Phospho-regulation accommodates Type III secretion and assembly of a tether of ER-Chlamydia inclusion membrane contact sites

Membrane contact sites (MCS) are crucial for non-vesicular trafficking-based inter-organelle communication. ER-organelle tethering occurs in part through the interaction of the ER resident protein VAP with FFAT-motif containing proteins. FFAT motifs are characterized by a seven amino acidic core surrounded by acid tracks. We have previously shown that the human intracellular bacterial pathogen Chlamydia trachomatis establishes MCS between its vacuole (the inclusion) and the ER through expression of a bacterial tether, IncV, displaying molecular mimicry of eukaryotic FFAT motif cores. Here, we show that multiple layers of host cell kinase-mediated phosphorylation events govern the assembly of the IncV-VAP tethering complex. CK2-mediated phosphorylation of a C-terminal region of IncV enables IncV hyperphosphorylation of a phospho- FFAT motif core and serine-rich tracts immediately upstream of IncV FFAT motif cores. Phosphorylatable serine tracts, rather than genetically-encoded acidic tracts, accommodate Type III-mediated translocation of IncV to the inclusion membrane, while achieving full mimicry of FFAT motifs. Thus, regulatory components and post-translational modifications are integral to MCS biology, and intracellular pathogens such as C. trachomatis have evolved complex molecular mimicry of these eukaryotic features.


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In naïve cells, membrane contact sites (MCS) are points of contact between the membrane of two 33 adjacent organelles (10-30 nm apart). They provide physical platforms for the non-vesicular 34 transfer of lipids and ions, and cell signaling events important for inter-organelle communication 35 and organelle positioning and dynamics (Prinz et al., 2020). Since their discovery and implication  IncV is modified by phosphorylation 124 The presence of a phospho-FFAT in IncV led us to investigate the phosphorylation status of IncV. 125 When subjected to anti-FLAG western blot analysis, lysates of HEK293 eukaryotic cells infected 126 with wild type C. trachomatis expressing IncV-3xFLAG displayed a doublet consisting of a 50kDa 127 and 60 kDa band ( Fig. 2A, middle lane, 293 + Ct). By contrast, IncV-3xFLAG ectopically 128 expressed in HEK293 cells had an apparent molecular weight that was shifted toward the 60k Da  This result led us to hypothesize that IncV is post-translationally modified by a host factor. 132 To determine if phosphorylation could account for the increase in the apparent molecular weight 133 of IncV, we performed a phosphatase assay. IncV-3xFLAG was immunoprecipitated, using anti-  The host kinase CK2 phosphorylates IncV 145 We next focused on identifying the host cell kinase(s) responsible for phosphorylating IncV. All 146 three subunits of Protein Kinase CK2 were identified as potential interacting partners of IncV in 147 an Inc-human interactome (Mirrashidi et al., 2015). To determine if CK2 associated with IncV at  Having established that IncV is phosphorylated and that CK2 localizes to ER-Inclusion MCS in 161 an IncV-dependent manner, we next tested if CK2 phosphorylates IncV. We performed an in vitro 162 kinase assay using recombinant CK2 and the cytosolic domain of IncV (amino acids 167-363 of 163 IncV) fused to GST (GST-IncV167-363) or GST alone, purified from E. coli. To detect 164 phosphorylation, we used ATPγS, which can be utilized by kinases to thiophosphorylate a 165 substrate, followed by an alkylation reaction of the thiol group to generate an epitope that is 166 detected using an antibody that recognizes thiophosphate esters (Allen et al., 2007). When GST 167 alone was provided as a substrate, there was no detectable phosphorylation, regardless of the 168 presence of CK2 and ATPγS (Fig. 2D, lanes 1 and 2). A similar result was observed with  IncV167-363 in the absence of CK2 and/or ATPγS (Fig. 2D, lanes 3 -5). However, in the presence 170 of both ATPγS and CK2, GST-IncV167-363 was phosphorylated (Fig. 2D, lane 6). Altogether, these 171 results demonstrate that CK2 directly phosphorylates IncV in vitro.

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Phosphorylation of IncV is necessary and sufficient to promote the IncV-VAP interaction in 174 vitro 175 We have previously reported an IncV-VAP interaction in vitro upon incubation of IncV167-363 with 176 the cytosolic MSP domain of VAP (GST-VAPMSP) purified from E. coli (Stanhope et al., 2017).

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However, this interaction was only detected when IncV167-363 was produced in eukaryotic cells, 178 which, based on the above results, led us to hypothesize that IncV phosphorylation is required for 179 the IncV-VAP interaction. We assessed the role of phosphorylation in the IncV-VAP interaction 180 by performing lambda (λ) phosphatase dephosphorylation of IncV coupled with a GST-VAPMSP 181 pull-down assay (Fig. 3A). IncV-3xFLAG was immunoprecipitated from lysates of HEK293 cells 182 using anti-FLAG-conjugated Sepharose beads, released from the beads using FLAG peptide 183 competition, and treated with λ phosphatase or buffer alone. Treated and untreated IncV-3xFLAG 184 samples were then incubated with GST-VAPMSP or GST alone bound to glutathione Sepharose 185 beads. The protein-bound beads were subjected to western blot analysis using an anti-FLAG 186 antibody (Fig. 3B). Untreated IncV-3xFLAG was pulled down by GST-VAPMSP but not by GST 187 alone, demonstrating a specific interaction between IncV and VAP (Fig. 3B, lanes 1 -3). However, 188 when the eluate containing IncV-3xFLAG was treated with λ phosphatase prior to incubation with 189 GST-VAPMSP, the two proteins failed to interact (Fig. 3B, lane 4), indicating that phosphorylation 190 of IncV is necessary for the IncV-VAP interaction in vitro.
We next determined if IncV phosphorylation by CK2 was sufficient to promote the  interaction in an in vitro binding assay (Fig. 3C). MBP-tagged VAPMSP (MBP-VAPMSP) and  IncV167-363 were expressed separately in E. coli and purified using amylose resin and glutathione 194 Sepharose beads, respectively. GST-IncV167-363 was left attached to glutathione Sepharose beads 195 and was phosphorylated by incubation with recombinant CK2 and ATP before being combined 196 with purified MBP-VAPMSP. GST-IncV167-363 was pulled down and the samples were subjected to 197 western blot using anti-MBP antibodies (Fig. 3D). Neither the beads alone, nor GST alone pulled 198 down MBP-VAPMSP, regardless of whether CK2 and ATP were present or not (Fig. 3D, lanes 1 -199 4). In the absence of CK2 and ATP, we observed minimal binding of MBP-VAPMSP to GST-   207 We next determined the contribution of CK2 to IncV phosphorylation and the subsequent assembly 208 of the IncV-VAP tether during Chlamydia infection. We first used a genetic approach to deplete

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To complement the genetic approach described above, we conducted a pharmacological approach to unphosphorylated IncV at the 10 μM concentration. These results demonstrate that CK2 activity 238 is essential for IncV phosphorylation during infection. 239 We next determined whether inhibition of CK2 affected the IncV-dependent VAP recruitment to 240 the inclusion and, therefore, the assembly of the IncV-VAP tether. We used the same experimental    these results indicate that serine residues S345, S346, and S350 located in a C-terminal motif of IncV, 283 are critical for CK2 recruitment to the inclusion and the CK2-dependent assembly of the IncV-284 VAP tether.

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To determine if IncVS3A failed to interact with VAP because of a lack of IncV phosphorylation, 286 we assessed IncVS3A apparent molecular weight by western blot analysis of lysates from HeLa 287 cells infected with a C. trachomatis incV mutant expressing IncVWT-or IncVS3A-3xFLAG.

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Compared to IncVWT-3xFLAG, which as previously observed ran as a doublet corresponding to IncVWT-3xFLAG upon treatment with the CK2 inhibitor CX-4945 (Fig. 5F, lane 3). These results 292 indicated that IncVS3A is unphosphorylated and suggested that phosphorylation of S345, S346, and 293 S350 may be sufficient to mediate the in vitro IncV-VAP interaction observed upon CK2 294 phosphorylation of IncV (Fig. 3D). To test this, S345, S346, and S350 were substituted to for 295 phosphomimetic aspartic acid residues. The corresponding IncV construct, referred to as IncVS3D, 296 was purified from E. coli and tested for VAP binding in vitro. IncVS3D did not result in a significant 297 increase in VAP binding compared to IncVWT (Fig. S4). Altogether, these results indicate that, 298 although critical for CK2 recruitment, assembly of the IncV-VAP tether at the inclusion, and IncV 299 phosphorylation status, phosphorylation of S345, S346, and S350 alone is not sufficient to promote 300 VAP binding in vitro, suggesting that additional IncV phosphorylation sites are required to 301 promote optimal interaction between IncV and VAP. In addition to the seven amino acid core of the FFAT motif, VAP-FFAT mediated interactions 306 also rely on the presence of acidic residues upstream of the core sequence, referred to as the acidic 307 tract. It allows for the initial electrostatic interaction with VAP by interacting with the 308 electropositive charge of the MSP domain before the FFAT core motif locks into its dedicated 309 groove (Furuita et al., 2010). We noted that, instead of typical acidic residues, the primary amino 310 acid structures upstream of the IncV FFAT motifs are highly enriched in phosphorylatable serine 311 residues (Fig. 6A). We hypothesized that, if phosphorylated, these serine residues could serve as  We next determined if phosphomimetic mutation of the serine-rich tracts of IncV to aspartic acid 328 residues (referred to as IncVS/D) was sufficient to rescue the ability of the cytosolic domain of IncV 329 expressed in E. coli to interact with the MSP domain of VAP in our VAP binding in vitro assay.

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As observed before, there was minimal binding of VAPMSP to IncVWT (Fig. 6D, lane 2). However, 331 we observed a 20-fold increase in VAPMSP binding to IncVS/D compared to IncVWT (Fig. 6D, lane   332 3), indicating that phosphomimetic mutation of the serine-rich tracts is sufficient to promote the  residues that are part of CK2 recognition sites (Fig. 7, Step 1). As a consequence, IncV becomes 352 hyper-phosphorylated, including phosphorylation of T265 of the phospho-FFAT and serine tracts 353 directly upstream of the FFAT motifs (Fig. 7, Step 2). We note that kinases other than CK2 must 354 be involved in this second step, since the phospho-FFAT is not a CK2 target. Phosphorylation of 355 the serine tract and of the phospho-FFAT result in full mimicry of eukaryotic FFAT motifs, leading 356 to IncV interaction with VAP and tether assembly (Fig. 7, Step3). Importantly, the post-    residues seems to be brought to the extreme, since the eight to ten amino acid stretch directly 416 preceding each FFAT motif include 80 to 87% of serine residues, the remaining residues being 417 acidic. Except for OSBP2/ORP4, which contains 6 acidic residues (including a phosphorylatable   Plasmid construction. 455 Plasmids were constructed using the primers (IDT) and templates listed in Table S1, Herculase 456 DNA polymerase (Stratagene), restriction enzymes (NEB), and T4 DNA ligase (NEB).

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Prior to blocking, membranes were stained with Ponceau S in 5% acetic acid and washed in dH2O.

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Membranes were incubated for 1 hour with shaking at room temperature in blocking buffer (5%   EDTA-free (Roche)) was added per well. Cells were lysed for 20 minutes at 4˚C with rotation.

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Lysates were centrifuged at 16,000xg for 10 minutes at 4˚C to pellet nuclei and unlysed cells.

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Quantification of YFP-CK2β, CFP-VAP, or YFP-VAP association with IncV-3xFLAG on the 553 inclusion membrane was performed using the Imaris imaging software. First, three-dimensional 554 (3D) objects were generated from the raw signal of IncV-3xFLAG on the inclusion membrane.

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Objects were edited such that IncV-3xFLAG colocalizing with the mCherry bacteria was removed.

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Within the resulting IncV object, the mean intensity of YFP-CK2β, CFP-VAP, or YFP-VAP was calculated by the Imaris software and normalized to the mean intensity of YFP-CK2β, CFP-VAP, 558 or YFP-VAP within the cytosol surrounding the inclusion.

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Quantification of the IncV-3xFLAG volume was performed to ensure there was no defect in 560 inclusion localization. Using the Imaris imaging software, the sum of the voxels corresponding to 561 the IncV-3xFLAG signal above the threshold set by the signal within the cytosol was calculated 562 for IncV-3xFLAG and mCherry. The IncV-3xFLAG volume was normalized to its corresponding 563 inclusion volume.  In vitro binding assay with IncV dephosphorylation. 601 First, the phosphatase assay was performed with the following changes: 1,000,000 HEK293 cells 602 stably transfected with pCMV-IE-N2-IncV-3xFLAG were seeded per 6 well. 6 wells were lysed in 500μL lysis buffer each and lysates from two wells were combined. 10μL of anti-FLAG beads 604 were added per 1000μL cleared lysate for 2 hours at 4˚C with rotation. All beads were combined 605 after the first wash, and proteins were eluted in 150μL elution buffer (130μL eluate collected). 606 Next, GST and GST-VAPMSP were purified as described in protein purification. Per phosphatase 607 assay tube: 1.5μg of GST or GST-VAPMSP attached to beads (determined empirically by 608 comparison of Coomassie stained gel to BSA standard curve) were suspended in 500μL lysis 609 buffer then added to tubes containing the IncV-3xFLAG-containing eluate (+/-phosphatase 610 treatment). Binding was allowed to occur overnight at 4˚C with rotation.

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To confirm that IncV dephosphorylation was successful, a set of control tubes were incubated with 612 beads alone (no GST construct) in lysis buffer to mimic experimental conditions.
Step 3: IncV phosphorylation leads to full mimicry of FFAT motifs and binding to 833 VAP (yellow). The dark and light grey bars represent the transmembrane and cytosolic domain of 834 IncV, respectively. P represent the phosphorylation of specific residues.    Figure S4 -source data 1: Quantification of blot densities for Figure S4.
885 Figure S4 -source data 3: Raw data for MBP blot in Figure S4B.
886 Figure S4 -source data 4: Raw data for Ponceau S blot in Figure S4B.