Actin polymerization in the endosomal pathway, but not on the Coxiella-containing vacuole, is essential for pathogen growth

Coxiella burnetii is an intracellular bacterium that replicates within an expansive phagolysosome-like vacuole. Fusion between the Coxiella-containing vacuole (CCV) and late endosomes/multivesicular bodies requires Rab7, the HOPS tethering complex, and SNARE proteins, with actin also speculated to play a role. Here, we investigated the importance of actin in CCV fusion. Filamentous actin patches formed around the CCV membrane that were preferred sites of vesicular fusion. Accordingly, the mediators of endolysosomal fusion Rab7, VAMP7, and syntaxin 8 were concentrated in CCV actin patches. Generation of actin patches required C. burnetii type 4B secretion and host retromer function. Patches decorated with VPS29 and VPS35, components of the retromer, FAM21 and WASH, members of the WASH complex that engage the retromer, and Arp3, a component of the Arp2/3 complex that generates branched actin filaments. Depletion by siRNA of VPS35 or VPS29 reduced CCV actin patches and caused Rab7 to uniformly distribute in the CCV membrane. C. burnetii grew normally in VPS35 or VPS29 depleted cells, as well as WASH-knockout mouse embryo fibroblasts, where CCVs are devoid of actin patches. Endosome recycling to the plasma membrane and trans-Golgi of glucose transporter 1 (GLUT1) and cationic-independent mannose-6-phosphate receptor (CI-M6PR), respectively, was normal in infected cells. However, siRNA knockdown of retromer resulted in aberrant trafficking of GLUT1, but not CI-M6PR, suggesting canonical retrograde trafficking is unaffected by retromer disruption. Treatment with the specific Arp2/3 inhibitor CK-666 strongly inhibited CCV formation, an effect associated with altered endosomal trafficking of transferrin receptor. Collectively, our results show that CCV actin patches generated by retromer, WASH, and Arp2/3 are dispensable for CCV biogenesis and stability. However, Arp2/3-mediated production of actin filaments required for cargo transport within the endosomal system is required for CCV generation. These findings delineate which of the many actin related events that shape the endosomal compartment are important for CCV formation.


Introduction
Coxiella burnetii is an intracellular Gram-negative bacterium that causes a severe flu-like disease called Q fever. Q fever is a zoonosis, and transmission to humans typically occurs by inhalation of contaminated aerosols generated by infected ruminant livestock [1]. Following deposition in the lungs, C. burnetii invades and replicates within alveolar macrophages [2].
Several intracellular pathogens exploit the host cytoskeleton for generation, maintenance, and/or stability of their respective replication vacuoles [19][20][21][22][23][24]. Cytoskeletal requirements for CCV formation are poorly defined, but some information exists on the role of filamentous (F-) actin. Colonne and co-workers [25] described an actin ring circumscribing the mature CCV in THP-1 macrophages that labels with vasodilator-stimulated phosphoprotein (VASP), an actin regulatory protein. Depletion of VASP disrupts CCV formation and heterotypic fusion

CCV actin patches are enriched for late endocytic vesicles and fusion regulatory proteins
Investigating the role of actin in CCV biogenesis revealed patches of F-actin juxtaposed with the CCV membrane (Fig 1A, S1A Fig). CCV actin patches have been previously described [26], but their association with the CCV and function during infection is unclear. Formation and enlargement of the CCV occurs primarily through fusion with late endocytic vesicles [34]. To investigate the role of actin patches in CCV biogenesis, Vero and THP-1 cells were fixed at 3 days post-infection (dpi), a time of accelerated vacuole growth, and stained for F-actin and markers of late endosomes (CD63 + ) and lysosomes (LAMP1 + ). CCV actin patches were associated with CD63 + and LAMP1 + vesicles that frequently appeared as clusters. Large, individual vesicles were also observed with patches (Fig 1A and 1B, S1A Fig). In contrast, minimal colocalization was observed between CCV actin patches and EEA1, a marker of early endosomes (Fig 1A and 1B).
The interaction between CCV actin patches and late endocytic vesicles suggested that actin patches function in endosomal fusion. Therefore, localization between CCV actin patches and late endosome fusion proteins was assessed. Rab7, a GTPase involved in regulating late endocytic membrane trafficking and fusion, localizes on the CCV membrane and is required for CCV formation and pathogen growth [11,35]. Staining for Rab7 in Vero cells at 3 dpi revealed Rab7 puncta on the CCV localized with F-actin patches (Fig 1C and 1D). The late endocytic SNAREs, VAMP7 and its partner syntaxin 8, also associate with the CCV membrane, with the former essential for CCV growth [36,37]. Similar to Rab7, both VAMP7 and syntaxin 8 formed clusters that localized with CCV actin patches, in contrast to VAMP8, a v-SNARE previously shown to be absent on developed CCVs [36] (Fig 1C and 1D). Comparable results were seen in THP-1 cells (S1B Fig). Thus, actin patches of expanding CCV accumulate proteins predicted to promote fusion between the CCV and late endosomes. To examine actin patches of more mature CCVs, Vero cells were stained for F-actin and CD63 or VAMP7 at 5 dpi, a time when C. burnetii is entering stationary phase growth and the CCV has finished expanding [38]. CCV actin patches colocalized with CD63 and VAMP7, but compared to 3 dpi, CCVs had smaller actin patches with decreased CD63 and VAMP7 intensity (S2A-S2E Fig).

Late endocytic vesicles cluster and fuse with CCV actin patches
The role of actin in membrane fusion is not defined; however, actin polymerization is associated with tethering late endocytic vesicles to target membranes [39]. Insight into CCV actin patch function can be gained by live cell imaging. Fusion between endocytic vesicles and CCV actin patches was examined by spinning disk confocal fluorescence microscopy. CellLight reagents were used to express fluorescent proteins that label lysosomes (LAMP1-GFP) and actin (actin-RFP). In Vero cells at 3 dpi, live cell imaging revealed docking of LAMP + vesicles with CCV actin immediately before fusion (Fig 2A, S1 Movie). To investigate whether CCV actin patches are needed for concentration of late endocytic vesicles, 3 dpi Vero cells were treated with latrunculin A (LatA) to depolymerize F-actin. Prior to LatA treatment, clusters of LAMP1 + vesicles were associated with F-actin patches punctuating the CCV. After LatA treatment, vesicles translocated to the juxta-nuclear region of the cell, leaving a large portion of the CCV devoid of LAMP1 + vesicles (Fig 2B, S2 Movie). CCV CD63 was also redistributed although EEA1-labeled early endosomes were not (S3A and S3B Fig). These results suggest actin patches serve as a scaffold that supports fusion of late endocytic vesicles with the CCV. To resolve whether actin polymerization is also needed for SNARE puncta, Vero cells at 3 dpi were treated with LatA, then stained for VAMP7. Consistent with LatA-mediated detachment of late endocytic vesicles, VAMP7 was depleted on the CCV membrane (Fig 2C and 2D). These data indicate actin polymerization promotes late endocytic SNARE clustering on the CCV membrane.

CCV actin patches depend on type IV secretion of C. burnetii effectors
The C. burnetti Dot/Icm type IVB secretion system (T4BSS) translocates effectors into the host cytoplasm that direct biogenesis of the CCV [17]. To test whether actin patch generation requires C. burnetii protein synthesis, Vero cells at 2 dpi were treated with chloramphenicol for 24 hr, then stained. Compared to untreated cells at 3 dpi, treated cells exhibited collapsed CCVs that lacked actin patches. Clustering of CD63 + vesicles on the CCV was also lost compared to untreated 3 dpi controls. The effects were reversible following washout of chloramphenicol and a 24 hr recovery period (Fig 3A-3C). Similarly, chloramphenicol treatment reversibly decreased CCV clustering and colocalization of VAMP7 and Rab7 with actin (S4A-S4D Fig). To confirm that CCV actin patch formation is T4BSS-dependent, Vero cells were infected with a C. burnetii dotA mutant and stained for F-actin and CD63 (Fig 3D-3F) [17]. At 1 dpi, the dotA mutant failed to induce formation of CCV F-actin patches associated with CD63 + vesicles. These results demonstrate that polymerization of CCV actin patches requires C. burnetii protein synthesis and Dot/Icm T4BSS effectors.
To identify regulators of actin polymerization at the CCV, Vero and THP-1 cells at 3 dpi were stained for vesicle-related actin regulators. VPS35, FAM21, WASH, Arp3, and cortactin clustered with CCV actin patches, suggesting CCV actin polymerization is regulated by the retromer-WASH complex, with filaments stabilized by cortactin (Fig 4A and 4B, S5 Fig).
Arp2 phosphorylation, which is required for activation, was equivalent in infected and uninfected cells [45]  To test whether the retromer-WASH complex association with CCV actin was directed by C. burnetii, Vero cells 2 dpi were treated with chloramphenicol for 24 hr, then fixed and stained for WASH or VPS35 (S8A and S8B Fig). Corresponding to the loss of CCV actin patches following treatment, WASH and VPS35 clusters were also lost, an effect that was reversed following antibiotic washout. Furthermore, CCVs harboring a dotA mutant failed to recruit Arp3 (S9 Fig). These data suggest C. burnetii effectors are required for recruitment and clustering of the retromer-WASH complex to mediate polymerization of CCV actin patches.

Retromer association with late endosomal/lysosomal markers is enriched in infected cells
Retromer sequesters membrane receptors for their retrieval from endosomes to the trans-Golgi or plasma membrane, a function that typically involves membrane tabulation and fission treatment redistributes LAMP1 + clusters at actin patches around the CCV to the juxta-nuclear region (white arrows). Live cell images of a Vero cell at 3 dpi expressing lysosome-GFP (LAMP1) and actin-RFP. (C and D) LatA treatment redistributes VAMP7 + clusters at actin patches to the juxta-nuclear region (white arrows). Vero cells at 3 dpi were treated with LatA for 10 min prior to fixation and immunostaining for F-actin, VAMP7, and NMII. Histogram depicts the mean intensity ± SD of ! 60 cells for at least 3 independent experiments. Statistical significance was determined using Student's t-test ( ÃÃÃÃ P <0.0001). NMII, C. burnetii Nine Mile phase II strain. Scale bar, 2.5 μm. Chloramphenicol reversibly causes CCV shrinkage and elimination of actin patches and associated clusters of CD63 + vesicles. Vero cells at 2 dpi were treated with chloramphenicol for 24 hr (+chlor) then fixed along with the corresponding 3 dpi untreated control (-chlor). Treated cells at 3 dpi were also washed to remove chloramphenicol and allowed an additional 24 hr recovery before fixation (recovery). Cells were fluorescently stained for F-actin and CD63. (D, E, and F) CCV actin patches and colocalization with CD63 are lost in cells infected with a C. burnetii dotA mutant. Vero cells infected for 24 hr with wild type C. burnetii or a dotA mutant were fixed and stained for F-actin and CD63. Asterisks mark the C. burnetii vacuole in the actin panels. Histograms depict the mean intensity of CCV actin or area ± SD of ! 60 cells for at least 3 [31]. In infected cells, retromer also localizes with CCV actin patches associated with endosome fusion. To determine how retromer interacts with vesicles of the endosomal pathway independent experiments. Statistical significance was determined using Student's t-test ( ÃÃÃÃ P <0.0001). Colocalization was determined using Pearson's correlation coefficient. NMII, C. burnetii Nine Mile phase II strain. Scale bar, 5 μm.

Sorting nexins partially colocalize with actin patches
Sorting nexins (SNXs) are phosphatidylinositol 3-monophosphate (PI3P) binding proteins that coordinate with retromer to recycle receptors from endosomes. SNX1 or SNX2 dimerize with SNX5 or SNX6 and regulate canonical retrograde trafficking. SNX3 mediates an alternative route of cargo trafficking to the trans-Golgi in a WASH and actin-independent manner. SNX27 is involved in recycling plasma membrane receptors. [31,48]. In contrast to the near complete localization of VPS35 with CCV actin patches, SNXs moderately colocalized (S10A and S10B Fig). To validate the specificity of antibodies directed against SNX1 and SNX2, Vero cells infected for 1 day with Chlamydia trachomatis (L2 serotype) were immunostained. As previously reported [49], SNX1 and SNX2 decorated the chlamydial inclusion (S10C Fig).

Retrograde recycling of the cation-independent mannose-6-phosphate receptor (CI-M6PR) is normal in infected cells
C. trachomatis and Legionella pneumophila inhibit retrograde trafficking via sequestration of SNX5/SNX6 and VPS29, respectively, to the pathogen vacuole [50,51]. Because retromer and SNXs localized to the CCV membrane, we examined whether C. burnetii infection disrupts normal retrograde trafficking of CI-M6PR from endosomes to the trans-Golgi network. Inhibition of retrograde trafficking traps CI-M6PR in dispersed, early endosomes as opposed to a focused, juxta-nuclear localization within the trans-Golgi [47,52]. CI-M6PR dispersal did not occur upon knockdown (KD) by siRNA of VPS35 [53,54] (Fig 6A, S11 Fig). Therefore, the chemical inhibitor Retro-2 was used which prevents vesicle fusion with the trans-Golgi network [55]. Without treatment, uninfected and infected Vero cells after 3 days of incubation showed focused CI-M6PR staining (S12A and S12B Fig). In Retro-2 treated cells, CI-M6PR showed dispersed staining. Collectively, these data indicate infection does not disrupt retrograde recycling of CI-M6PR.

Knockdown of VPS35 reduces CCV actin patches and redistributes Rab7 with no effect on C. burnetii growth
Retromer recruits the WASH complex to endosomal membranes where it promotes actin polymerization required for scission of membrane tubules containing cargo for recycling [31,33,42]. To investigate whether retromer is necessary for CCV actin patch and vacuole formation, KD of VPS35 was performed (Fig 6A). VPS35 KD inhibited CCV actin patch formation as witnessed by decreased actin patch size and intensity (Fig 6B and 6C). Similar results were seen with KD of VPS29 in HEK 293 cells (S13A-S13D Fig). VPS35 KD cells lacked FAM21 and WASH on the CCV membrane (S14A-S14D Fig), indicating retromer is required for recruitment of the WASH complex to the CCV membrane and for efficient polymerization of CCV actin. Compared to cells treated with non-targeting (NT) siRNA, VPS35KD had no effect on C. burnetii replication but slightly decreased the size of the 3 day CCV (Fig 6D  and 6E).
In addition to regulating fusion of late endosomes, Rab7 recruits retromer by binding VPS35 [56,57]. Focalized clusters of CD63 and Rab7 on the CCV in control cells were absent in VPS35 KD cells, where the markers localized uniformly around the CCV (Fig 6B, S15A  Fig). Diminished actin patches of VPS35 KD cells exhibited reduced colocalization with CD63, suggesting CD63 + vesicles are not docking to CCVs at actin patches (S15A and S15B
https://doi.org/10.1371/journal.ppat.1007005.g005  Similarly, VAMP7 exhibited decreased clustering and colocalization with CCV actin patches compared to NT cells (S15C and S15D Fig). Collectively, these results suggest retromer depletion allows Rab7 to uniformly distribute on the CCV, which promotes a similar distribution of late endocytic vesicles/fusion regulators.
KD of VPS35 perturbed recycling of the glucose transporter 1 (GLUT1) from endosomes to the plasma membrane, with trafficking redirected to lysosomes (S16A Fig) [58]. C. burnetii infection did not disrupt GLUT1 recycling to the plasma membrane. However, KD of VPS35 in infected cells dramatically increased the percentage of GLUT1 + CCVs (S16B and S16C Fig). This indicates CCV growth is not dependent on retromer recycling of plasma membrane receptors.

WASH deficiency prevents CCV actin patch formation without affecting C. burnetii or CCV growth
In VPS35 KD cells, the CCV lacks WASH and has reduced actin patches. To determine whether WASH is necessary for CCV actin patch formation and vacuole growth, we performed infections of inducible WASH knockout MEFs. Treatment with 4-hydroxy-tamoxifen (4-OHT) of WASH (f/f) MEFs eliminates expression of WASH (WASHout) as determined by immunoblot and immunofluorescence (S17A and S17B Fig). Similar to VPS35 KD cells, CCVs in WASHout cells at 3 dpi lacked actin patches and exhibited uniform distribution of Rab7 around the CCV (Fig 7A and 7B). Rab7 redistribution was anticipated as retromer binds Rab7 [47]. CCV size and C. burnetii replication remained similar to untreated control cells (WASH f/f ) (Fig 7C and 7D). WASHout cells also displayed uniform staining of VPS35 around the CCV (Fig 7E). Treatment of wild type MEFs (WASH +/+ ) with 4-OHT did not affect actin patch formation, Rab7 clustering, CCV size or C. burnetii replication (S18A-S18D Fig). Collectively, these results indicate that WASH depletion inhibits CCV actin patch formation, resulting in dispersion of retromer/WASH-generated actin sorting platforms.

Arp2/3-dependent endocytic trafficking is necessary for CCV biogenesis
The Arp2/3 complex nucleates formation of barbed actin filaments involved in phagocytic uptake and endosomal trafficking [29]. Expansion and maintenance of the CCV is normal without WASH, an NPF that activates Arp2/3, suggesting nucleation at the CCV is dispensable for C. burnetii growth [29]. To determine if Arp2/3-mediated actin polymerization in the endosomal pathway is necessary for CCV biogenesis, and to distinguish from phagocytic uptake of C. burnetii, Vero cells were treated at 1 dpi for 2 days with the specific Arp2/3 inhibitor CK-666, which stabilizes the inactive state of Arp2/3 [59]. Like chloramphenicol-treated cells, CCVs in CK-666-treated cells lacked actin patches and failed to develop into spacious vacuoles supporting C. burnetii growth (Fig 8A and 8B). Additionally, CCVs in Vero cells infected for 2 days, then treated for 1 day with CK-666, displayed shrunken CCVs, an effect that was reversible (Fig 8C and 8D). Loss of actin patches also correlated with loss of CCV Arp3 labeling (S19A and S19B Fig). KD of Arp 3 also resulted in significantly smaller CCVs (S19C, S19D and S19E Fig). Collectively, these results suggest CCVs of treated cells have a severe reduction in fusion with endosomes. To confirm inhibitory effects were not cell type specific, or related to retromer-WASH regulated trafficking or actin patch formation, WASHout cells at 2 dpi were treated for 1 day with CK-666. Infected WASHout cells also had shrunken CCVs (S20A and S20B Fig). This result indicated Arp2/3-mediated endocytic events upstream of vesicle fusion with CCVs are being disrupted.
Fusion with late endosomes is required for CCV biogenesis [36]. Transferrin traffics to the CCV via the endosomal pathway [60]. Therefore, transferrin localization was examined in cells treated with CK-666 to determine if Arp2/3 is required for endosomal trafficking associated with CCV biogenesis. In Vero cells infected for 2 days, then treated with CK-666 for 24 hr, trafficking of 488-transferrin to CD63 + limiting membrane of CCVs was severely reduced https://doi.org/10.1371/journal.ppat.1007005.g007 (Fig 9A-9C). These data demonstrate that Arp2/3-mediated actin dynamics that regulate trafficking within the endosomal pathway are required for CCV formation and pathogen growth.

Fig 8. Arp2/3 is needed for CCV biogenesis. (A and B)
CK-666 treatment prevents CCV expansion. Vero cells at 24 hr post-infection were treated with CK-666 or chloramphenicol for 2 days. Cells were fixed and fluorescently stained for CD63 and F-actin. (C and D) CK-666 treatment causes CCV collapse. Vero cells at 2 dpi were treated with CK-666 for 24 hr, then fixed along with the corresponding 3 dpi DMSO-treated control or washed to remove CK-666 and allowed an additional 24 hr recovery before fixation. Cells were fluorescently stained for F-actin and CD63. Histograms depict the means of the percentage of cells with normal CCV expansion or CCV area ± SD of ! 60 cells for at least 3 independent experiments. Statistical significance was determined by the Student's t-test ( ÃÃÃÃ P <0.0001). NMII, C. burnetii Nine Mile phase II strain. Scale bar, 5 μm. https://doi.org/10.1371/journal.ppat.1007005.g008

Discussion
Exploitation of the actin cytoskeleton is a common theme among intracellular bacteria [61]. Here, we show that F-actin patches form on the CCV membrane in Vero cells and THP-1 macrophages. Patches decorate with late endosome fusion proteins CD63, Rab7, VAMP8, syntaxin 8, and to a lesser extent, LAMP1. Consistent with the presence of membrane fusion complexes, actin patches are preferred docking sites for vesicle fusion. Rapid disruption of CCV actin patches with LatA results in relocalization of LAMP1 + vesicles away from the CCV, without affecting vacuole size. Accordingly, this corresponds to the disappearance of CCV VAMP7. Actin patches are less pronounced on non-expanding, mature vacuoles where fusion processes are likely down-regulated.
Our data show that CCV actin patches also colocalize with retromer, Fam21 WASH, and Arp2/3, and that the function of these proteins is necessary for patch formation. Several sorting nexins are also proximal to the retromer, although none strongly colocalize. SNX1 and SNX2 have previously been shown to localize in proximity of WASH and VPS29 [33,62]. Despite an association with CCV membrane fusion sites, retromer-generated actin is dispensable for CCV formation and C. burnetii growth. These conclusions are supported by robust replication of C. burnetii in VPS29 or VPS35 KD cells and WASH knockout MEFs, which lack CCV actin patches. Our results contrast with a previous study that reported individual silencing of VPS29, VPS35, SNX2, SNX3, SNX5, or SNX6 inhibits C. burnetii infection. [11]. However, due to functional redundancy of SNX1 and SNX2, as well as SNX5 and SNX6, depletion of one of either pair will not disrupt retrograde trafficking [53,63]. Retromer is proposed to benefit C. burnetii by recycling beneficial host factors and/or removing harmful CCV determinants. Promoting proper CCV maturation was also suggested, although acidification of the CCV appears normal [11]. Disparate results may be due, in part, to different experimental readouts and cell lines (e. g. HeLa versus HEK 239 cells).
Unlike the neutral effect of retromer inhibition on C. burnetii growth, intracellular growth of Legionella pneumophila, C. trachomatis, and Salmonella typhimurium is negatively impacted by retrograde trafficking, and each pathogen secretes effector molecules to disrupt the process [49,51,64,65]. S. typhimurium inhibits retrograde trafficking of CI-M6PR through the activity of the type 3 secretion system (T3SS) effector SifA, which binds the host protein SifA-and kinesin-interacting protein (SKIP). SifA-Skip sequesters Rab9, which is necessary for late endosome to trans-Golgi transport of CI-M6PR. The end result is a Salmonella-containing vacuole with reduced lysosomal activity that enables pathogen growth [66]. The C. trachomatis T3SS effector IncE localizes to the chlamydial inclusion where it binds SNX5 and SNX6 [49,50,67]. Like S. typhimurium, subversion of retrograde trafficking is proposed to benefit chlamydial infection by disrupting lysosome function [50,67]. In L. pneumophila, the T4BSS effector RidL localizes to the Legionella-containing vacuole where it binds VPS29 and PI3P. These interactions restrict assembly of the retromer, thereby inhibiting retrograde trafficking [51]. The benefit to L. pneumophila is unclear. SNX1, SNX2, SNX5, and SNX6 contain a Bin/Amphiphysin/Rvs (BAR) domain with curvature sensing, inducing, and stabilizing activities that promote endosomal tubulation associated with recycling of receptors from endosomes to the trans-Golgi (retrograde transport) or plasma membrane [48,63]. These SNXs, as well as SNX3 and SNX27, contain a phox homology (PX) domain that preferentially binds PI3P on early endosomes. The canonical retromer involved in retrograde trafficking of cargo, such as CI-M6PR, is recruited to membranes by binding of VPS35 to both Rab7 and SNX3 [56,57]. The retromer involved in recycling plasma membrane receptors, such as GLUT1, is recruited via binding of VPS26A or VPS26B to SNX27 [58,[68][69][70]. Both of these retromer complexes utilize SNX-BAR and WASH-Arp2/ 3-mediated actin polymerization for tubule formation and scission [31]. The SNX3 retromer traffics cargo such as Wntless to the trans-Golgi where it is loaded with Wnt for transport to the plasma membrane [46,71,72]. The SNX3 retromer lacks the WASH complex and SNX-BARs, with cargo vesicles budding from the donor membrane through a process involving clathrin [46,56]. The appearance of GLUT1 on the CCV membrane following VPS35 KD is consistent with trafficking of the receptor to lysosomes for degradation following retromer disruption [58]. Thus, disruption of this recycling pathway, which controls trafficking of additional receptors, such as the monocarboxylate transporter [73], is of little consequence to C. burnetii. Surprisingly, VPS35 KD did not disperse CI-M6PR to endosomes, suggesting retrograde trafficking is unaltered, and that delivery of hydrolases to the CCV occurs normally.
In HeLa cells, a 6.5 h treatment with Retro-2 inhibits endosome-to-Golgi retrograde trafficking of toxins without affecting trafficking of endogenous cargos, like CI-M6PR [55]. The precise mechanism of inhibition is unknown but is speculated to involve direct inhibition or altered localization of Golgi syntaxin 5. Based on a dispersed endosomal location of CI-M6PR, our results show trafficking of CI-M6PR in Vero cells is inhibited by Retro-2 following a 48 hr treatment. Retro-2 also did not inhibit CCV formation, which further supports the notion that canonical retrograde trafficking is dispensable for C. burnetii growth. This result also suggests C. burnetii does not require CI-M6PR-mediated delivery of hydrolases for growth.
F-actin patches generated in a retromer-WASH complex and Arp2/3-dependent fashion are thought to generate force for scission of endosomal tubules [32,62]. VPS35-bound FAM21 recruits WASH to endosomal membranes [33,62]. On endosomes, retromer still binds in the absence of WASH, but is redistributed on the endosome membrane. We observed a similar redistribution of VPS35 on the CCV of WASH knockout MEFs.
Activation of Arp2/3 by WASP family NPFs and subsequent generation of branched actin filaments modulates several membrane-associated functions including lamellipodia formation, receptor trafficking, endosome tubulation and shape, retrograde trafficking, autophagosome formation, and endocytosis/phagocytosis [30]. Accordingly, KD of Arp3 prior to infection of HeLa cells inhibits phagocytosis of C. burnetii [11]. Here, we show that treatment with the specific Arp2/3 inhibitor CK-666, or Arp3 KD, inhibits CCV formation. Moreover, CK-666 treatment of cells harboring mature CCV causes vacuole collapse. Because actin filamentation mediates endocytic trafficking [29], it is predicted to contribute to CCV maturation. Indeed, CK-666 inhibition of Arp2/3 inhibits endocytic trafficking of transferrin to the CCV.
A previous study demonstrated C. burnetii protein synthesis is required for CCV recruitment of VAMP7 [36]. We show that chloramphenicol reversibly inhibits recruitment of VAMP7, Rab7, VSP35, and WASH, as well as actin patch formation. Moreover, vacuoles harboring a dotA mutant lack actin patches. Collectively, these data suggest that recruitment of retromer and membrane fusion complexes is mediated by a C. burnetii T4BSS effector protein (s). The CCV is unusual in being positive for both Rab7, a marker of late endosomes/lysosomes, and enriched for PI3P, a characteristic of early endosomes [9]. Indeed, enrichment of PI3P on the CCV is associated with the activity of the C. burnetii effector CvpB. CvpB inhibits phosphotidylinositol 3-phosphate 5-kinase, the enzyme responsible for converting PI3P to phosphatidylinositol 3,5-bisphosphate on late endosomes [9]. Treatment with wortmannin, a phosphatidylinositol 3-kinase inhibitor, releases retromer and SNX1 from endosomes [47,63]. Thus, the possibility exists that retromer is aberrantly targeted to the CCV based on cooperative binding of VPS35 and SNX3 to Rab7 and PI3P, respectively, without serving a functional role [42,70].
The ability of CCVs devoid of actin patches, such as those in WASH knockout MEFs, to expand normally may be based on the dual function of Rab7 in membrane fission and fusion.
In yeast, the Rab7 homolog Ypt7 is released upon membrane tubulation mediated by the SNX-BAR retromer, allowing interaction with the HOPS complex and membrane fusion [74]. Perhaps dispersal of Rab7 following depletion of VPS29 or VPS35, or in WASH knockout cells, reflects the lack of F-actin organizing platforms that concentrate Rab7 at membrane fusion complexes [70]. Indeed, actin polymerization organizes WASH in discrete membrane domains [75]. Fusion of late endosomes/MVBs with the CCV would then occur in a dispersed manner, as opposed to localized fusion associated with actin patches. The ability of C. burnetii to prosper without CCV actin may also be related to the hyper-fusogenic nature of the vacuole [76].
During the preparation of this manuscript, two controversial studies were coordinately published indicating SNX-BAR without retromer can mediate retrograde retrieval of CI-M6PR [53,54]. Data derived by quantitative proteomics and protein binding assays show CI-M6PR directly binds SNX5 and SNX6 [53,54]. Moreover, retrograde retrieval of CI-M6PR is unaffected by KD or knockout of retromer components [53,54]. This behavior is associated with SNX-BAR and retromer occupying separate microdomains on endosomes, as suggested by a previous report [77,78]. As with our investigation, these studies employed the broadly used and accepted assay for dysfunctional retrograde sorting, i. e., dispersal of CI-M6PR into endosomes. Others have suggested that disparate results arise from different measures of CI-M6PR dispersal [79]. Moreover, a two-step model is proposed that invokes a role for retromer in transiently concentrating cargo, which is then rapidly transferred to SNX-BAR for tubule-mediated retrieval to the Golgi [53,77]. Clearly, more study is needed, although our data showing poor colocalization of SNXs with retromer, and the lack of CI-M6PR dispersal following VSP35 KD, agrees with the newly proposed model. Nonetheless, plasma membrane recycling of GLUT1 is clearly inhibited upon retromer disruption to result in aberrant trafficking of the receptor to the CCV membrane.
Based on the bulk of published data, we propose a model, similar to one recently proposed by Jimenez-Orgaz et al. [80], that invokes a dual regulatory role for CCV Rab7 in endosome fusion and retromer recruitment (S21 Fig). Membrane receptors, such as GLUT1, are recycled from transitioning early to late endosomes by retromer. This can occur coincidently with late endosome engagement with the CCV, but prior to vesicle fusion. Retromer recruitment by CCV Rab7, and subsequent WASH-Arp2/3 generation of actin sorting platforms, concentrates Rab7 and impedes its lateral diffusion. Consequently, recruitment of HOPs/SNARE complexes by Rab7 results in focused fusion of late endosomes with the CCV. In the absence of retromer and sorting platforms, membrane receptors are not recycled but instead deposit on the CCV in a dispersed manner due to the unfocused nature of Rab7. While retromer-derived F-actin is dispensable for C. burnetii growth, additional Arp2/3-generated F-actin structures are essential for CCV biogenesis and pathogen replication, including those that mediate endocytic trafficking. An intriguing question is the identity and function of TBSS effector(s) modulating this fusogenic behavior. In summary, this study illustrates the complex interplay between the CCV and actin-mediated vesicular trafficking pathways. It also describes a large vacuole model for studying retromer function, reminiscent of vacuoles generated by ectopic expression of constitutively-activated Rab5 [54].

Cell culture and infection
HEK 293 (ATCC CRL-1573, human embryonic kidney epithelial) cells were cultured in Delbecco's Modified Eagle Medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS). THP-1 (ATCC TIB-202, human monocytic) and Vero (ATCC CCL-81, African green monkey epithelial) cells were cultured in RPMI medium 1640 (Life Technologies) supplemented with 10% FBS. WASH conditional knockout mouse embryonic fibroblasts (MEFs) (Daniel Billadeau, Mayo Clinic) were grown in DMEM containing 10% FBS. Generation and characterization of these cells are described in Gomez et al. [81]. MEFs were grown in DMEM containing 10% FBS, and knockout of WASH was obtained with two sequential 24 hr treatments of 3 μM 4-OHT (Sigma, H7904). After treatment, MEFs were washed and cultured for an additional 6-7 days before use in experiments [81]. All cell lines were incubated at 37˚C with 5% CO 2 . Infection of Vero cells with C. trachomatis was at a multiplicity of infection (MOI) of 50 based on inclusion forming units.
C. burnetii Nine Mile phase II, RSA439 (NMII) was propagated in Vero cells and purified as described [82]. The C. burnetii dotA mutant was propagated in the synthetic medium ACCM-2 as described [17]. C. trachomatis (LGV-434, serotype L2) was propagated in HeLa cells and purified as described [83]. For infection, HEK 293, Vero, or MEF cells were seeded on coverslips in 24-well plates at a density of 4 x 10 4 cells per well. THP-1 cells were seeded on coverslips in 24-well plates at a density of 3 x 10 5 cells per well and stimulated with 200 nM phorbol myristate acetate (Sigma) for 24 hr for differentiation into macrophage-like cells and attachment to coverslips. For immunofluorescence staining, cells were infected at an MOI of 100 based on genome equivalents (GE) quantified by TaqMan qPCR using a StepOnePlus Real-Time PCR system (Applied Biosystems) and primers specific to C. burnetii groEL [38]. For C. burnetii growth analysis, cells were infected at an MOI of 10.

Live imaging
Vero cells on glass bottoms of 24-well SensoPlates (Greiner bio-one, 662892) were transfected with CellLight Actin-RFP, BacMam 2.0 (Molecular Probes, C10502) 1 dpi and CellLight Lysosomes-GFP, BacMam 2.0 (Molecular Probes, C10507) the night before imaging at 3 dpi. Cell-Light reagents were used at concentrations recommended by the manufacturer. Cells were washed to remove CellLight reagents before imaging on a Nikon ECLIPSE Ti spinning disk confocal fluorescence microscope. For imaging of LAMP1 + vesicles fusing with CCV membranes, cells were incubated at 37˚C with 5% CO 2 and imaged at 1 frame every 2 sec. For LatA (Sigma) treatment, cells were imaged at room temperature at 1 frame per min. Imaging started 15 min before adding LatA followed by 30 min post-treatment.

siRNA transfection
Dharmacon ON-TARGETplus SMARTpool siRNAs for VPS29 (L-009764-01-0005), VPS35 (L-010894-00-0005), Arp3 (L-012077-00-0005), and non-targeting (D-001810-10-5) were used. HEK 293 cells were used as opposed to HeLa cells because more complete and consistent KD was obtained. For KDs, HEK 293 cells were grown on coverslips in 24-well plates at a density of 4 x 10 4 cells per well for 1 day before transfection. Dharmafect 1 (Dharmacon) was used to transfect cells with siRNA complexes at a concentration of 2 μM. For KD of VPS29 and VPS35, cells were infected two days post-transfection. For KD of Arp3, cells were transfected 1 and 3 days before infection. Cells were fixed 3 dpi and processed for immunofluorescence. Immunoblots of lysates were used to confirm efficiency of KD. Actin served as a loading control.

C. burnetii growth in MEFs and HEK 293 cells
MEF or HEK 293 cells seeded at 4 x 10 4 cells per well in 24-well plates were infected with C. burnetii at an MOI of 10 by centrifuging at 500 x g for 30 min at room temperature. Cells were then washed and incubated with growth medium. Samples were collected by trypsinization, bead beaten with 0.1 mm Zirconia/silica beads (BioSpec) in a homogenizer (ThermoElectron FastPrep FP120), and boiled 10 min. GE were quantified by qPCR.

CI-M6PR retrograde trafficking
CI-M6PR retrograde trafficking assays were performed as described in Osborne et al. [52]. HEK 293 cells were incubated with 10 μg/ml anti-CI-M6PR (BioRad, MCA2048) in serumfree medium for 1 hr at 37˚C. Cells were washed with PBS prior to immunostaining. Vero cells were treated with DMSO or Retro-2 for 2 days, followed by incubation for 1 hr at 37˚C with 10 μg/ml anti-CI-M6PR in serum free medium. Cells were washed with PBS, then fixed and fluorescently stained.

Transferrin endocytosis assay
Vero cells seeded on glass coverslips at 4 x 10 4 cells per well in 24-well plates were infected at an MOI of 100. Two dpi cells were treated with DMSO or CK-666 for 24 hr. Cells were incubated with 25 μg/ml of Alexa Fluor-488 transferrin (ThermoFisher, T13342) for 1 hr, washed three times with PBS, fixed with 4% PFA, then fluorescently immunostained for CD63 and C. burnetii.

Microscopy and analysis
Fixed and stained cells were imaged using Zeiss LSM-710 confocal fluorescence microscope (Carl Zeiss). For compiling images, z-sections of 0.32 μm slices were taken. Depicted images of cells are maximum intensity projections of 3 slices, totaling 1 μm thickness. Fiji (Image J, National Institutes of Health) was used for all image analysis. The entire CCV was selected for co-localization and fluorescence intensity analysis and included a combined measurement of 3 z-sections from the vertical center (z-axis) of the CCV. The area of CCVs was measured using CD63, LAMP1, or Rab7 as CCV membrane markers. For determining disruption of CI-M6PR retrograde trafficking, the percentage of cells with visually compact versus scattered CI-M6PR staining was quantitated. Correlation coefficients (Pearson's Correlation Coefficient) were determined using ImageJ Coloc 2. Unless otherwise stated, a minimum of 60 cells for each condition from 3 independent experiments was used for analyses. GraphPad Prism (GraphPad Software) was used for all graphing and statistics. The colocalization of CCV actin patches with Rab7 is reduced with chloramphenicol treatment. Colocalization analysis of CCVs was determined using Pearson's correlation coefficient. Graphs represent the means ± SD of ! 60 cells from at least 3 independent experiments. Statistical significance determined by Student's t-test ( ÃÃÃÃ P <0.0001). NMII, C. burnetii Nine Mile phase II strain. Scale bar, 5 μm.  A and B) Three dpi Vero cells were fixed, fluorescently stained for F-actin, and immunostained for the indicated sorting nexins (SNXs). SNXs show partial colocalization with CCV actin patches. (C) For a positive SNX control, Vero cells 1 dpi with Chlamydia trachomatis L2 were stained for SNX1 or SNX2. Similar to a previous report [48], SNX1 and SNX2 stain the C. trachomatis inclusion membrane, validating the antibodies. Colocalization analysis of CCVs was determined using Pearson's correlation coefficient. Graphs represent the means ± SD of ! 60 cells from at least 3 independent experiments. Statistical significance was determined by the Student's t-test ( ÃÃÃÃ P <0.0001). NMII, C. burnetii Nine Mile phase II strain. Scale bar, 5 μm. (TIF) S11 Fig. CI-M6PR is not dispersed in VPS35 knockdown cells. HEK 293 cells knocked down for VPS35 or non-targets and infected for 3 days were incubated with anti-CI-M6PR antibody for 1 hr at 37˚C, then fixed and immunostained. The trans-Golgi (TNG46) was stained to evaluate retrograde trafficking of CI-M6PR. Knockdown of VPS35 (siVPS35) has no effect on CI-M6PR staining which shows focused colocalization with TGN46 similar to the non-targeted control. Images are representative of 2 independent experiments. NMII, C. burnetii Nine Mile phase II strain. Scale bar, 10 μm.  A and B) VPS35 knockdown or control HEK 293 cells at 3 dpi were fluorescently stained for F-actin and CD63. VPS35 knockdown cells have uniform staining of CD63 around the CCV and lack colocalization of CD63 clusters with reduced CCV actin patches. (C and D) Same as (A and B) but with VAMP7 staining. VAMP7 staining is still observed around CCVs of VPS35 knockdown cells but lacks colocalization with reduced CCV actin patches. Graphs represent the means ± SD of ! 60 cells from 3 independent experiments. Colocalization was determined using Pearson's correlation coefficient. Statistical significance was determined by the Student's t-test ( ÃÃÃÃ P <0.0001). NMII, C. burnetii Nine Mile phase II strain. Scale bar, 5 μm. (TIF) S16 Fig. C. burnetii infection does not disrupt GLUT1 retromer-dependent trafficking to the plasma membrane. (A) Uninfected VPS35 knockdown or NT-treated HEK 293 cells were stained for GLUT1 and CD63. As previously reported [57], knockdown of VPS35 disrupts recycling from early endosomes to the plasma membrane, redirecting GLUT1 trafficking to late endosomes/lysosomes. (B and C) Three dpi VPS35 knockdown or NT-treated HEK 293 cells were fixed and stained for GLUT1. Infection alone does not affect GLUT1 trafficking. However, knockdown of VPS35 results in trafficking of GLUT1+ vesicles to the CCV, increasing GLUT1 intensity on the CCV membrane. Graphs represent the means ± SD of ! 60 cells from 3 independent experiments. Statistical significance was determined by the Student's ttest ( ÃÃÃÃ P <0.0001). NMII, C. burnetii Nine Mile phase II strain. Scale bar, 5 μm. The proposed model depicts a dual regulatory role for Rab7 in mediating recruitment of retromer for membrane receptor recycling and fusion of late endosomes with the Coxiella-containing vacuole (CCV). In the presence of retromer and WASH (left of dashed line), GLUT1 on transitioning early to late endosomes is normally recycled to the plasma membrane. GLUT1 is recognized by SNX27, which along with Rab7, recruits the retromer-WASH-Arp2/3 complex that generates F-actin required for recycling. Simultaneously, Rab7 recruits the retromer-WASH-Arp2/3 complex which generates F-actin sorting platforms that cluster Rab7 on the CCV. In turn, HOPs/SNARE complexes (VAMP7 and syntaxin 8) are recruited to platforms which results in focused fusion of late endosomes with the CCV. In the absence of retromer and actin sorting platforms (right of dashed line), the unfocused nature of Rab7 results in GLUT1 receptors that are not recycled but instead deposit on the CCV. Late endosomal markers also disperse in the CCV due to the unfocused nature of Rab7. (TIF) S1 Movie. Actin patches tether late endocytic vesicles to the CCV membrane for fusion. Live cell imaging of a 3 dpi Vero cell transfected with CellLight reagents (BacMam) lysosome-GFP (LAMP1) and actin-RFP. Cells were incubated at 37˚C with 5% CO 2 and imaged at 1 frame every 2 sec.