The novel bacterial effector protein Cb EPF1 mediates ER-LD membrane contacts to regulate host lipid droplet metabolism

  1. Rajendra Kumar Angara
  2. Arif Sadi
  3. Stacey D. Gilk

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Abstract

Effective intracellular communication between cellular organelles is pivotal for maintaining cellular homeostasis. Tether proteins, which are responsible for establishing membrane contact sites between cell organelles, enable direct communication between organelles and ultimately influence organelle function and host cell homeostasis. While recent research has identified tether proteins in several bacterial pathogens, their functions have predominantly been associated with mediating inter-organelle communication specifically between the bacteria containing vacuole (BCV) and the host endoplasmic reticulum (ER). However, this study reveals a novel bacterial effector protein, Cb EPF1, which acts as a molecular tether beyond the confines of the BCV and facilitates interactions between host cell organelles. Coxiella burnetii , an obligate intracellular bacterial pathogen, encodes the FFAT motif-containing protein Cb EPF1 which localizes to host lipid droplets (LDs). Cb EPF1 establishes inter-organelle contact sites between host LDs and the ER through its interactions with VAP family proteins. Intriguingly, Cb EPF1 modulates growth of host LDs in a FFAT motif-dependent manner. These findings highlight the potential for bacterial effector proteins to impact host cellular homeostasis by manipulating inter-organelle communication beyond conventional BCVs.

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    Referee #3

    Evidence, reproducibility and clarity

    Angara et al describe a secreted Coxiella burnetii effector with an FFAT motif, which they name CbEPF1, that localizes to host cell LDs and is able to interact with VAP domain containing host proteins. Furthermore, this interaction is able to impact LD size. The study has several interesting findings that suggest that this protein can impact ER structuring around lipid droplets. The use of bacterial 2-hybrid approach to demonstrate binding between CbEPF1 and VAP domain containing proteins is convincing, further supported by co-ip experiments that go on to establish specificity of each of the FFAT motifs for distinct VAP domain containing proteins. However, the relationship between LD localization of the protein and ER-restructuring to the outcome of infection is not clear from the data presented here. In addition, there are many analyses that need to be performed for the data to convincingly support the claims made in the manuscript.

    My major concerns are:

    1. The authors claim that CbEPF1 localizes to lipid droplets in Coxiella burnetii infected epithelial cells. Towards this, the authors express CbEPF1-GFP in C. burnetii infected cells expressing BFP-KDEL. The data in Figure 1B and 1C indicate that the protein localizes to lipid droplets in oleic acid treated cells. It is interesting to note that without addition of oleic acid, the protein localizes to the ER. What is surprising is that the BFP-KDEL signal is also localizing to the LD surface (Figure 1B and 2A). While in this section it seems that the protein migrates from ER to LDs, in the later sections, similar data is used to make the claim that the protein induces ER apposition to the LD and localizes to regions of LD-ER contact. Therefore, it raises several questions about the localization of the protein: (i) is it an ER-localized protein that migrates to LDs. In that case, what features of the protein enable its LD binding? In addition, the authors must perform LD isolation to validate that the protein indeed localizes to lipid droplets. (ii) Is it an ER-localized protein, that like BFP-KDEL has the ability to localize to ER-LD contact sites, but remains on the ER membrane? Again, biochemical evidence supporting membrane specification is important to understand the localization of the protein. The authors have focussed only on the FFAT motifs of CbEPF1 and not described the overall domain analysis of the protein. It is therefore difficult to understand at this point how the protein localizes to the ER and the ER-LD junction or LD.
    2. Quantitative image analysis:

    (i) Authors must perform colocalization analyses to substantiate the claims for ER/LD localization. The authors refer to "extended ER-LD contacts" in figure 2B and the text. These data need to be supported with colocalization analysis between BFP-KDEL and the GFP channel.

    (ii) Related to Figure 4A: Mander's Colocalization analyses with Costes correction are required to convincingly demonstrate that the dual FFAT motif is required for ER-LD contacts.

    (iii) Related to Figure 4B: Please show the LD phenotype of untransfected, and CbEPF1-GFP transfected cells also. Can the authors provide a means to quantify the clustering of LDs.

    (iv) Figure 5A and B. It is not clear from the figure legend whether the data are pooled from multiple experiments or a single experiment. Experimental replicates must be incorporated in the final analysis.

    1. The data presented in this manuscript depends largely on overexpression of the protein in uninfected cells. Given that C. burnetii induces LD formation, there are three main areas that need clarity:

    (i) What is the localization of the protein in infected cells without the addition of oleic acid under conditions where infection itself induces LD biogenesis.

    (ii) What happens to ER-LD contacts upon infection with C. burnetii?

    (iii) Does the presence of CbEPF1 play any role in infection induced LD biogenesis? This may be an optional experiment to undertake at this point as this would involve significant amount of time investment in generating bacterial strains.

    Minor comments:

    1. Line 36: "maintain" instead of "maintains"
    2. The introduction cites mainly reviews with overlapping concepts but does not cite primary literature in the area of organelle-organelles contacts and inter-organelle communication (lines 39-40 and line 49). It would be good to cite key primary research articles in these areas.
    3. There are some crucial references related to bacterial secreted effectors that target host lipid droplets, that are missing from the introduction. For example, Chlamydia trachomatis is known to secrete effectors that localize to host lipid droplets (PMID: 18591669). Legionella pneumophila secretes a small GTPase LegA15 to lipid droplets impacting vesicle secretion of the host cell (PMID:36525490).
    4. For all figures, please show all individual channels in monochrome and a merge of BFP-KDEL+LD marker and merge of CbEPF1-GFP+LD marker and/or BFP-KDEL+CbEPF1 (wherever appropriate).

    Significance

    The study by Angara et al reports a dual FFAT motif containing protein of Coxiella burnetii that impacts ER-LD association. The strengths of the study lie in characterization of the FFAT motifs in VAP domain binding, and the role of these FFAT motifs in mediating ER-LD contacts. However, the claims made towards LD localization versus localization to ER-LD contact sites by this protein are not well supported by the data. In addition, the relevance of these findings in infected cells are not addressed in this study as the data presented here pertain to an over-expression system.

    Bacterial proteins that exit the bacterial containing vacuole and impact organelles outside the endocytic compartments are fascinating as they have the potential to impact global processes such as transcription, metabolism, and protein secretion. Lipid droplet (LD) homeostasis is dysregulated in many bacterial infections and also plays a crucial role in host defense against infection. Therefore, the knowledge of bacterial effectors in this dysregulation will also potentially provide means of countering bacterial strategies to affect host LD homeostasis. Therefore, the findings presented by Angara et al will provide conceptual and mechanistic advances to the specialized audience in infection and immunity. However, I can foresee that the findings have the potential for interesting tools to be developed for organelle-organelle contacts to be studied, provided there is more clarity on how the protein localizes to the ER/ER-LD contacts/LDs.

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    Referee #2

    Evidence, reproducibility and clarity

    This is a strong manuscript about the existence of proteins coded by intracellular parasites (here Coxiella) that have evolved to parasitise the lipid transport machinery of their hosts. This is a first in that the parasite protein acts at a distance from the parasite itself, manipulating two of the host organelles - and not acting at their site of contact with PVs. There is considerable research into one protein and its effect when expressed by itself.

    Despite all the advances there are a couple of areas where the manuscript can be improved, and a few extra fairly straightforward experiments added about the amphipathic helix. Even though these are unlikely change the overall message, they would make the story more complete.

    Major points

    More details are required about the amphipathic helix. Check that the AH does target LDs by expression of the AH alone in a GFP chimera +/- oleate and then mutagenesis. Also show the AH in a helical wheel projection (eg by Heliquest) and say if it aligns with similar AHs in homologs (see my point below)

    Fig 1B: In infected cells, do the affected LDs tend to be close to the PVs?

    Also in Fig 1B: highlight small KDEL+ve ER rings around LDs here. Study whether LDs have these in infected cells without the confounder (?artefact) of EPF1 over-expression

    Fig 2A the ER looks quite different here from Fig 1B, even at t0. Grossly the strands are spaced wider apart. In detail there are no rings around LDs. Can the authors explain this? Which morphology is common, especially in cells early in infection without co-expressed protein?

    Fig 6 & Line 237: "As the N-terminal region of CbEPF1 is undefined": I suggest that the authors could do more here. At minimum change model to highlight the strong probability that the N term is a globular domain that functions at the LD ER interface. (What are three other unidentified LD proteins? I suggest omitting them).

    Although the Alphafold prediction for EPF1 is low confidence only, in a few minutes of BLAST searching I found the homolog A0A1J8NR10_9COXI (also FFAT+ve) which has a moderately confident structural prediction for its N terminus. This model has a quite large internal hydrophobic cavity, indicating lipid transfer capability and function similar to known LTPs. This means that action as a "tether" possibly results from experiments with viral promoters (see minor point on terminology).

    Minor:

    Fig 2B: add more arrowheads/arrows to fit legend (says they are both multiple)

    FFAT selectivity for MOSPD2: say if this fits the di Mattia or (as appears likely) it extends the known differences between VAPA/B and MOSPD2. Also say if VAPA is expected to behave as VAPB

    Explain how "Mutations in the CbEPF1 FFAT motif(s) did not influence CbEPF1-GFP localization to either the host ER (Supplementary Fig. 1)". In F3mt this shows that EPF1 has a way to target ER other than FFAT/VAP. Discuss if that is via AH insertion in ER.

    Also, the (admittedly low) level of ER targeting is possibly slightly reduced by F3mt, as shown by greater GFP in the nucleus in the single cells shown. If this is a feature of the whole field of cells, it implies that the FFATs normally work with the AHs to target EPF1 to the ER.

    "clustering" LDs w F3mt: could this indicate dimer formation by CbEPF1? Note: to me it appears wrong to describe fig 4A as showing ER exclusion. LD proximities to each other dominate. It's not 100% clear that LDs cluster as their proximities are not universal: "LD-LD interactions" may be (very) weak.

    Fig 5: can levels of EPF1 here be compared to those in cells undergoing natural infection(approximate comparison by qPCR better than nothing if no antibodies are available)? Fig 5a: would it be possible to increase the number of cells counted to attempt to make the reduced number of LD in F3mt significant?

    Minor

    Line 226: no sequence homology: misses the point- there is the common feature of an AH

    Issue to be discussed, as probably too difficult to experiment on: when EPF1 is on the ER does it engage vap only weakly (implying a means to mask its motifs), since if the interaction is strong vap is then unable to bind other partners?

    Line 245: "MOSPD2, a sole VAP that is known to localize on LD surfaces" (worth citing Zouiouich again here). Do the cells/tissues infected by Coxiella express MOSPD2?

    Line 259/260: this "suggestion" about cholesterol should be toned down. It is a speculation that could be tested in future, but the data here do not suggest it.

    'Tether' this word implies more than just bridging but also a role in the physical formation of the contact. Since EPF1 most likely has an LTP domain, it seems linguistically confusing to refer to it as a tether, especially since the experiments that physically later LD-ER contact involve probable over-expression.

    Discuss whether it is 100% certain that EPF1 is in the host cytosol or whether some experiment(s) at a future date (proteomic/western blotting) will be needed to make that conclusion 100% secure.

    Referees cross-commenting

    COMMENT 1

    I realise that both reviewer 1 and reviewer 3 have considered this MS carefully, but I think that their reviews could be improved in some respects. I will add two comments, one for each of the other reviews.

    Reviewer 1. The review poses multiple questions to the authors suggesting that answering these questions experimentally would strengthen the paper. Some of the points seem to misunderstand what is the accepted standard for membrane cell biological research into membrane contact sites. While it might be that the authors can rebut these points, I think it is preferable to use Cross Commenting as an opportunity to address these issues beforehand.

    Major Comment 1: CbEPF1 and ER-LD contact

    Looking at endogenous proteins: I wondered about the same point, but I concluded that this is not likely to be possible in the scope of this submission. If it were possible then I guess the authors would have attempted it. Looking on Google Scholar I could find no example of an endogenous Coxiella proteins being tagged in the bacterial genome. So the only way to find the portion is via an antibody. Assuming the authors do not have one, I do not think we should ask for one at this stage in the publication process.

    Electron microscopy: the reviewer is incorrect to say that this is necessary. It may be the gold standard, but it is a huge amount of extra work. Furthermore it is not at all necessary when the protein in question localises clearly to the interface between organelles identified by confocal microscopy.

    Can a specific CbEPF1 domain be identified? Here a Amphipathic Helix has been identified, but the lack of dissection of that region by the authors explains this question by the reviewer, which is also shared by Reviewer 3. I agree with the implication that more should be done to dissect that.

    Major Comment 2: CbEPF1 FFAT motifs and VAP binding

    Are the two FFAT motifs redundant or synergistic? I would say that the authors have addressed that to a reasonable extent

    CbEPF1 binding specificity towards a VAP/MOSPD2 Ditto

    Major Comment 3: LD clustering

    Since this is an effect of mutated protein only, I think that the 3 questions posed at the end here need only be addressed in Discussion.

    Major Comment 4: CbEPF1-mediated increase in LD number and size

    less LD upon expression of F1mt or F2mt, compared to WT: this seems wrong. The numbers are the same. The comment about IF images are unjustified as they have been quantified and do show a difference. I agree that the biological relevance is unclear, and that this might be addressed. That would require making a mutant Coxiella strain. While that would make a big different to this work, my feeling is that this is well over a year's work.I would be guided by the authors on that and I would not suggest it as required for this MS.

    De novo LD production at the ER is unlikely: This statement is ill-considered as the FFAT motifs ARE required (Fig 5). Furthermore, in all systems ever reported de novo LD production takes place at the ER, so any alternative would be quite extraordinary.

    Altogether, strengthening this aspect of the study: In my view, this area does not need more work and it would not be constructive to ask for more.

    Major Comment 5: Functional relevance

    assessing the phenotype of a Coxiella CbEPF1 mutant I agree that this would be good, but it mightn't be feasible within the confines of this one paper. In the various projects that have made transposon mutants of Coxiella, has a strain been made that affects EPF1? If not, then the authors should state this and discuss it as work for the future. The reviewers cannot expect any experiments!

    Is VAP required for Coxiella intracellular growth/vacuole maturation? On the surface this suggestion seems to offer an experimental route to understanding EPF1. However, VAP binds to >100 cellular proteins, many relating to lipids traffic and a considerable number of these already lcoalised to lipids droplets (ORP2, MIGA2, VPS13A/C). It is therefore unlikely that such an experiment would be interpretable, and I recommend that this request be reconsidered.

    Are LD formation induced upon infection? Are ER-LD contact increased upon infection? These are very reasonable ideas and the results would be interesting additions to this paper.

    COMMENT 2 I have given one set of comments already. Here are my comments for Reviewer 3.

    The review makes a few assumptions that I question. While it might be that the authors can rebut these assumptions, I think it is preferable to use Cross Commenting as an opportunity to address these issues beforehand.

    Major Point 1: What is surprising is that the BFP-KDEL signal is also localizing to the LD surface: "Surprising" is misguided, as it seems to deny the probability that there is a class of proteins that sit at organelle interfaces binding to both partners simultaneously. Maybe the reviewer means "significant" here, in which case I would agree.

    The authors must perform LD isolation the reviewer is incorrect to say that this must be done. It is a huge amount of extra work. Furthermore it is not at all necessary when the protein in question localises clearly to the structures, and its may not even work as the protein may need a reasonably high general concentration to avoid gradual dissociation (wit any re-association) during organelle purification.

    what features of the protein enable its LD binding? Here an Amphipathic Helix has been identified, but the lack of dissection of that region by the authors explains this question by the reviewer, which is also shared by Reviewer 1. I agree with the implication that more should be done to dissect that.

    Major Point 2: Quantitative image analysis:

    Mander's Colocalization analyses with Costes correction are required No. The images in Figure 4 speak for themselves.

    Please show the LD phenotype of untransfected, and CbEPF1-GFP transfected cells also This s a good idea.

    provide a means to quantify the clustering of LDs Unnecessary. Not all findings need to be quantified.

    Major Point 3:

    Data depends largely on overexpression of the protein in uninfected cells. I agree

    What is the localization of the protein in infected cells? I wondered about the same point, but I concluded that this is not likely to be possible in the scope of this submission. If it were possible then I guess the authors would have attempted it. Looking on Google Scholar I could find no example of an endogenous Coxiella proteins being tagged in the bacterial genome. So the only way to find the portion is via an antibody. Assuming the authors do not have one, I do not think we should ask for one at this stage in the publication process.

    What happens to ER-LD contacts upon infection with C. burnetii? This is a very valid question, and answering it would not only strengthen the manuscript but should be achievable in 1-3 months.

    Significance

    This work takes a reasonably big step towards uncovering how parasites have mimicked the molecular machinery of contact sites, here in the context of ER-LD interactions and tantalizingly suggestive of lipid transfer at that contact site (although hard to get strong evidence for that at this stage). This provides yet more evidence for the conservation and overall importance to cells of lipid transfer at contact sites, as well as reminding us of the ability of parasites to attack every aspect of cell function.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The authors report a Coxiella burnetii effector protein, CbEPF1, which associate with lipid droplets (LD) at points of contact with the endoplasmic reticulum (ER). The presence of two FFAT motifs in CbEPF1 mediates CbEPF1 interaction with the ER protein VAP, LD-ER interaction, and an increase in LD size. Based on these results the authors put forward a model by which translocation of CbEPF1 into the host cell cytosol results in regulation of host cell lipid metabolism via the formation of ER-LD contact. The study relies heavily on fluorescence microscopy of ectopic (over)expression of CbEPF1 in eukaryotic cells.

    Major comments:

    CbEPF1 and ER-LD contact:

    The immunofluorescence analysis of cell ectopically expressing CbEPF1-GFP provides convincing evidence that CbEPF1-GFP associates with LD and that CbEPF1-positive LD are positive for ER markers such as BFP-KDEL and VAPB (Fig 1&2). However, the study would benefit from looking at endogenous proteins to rule out any potential overexpression artefacts. Does endogenous VAP localize to CbEPF1-positive LD? Does CbEPF1 expressed from Coxiella (endogenous protein, or at least a tagged protein expressed from the bacteria) localize to LD? While the immunofluorescence images are of very high quality and convincing, electron microscopy is necessary to ascertain that membrane contacts between LD and the ER are induced upon CbEPF1 overexpression. CbEPF1 interaction with the ER is linked to the FFAT motifs (see below); what is known about CbEPF1 association with LD? Can a specific CbEPF1 domain be identified? In other words, does CbEPF1 contains 2 distinct membrane targeting domains that confer specificity to each of the contacting organelle (ER and LD in the present case) and thereby resemble other contact site localizing proteins.

    CbEPF1 FFAT motifs and VAP binding:

    The similarity of the sequence of the CbEPF1 FFAT motifs to the canonical sequence (Fig 1A), combined with data with alanine substitution mutation of the essential residue in position 2 of the FFAT motifs (Fig 3, 4), strongly support that CbEPF1 contains 2 functional FFAT motifs that confer VAP binding. FFAT motifs can mediate binding to VAPA, VAPB, and/or MOSPD2. Moreover, in addition to forming homodimer, VAPA, VAPB, and MOSPD2 can form heterodimers. What is the rationale for using VAPB (Fig 3CDE)? Do VAPA and/or MOSPD2 also yield positive results using the assays performed in Fig 3CDE, or is the CbEPF1-VAP interaction specific to VAPB? In the same line, in Fig 3E, is it possible that the CbEPF1-MOSPD2 is indirect and due to VAP- MOSPD2 interaction? Regarding the two FFAT motifs: are the two FFAT motifs redundant or synergistic? Although the data is not quantified, Figure 3E suggest a synergistic effect for VAP binding, however most IF data suggest redundancy. On the other end, the second FFAT motif seems necessary for MOSPD2 biding. Overall, clarifying CbEPF1 binding specificity towards a VAP/MOSPD2 and the role of each FFAT motif in this process could elevate the study by providing mechanistic insights into the hijacking of ER-LD contact sites by Coxiella and comparing and contrasting with the formation of ER-LD in naïve cells and/or the hijacking of VAP-dependent contact by other microbial pathogens.

    CbEPF1-mediated LD clustering in the absence of VAP binding

    A CbEPF1 FFAT motif mutant (F3mt) associates with LD that do not associate with the ER marker KDEL, and causes LD clustering (Fig 4). The authors speculate that the lack of LD-ER interaction results in LD-LD interaction, potentially via interaction of unidentified protein(s) on the LD surface. What is the biological relevance of the LD clustering phenotype? What is known about the role of LD clustering vs ER-LD contact, in the context of lipid metabolism? Could mechanistic characterization of this phenomenon provide insights in LD biology and/or the role of CbEPF1/ER-LD contacts in the context of Coxiella infection?

    CbEPF1-mediated increase in LD number and size

    CbEPF1-GFP overexpression result in an increase in the number of LD per cell independently of the FFAT motifs (Fig 5A). CbEPF1-GFP overexpression also result in an increase in LD diameter; however, this phenotype is dependent on wild-type FFAT motifs (Fig 5B). Quantification and corresponding statistical analysis support these conclusions. However, the representative images are not necessary in line with the bar graphs. For example, there appear to be less LD upon expression of F1mt or F2mt, compared to WT. Additionally, the increase in size is moderate and hard to appreciate in the IF images. It is also unclear, if/how an increase in LD number and/or size is biologically relevant in the context of Coxiella infection. Regarding potential mechanism(s), it is also unclear how CbEPF1 is promoting an increase in LD number. De novo LD production at the ER is unlikely given that the FFAT motifs, and therefore ER association, are not required. What would an alternative mechanism be, and can it be experimentally tested? Regarding the increase in LD size, the author suggest that the phenotype could be due to impaired lipid transfer from the ER to LD. This is an interesting model. Do the authors envision that CbEPF1 is a lipid transfer protein and/or act on ER-LD associated lipid transfer? Can either be experimentally tested? Altogether, strengthening this aspect of the study would clarify the proposed model and significantly increase the impact of the study.

    Functional relevance:

    One aspect that is not addressed in the study, is what are the benefit(s), if any, of CbEPF1 translocation into the host cytosol and targeting to ER-LD contact? This could be addressed by assessing the phenotype of a Coxiella CbEPF1 mutant? On the flip side, is VAP required for Coxiella intracellular growth/vacuole maturation? Another avenue would be to investigate if CbEPF1 affects the lipid composition of the CCV. The present study suggest that LD may be important for Coxiella intracellular life cycle. Are LD formation induced upon infection? Are ER-LD contact increased upon infection? One may also expect that inhibition of LD formation would affect bacterial replication and that stimulation may promote growth/vacuole maturation? Any experiments addressing the biological relevance of the present findings in the context of Coxiella infection would tremendously increase the impact of the study.

    Minor comments:

    None. The manuscript is well written and easy to follow.

    Significance

    Inter-organelle communication through the formation of membrane contact between two apposing organelles is critical to maintain host cell homeostasis via the transfer of small molecules such as lipid and ions. Bacterial and viral pathogens have been shown to manipulate proteins that localize to cellular membrane contact and to promote membrane contact between their intracellular vacuole and the ER. Both viral and bacterial proteins that contains FFAT motifs and interact with VAP have been described in the context of tethering of the ER to the membrane compartment in which the pathogen replicate. The present study stands out by the characterization of a FFAT motifs-containing bacterial effector protein that targets cellular contact, ER-LD more specifically. The significance is 2-fold. It expands the list of FFAT-motif containing proteins in pathogens, reinforcing the idea that VAP/membrane contact may be a universal mechanism of host-pathogen interaction, which will be of interest to those studying host-microbe interaction in basic or translational research settings. The study also has the potential to move forward research on LD biology and LD-ER contact, which will be of interest to cell biologists in general, and to the evergrowing membrane contact community, specifically.

  5. Version published to 10.1101/2023.12.11.571031v1 on bioRxiv