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

  1. Rachel J Ende
  2. Rebecca L Murray
  3. Samantha K D'Spain
  4. Isabelle Coppens
  5. Isabelle Derré

  • Curated by eLife

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    Evaluation Summary:

    This manuscript will be of interest to readers in the field of infection and cell biology. The authors follow up their previous study to further deepen our understanding of host-pathogen interactions that contribute to transkingdom contact sites. The authors show that the Chlamydia effector protein IncV tethers ER to the bacterial vacuole in a manner dependent on the phosphorylation of its C-terminus by the host kinase CK2. In addition, the authors show that IncV recruits the host kinase CK2 to the bacterial vacuole in manner required for its phosphorylation and ER tethering. Overall, the data justify most of the key claims put forward by the authors. Nonetheless, clarification is required concerning the chain of events and the phosphorylation events required for VAP binding.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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Abstract

Membrane contact sites (MCS) are crucial for nonvesicular trafficking-based interorganelle communication. Endoplasmic reticulum (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 and the formation of ER-Inclusion MCS. Via a C-terminal region containing three CK2 phosphorylation motifs, IncV recruits CK2 to the inclusion leading to IncV hyperphosphorylation of the noncanonical 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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  1. Version published to 10.7554/elife.74535 on eLife
  2. eLife

    Evaluation Summary:

    This manuscript will be of interest to readers in the field of infection and cell biology. The authors follow up their previous study to further deepen our understanding of host-pathogen interactions that contribute to transkingdom contact sites. The authors show that the Chlamydia effector protein IncV tethers ER to the bacterial vacuole in a manner dependent on the phosphorylation of its C-terminus by the host kinase CK2. In addition, the authors show that IncV recruits the host kinase CK2 to the bacterial vacuole in manner required for its phosphorylation and ER tethering. Overall, the data justify most of the key claims put forward by the authors. Nonetheless, clarification is required concerning the chain of events and the phosphorylation events required for VAP binding.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    This interesting manuscript provides in vitro and in vivo evidence that a bacterial factor (IncV) requires phosphorylation by host kinase(s) to interact with VAP and establish a contact site between the ER and the bacterial inclusion membrane.

    Three different phosphorylation events are required. First CK2 is recruited to and phoshphorylates the c-terminus of IncV. Second, this leads to further phosphorylation of residues surrounding two FFAT motifs, strengthening the interaction with VAP. Third, one of the FFAT motifs is a phospho-FFAT and requires phosphorylation of a specific threonine by an unknown kinase.

    The story is interesting and the experiments carefully executed.

    The phosphorylation of IncV by CK2 and its effect on VAP binding are clearly demonstrated using phosphonull and phosphomimic mutants.

    Clarification is nonetheless required concerning the chain of phosphorylation events required for VAP binding, and whether they are successive or unrelated but converging on changing affinity for VAP.

  4. eLife

    Reviewer #2 (Public Review):

    This study investigates the membrane contact sites (MSCs) between the C. trachomatis inclusion and the host endoplasmic reticulum (ER). The MCS is mediated by the interaction between the pathogen factor IncV (localising to the C. trachomatis inclusion) and the ER host factor VAP. The manuscript offers new insights into the biology of IncV and VAP interaction and, in addition, identifies a novel key factor critical for host ER association with the C. trachomatis inclusion.

    The manuscript presents its scientific findings in three major parts.

    First, the authors provide in vitro data indicating that the C. trachomatis factor IncV is phosphorylated, most likely by an unknown host factor. Based on data available in the literature, the authors hypothesised that the host kinase CK2 phosphorylates IncV.

    Second, the study proceeds to investigate the function of the host kinase CK2 in IncV phosphorylation in vitro and during C. trachomatis infection. In vitro binding assays show that phosphorylation of IncV is necessary and sufficient to promote the interaction between IncV and VAP. In addition, in vitro binding assays also suggest that CK2-mediated IncV phosphorylation is necessary and sufficient to promote interaction between IncV and VAP. Subsequently, the study presents experiments during C. trachomatis infection to illustrate the function of CK2 in mediating the interaction between IncV and VAP. Partial siRNA knock-down of host CK2 and inhibition of CK2 with CX4945 suggest IncV phosphorylation via CK2 in the host cell. When CK2 inhibitor CX4945 is applied at different concentrations, a dose-dependent reduction of VAP association to the C. trachomatis inclusion can be observed via IFA.

    Lastly, domains and residues of IncV that are phosphorylated and serve as binding domains for the CK2 kinase or VAP are identified. Experiments using IncV mutants identify three serine residues at the C-terminus of IncV to be critical for CK2 binding and IncV phosphorylation. However, in vitro data suggest that phosphorylation of the three serine residues alone is not sufficient to mediate IncV-VAP interaction. Indeed, the phosphorylation of serine tracts upstream of the IncV FFAT motifs appear to mediate the IncV-VAP interaction. Preventing phosphorylation of these serine tracts by substituting the endogenous serine residues with alanine abolished VAP recruitment to the C. trachomatis inclusion in the host cell, but not recruitment of CK2.

    Overall, I find the conclusions of this manuscript are mostly well supported by the presented data. Some aspects of the paper, however, would benefit from further experiments and clarification.

    Strengths of the manuscript:

    The manuscript is well written and the authors present their scientific reasoning in a clear manner. The experimental data presented here are put into the context of previous findings regarding IncV (Stanhope et al., 2017), making it easy for the reader to follow the path of scientific discovery.

    The in vitro binding and phosphorylation assays clearly define the requirements for the interaction between IncV and VAP. The need for CK2-dependent phosphorylation of IncV for VAP recruitment to the inclusion is convincingly demonstrated. Overall, the in vitro experiments are thoroughly executed and support the author's conclusions.
    It remains a constant question in the field of trans-kingdom MCS how proteins from the pathogen are able to interact with host factors. Murray and colleagues put forward a very relevant finding by providing data that would indicate pathogen mimicry of host protein features to enable host organelle recruitment. All in all, this paper significantly contributes to our understanding of trans-kingdom MCS between the pathogen and the host.

    Weaknesses of the manuscript:

    Although immunofluorescence combined with confocal microscopy represents one of major experimental tools to analyse the association of IncV, CK2 and VAP to the C. trachomatis inclusion, the methodology is not depicted in a comprehensible manner. The procedure itself is described in the Material and Methods section of the paper. However, it remains unclear how the authors conducted the analysis of their immunofluorescence data. A representative schematic demonstrating the complexity of the procedure, including the generation of 3D objects and the normalization of fluorescence intensity, would create more clarity about the methodology used in this study.

    The data for CK2 during C. trachomatis infection of a host cell is not entirely convincing. One, the knockdown of CK2 via siRNAs was not complete. Despite rigorous analysis, the conclusions drawn from these data are not very strong. Second, the inhibition of CK2 with CX4945 has an inherent weakness since secondary effects on other host cell processes cannot be excluded. Nonetheless, the entirety of the data provides logical indications for the function of CK2 during C. trachomatis infection, despite the weaknesses of the individual experimental set-ups.

    The manuscript lacks clarity regarding the details of CK2 function and IncV phosphorylation. The provided in vitro data can be interpreted such that CK2 is sufficient to cause IncV phosphorylation and to mediate interaction with VAP. However, in the discussion section, the authors argue that other kinases must be involved in the phosphorylation of the serine tracts upstream of the FFAT domain since phospho-FFAT is not a CK2 target (lines 354-355). However, no reference is provided to support this statement and no further insight into this reasoning is given. This makes it difficult for the reader to understand the final conclusion about CK2 phosphorylation.
    The final model proposes a sequential binding of CK2 and VAP to IncV during C. trachomatis infection. CK2 binds to IncV first to mediate IncV phosphorylation, thus promoting VAP binding. While in vitro data could be interpreted this way, data obtained from host cells infected with C. trachomatis does not strengthen the subsequential nature of the model. It would support the model to show an scenario during the infection with C. trachomatis in which VAP is absent from the C. trachomatis inclusion despite the presence of CK2. If the model stands true, this could be achieved with a CK2 variant unable to phosphorylate IncV.

    Interestingly, the manuscript offers an experimental set up that could show the above scenario. The paper presents an IncV S/A variant in which the serines from the serine-rich tracts upstream of IncV FFAT motifs are substituted with alanine, an amino acid that cannot be phosphorylated. When expressing the IncVS/A variant in C. trachomatis, CK2 still localises to the inclusion while VAP is absent. However, since the authors argue that CK2 cannot phosphorylate the serine-rich tracts upstream of IncV FFAT motifs, this observation is not suitable to support the model that the authors are proposing.

    Overall, the author's conclusions regarding the details of CK2-dependent phosphorylation of CK2 during C. trachomatis infection are difficult to follow in the current manuscript.

  5. Version published to 10.1101/2021.10.19.465067v1 on bioRxiv