IL-1β impairs retrograde flow of BDNF signaling by attenuating endosome trafficking.

Rerouting of plant late endocytic trafficking toward a pathogen interface.

Ride the wave: Retrograde trafficking becomes Ca2+ dependent with BAIAP3.

Uni-directional ciliary membrane protein trafficking by a cytoplasmic retrograde IFT motor and ciliary ectosome shedding.

Identification of Legionella pneumophila effectors regulated by the LetAS-RsmYZ-CsrA regulatory cascade, many of which modulate vesicular trafficking.

Copper transport and trafficking at the host-bacterial pathogen interface.

Subversion of Host Innate Immunity by Uropathogenic Escherichia coli.

Subversion of the Immune Response by Rabies Virus.

Mechanisms of bacterial morphogenesis and their subversion by phages.

Allelic barley MLA immune receptors recognize sequence-unrelated avirulence effectors of the powdery mildew pathogen.

A retrograde trafficking inhibitor of ricin and Shiga-like toxins inhibits infection of cells by human and monkey polyomaviruses.

Soybean NDR1-like proteins bind pathogen effectors and regulate resistance signaling.

Structural optimization of a retrograde trafficking inhibitor that protects cells from infections by human polyoma- and papillomaviruses.