Ultrastructural insights into the microsporidian infection apparatus reveal the kinetics and morphological transitions of polar tube and cargo during host cell invasion

  1. Himanshu Sharma
  2. Nathan Jespersen
  3. Kai Ehrenbolger
  4. Lars-Anders Carlson
  5. Jonas Barandun

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Abstract

During host cell invasion, microsporidian spores translocate their entire cytoplasmic content through a thin, hollow superstructure known as the polar tube. To achieve this, the polar tube transitions from a compact spring-like state inside the environmental spore to a long needle-like tube capable of long-range sporoplasm delivery. The unique mechanical properties of the building blocks of the polar tube allow for an explosive transition from compact to extended state and support the rapid cargo translocation process. The molecular and structural factors enabling this ultrafast process and the structural changes during cargo delivery are unknown. Here, we employ light microscopy and in situ cryo-electron tomography to visualize multiple ultrastructural states of the Vairimorpha necatrix polar tube, allowing us to evaluate the kinetics of its germination and characterize the underlying morphological transitions. We describe a cargo-filled state with a unique ordered arrangement of microsporidian ribosomes, which cluster along the thin tube wall, and an empty post-translocation state with a reduced diameter but a thicker wall. Together with a proteomic analysis of endogenously affinity-purified polar tubes, our work provides comprehensive data on the infection apparatus of microsporidia and uncovers new aspects of ribosome regulation and transport.

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  1. Version published to 10.1371/journal.pbio.3002533
  2. Review Commons

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    Reply to the reviewers

    Please see the attached pdf "Response to Reviewer" with all reviewer comments, our responses, and descriptions of the edits in the text supplementary information, and figures.

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    Referee #3

    Evidence, reproducibility and clarity

    This paper by Sharma et al describes ultrastructural changes in the polar tube (PT) of the microsporidian species Varimorpha necatrix upon PT firing. The relationship between cargo transport and the diameter of the PT, as well as the thickness of the PTP coat, are investigated. Moreover, low-resolution sub-tomogram averaging (STA) reveals that ribosome dimers occasionally arrange into spiral-like arrays on the inner surface of the bilayer lining the PTP coat. The data are well presented and, in most parts, appropriately interpreted. I have the following comments that I suggest should be addressed.

    Major:

    1. The authors suggest that the ribosomes in the arrays are dimers. Yet, figure 2c only shows a STA map of a 70S particle. A map of the dimer should be included to support this significant message of the paper.
    2. Do the authors see any 70S particles and if so, how common are they? 3D classification would clarify this.
    3. The authors put a lot of emphasis on the finding of array-like ribosomes within PTs. However, these appear to be present in only a minority of the cases. Moreover, similar ribosome arrays have previously not been seen in other microsporidian PTs. This raises the question of how significant these arrays are. Do they only occur in some microsporidia, or only at certain time points? This should be more clearly discussed, for example in lines 232 - 233. Also, do the authors suggest that this is a specific organisation or just a matter of close packing?
    4. As the arrays are only occasionally observed, the statement that ribosomes are transported through PTs in a spiral-like fashion should also be toned down in the abstract and throughout the manuscript. The fact that the arrays are only seen sometimes, makes the finding even more interesting, as it may infer a dynamic reorganisation process.
    5. The ribosome arrays appear to co-localise with the membrane. If this is the case, does the membrane show up in their STA? If so, it would be essential to show this.
    6. How do the ribosomes in the arrays differ from the free-floating ones? Are the latter not associated with the membrane, while the former are not? Can differences be visualised through 3D classification?
    7. The difference in the PTP coat in empty vs. filled PTs are very interesting. Can the authors clarify how this was measured and mention the number of measurements, mean, and standard deviation in the main text? Line plots would help substantiate the measurements.
    8. Do the authors observe any differences in the regularity of the array? This could be assessed by investigating power spectra of tomograms of STAs.
    9. How do the authors suggest such large changes in thickness come about? Is the PT coat "bunched up", as the PT compresses and stretched out, as the PT extends?
    10. How often are each of the described PT stages seen as a percentage of all data? Are some observed more often than others or is the distribution equal?
    11. Line 125. How do the authors know that they observe nuclei? Can they identify nuclear envelopes? Are nuclear pores evident?
    12. Line 168 - 169: How many measurements were taken from each state? What was the mean and SD for membranes and coats? This will be interesting, especially, as the thickness of the PT coat can vary along the length of one PT.

    Minor:

    1. Line 102 "optimal conditions" sounds obscure, please briefly mention what these are.
    2. Line 119. Are membrane-less PTs ever seen?
    3. Line 156, the word "remodelling" may be too specific, considering that only differences in thickness were measured.
    4. Line157: "Visualising PT sections ...." Sections sounds like physical cryosections were investigated. Perhaps better: "Inspecting tomograms of PT segments in different states..."
    5. Line 161: "subtomogram averaging particles picked on the tube wall from both states" better: "subtomogram averaging of the tube wall from both states"
    6. Line 162: delete "it be"
    7. Line 201: Is organ the right word here?
    8. Fig 2: Increase transparency to reveal the atomic model in C more clearly.
    9. Fig 2b. I suspect the beige ribosomes are ones that do not follow the array? If so, can you please clearly state it? Also, are these dimers too? And can you tell if they are different?
    10. Fig. 3e: The diagonal lines in the schematic infer that the data provide some level of insight into the PT lattice structure. As this is not the case, it would be better to remove these lines.
    11. A flow chart highlighting the sub-tomogram averaging workflows employed should be included.

    Significance

    This paper advances our knowledge of the microsporidian polar tube with regard to its structure, dynamics, and transported content. Ribosome arrays have not been described before in extended PTs, so this is an interesting discovery, which adds to the complexity of ribosome regulation in microsporidia.

    Strengths are the novelty of the findings, in particular the ribosome arrays, PT dynamics, and PT composition.

    As a weakness, I feel that the tomography data could have been analysed in more depth. For example, at least a low-resolution map of the ribosome dimer would be important to show that the ribosomes in the arrays are indeed dimers. In addition, 3D classification would be useful to understand, if all ribosomes occur as dimers or only a fraction.

    The paper is clearly written and well presented and thus suitable for a wider audience, including researchers studying microsporidia, infection biology, host-pathogen interactions, and ribosome biology.

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    Referee #2

    Evidence, reproducibility and clarity

    In this paper Sharma et al. use cryo-electron tomography to study structural properties of the polar tube invasion apparatus from the microsporidian parasite Vairimorpha necatrix. The main conclusions of the paper are related to the unique organization of ribosomes in the polar tube, and the organization of the surface layer of the tube. The cryo-ET data presented in this paper are of high quality, and add new insights into the structure of the polar tube, which have not been reported previously. The authors also purify an endogenous polar tube protein, PTP3, via a native cluster of histidines, and identify co-purifying proteins, which provides new insights into the proteins present in the polar tube that may interact directly or indirectly with PTP3. The endogenous purification was innovative and well carried out, a very nice result.

    Major comments:

    1. Our biggest comment on this manuscript is that we feel the cryo-ET data are often over-interpreted. We would like to request the authors to ensure that their conclusions are justified by the data. We realize that this is a general weakness of cryo-ET at the moment, and that often features that are observed may not be able to be unambiguously defined. The interpretation of the data does need to reflect this. Below are several of the main examples we found, but we urge the authors to keep this in mind as they revise the whole manuscript.
      • a) Assignment of densities: Ribosomes and lipid bilayer are reasonable to assign, because STA in Fig. S3 supports this. Fig. 1 and through the text, eg. lines 120, 126 - proteasomes, PTPs, assigning the outer layer to PTPs, is not justified based on the data. For these, it would be reasonable to speculate in the discussion, it is a reasonable hypothesis, but currently there are no data that directly support this assignment/interpretation. Statements such as in Line 184 "large-scale remodeling of the PTP layer" - are misleading, and do need to be worded with the appropriate level of certainty, currently it is only a hypothesis that this layer is, in fact, composed of PTPs.
      • b) Definition and classification of Cargo: The authors observe polar tubes with different cargos in them. From the cryo-ET data itself, it is not clear what the cargo is. Based on the timescale of the event, it is unlikely that one would catch a substantial number of tubes in the process of transporting cargo. The data are still valuable, but the authors should take care in how the cargo are interpreted, and what the relationship may be to transport of sporoplasm through the tube.
      • c) Time component in interpretation: The authors discuss data as a function of time, for example one section is entitled, "Remodeling of the polar tube protein layer during cargo transport". These data are simply 45 random snapshots of polar tubes, so currently there is no time component in these data. Such a section could be valuable to add to a discussion section, but since it is quite speculative, it would be misleading to a reader for these to be presented as results. Along these lines, Line 118 correlates a "germination phase" with tube thickness. As these experiments have no time component, there is no basis for this correlation. These data can of course be used to generate a hypothesis, which would be appropriate for the discussion section, or clearly indicated that it is speculative (not a direct conclusion from the data presented)
      • d) Line 156-158 - is an overinterpretation of the data, because in our understanding it is currently not known what is in the tubes, and what state they are in. Please re-word.
    2. One of the main conclusions of the paper is the arrangement of ribosomes in the PT. Yet, these are only observed in 5/45 tomograms. What is the authors' interpretation of this observation? Are they just stuck in the tube in some cases?
    3. We request the authors to please provide sufficient information in their methods for reproducibility of their experiments, specifically in these sections:
      • a) In the germination section please provide information on reproducibility of the spore preparation, and information on germination rates. Line 104: "with spores consistently displaying high germination efficiencies" - please clarify what "high" means.
      • b) Light microscopy: please specify rates of incomplete and complete germination, how this was evaluated, how many events were analyzed, and any differences between complete (sporoplasm visible) or incomplete (sporoplasm not visible) germination.
      • c) Line 325: please provide detailed information on CNN-based picking and segmentation, for example, parameters used for optimization
      • d) Line 330: please specify number of tomograms
      • e) Line 331: please specify how manual alignment and particle centering was achieved
      • f) Line 332: please provide information on template-matching options / thresholds used
    4. Supporting Fig 2c: we found it confusing to understand how Pempty is defined, it does not look empty in some cases, and the 3 shown look very different. On what basis is the tube labeled "empty"? The definition provided in line 131 does not seem to match the figures.

    Minor comments:

    Fig. 1: The lipid bi-layer in parts a to e seems different, and we found this confusing. Is the pink label in A not pointing to the correct layer? The corresponding segmentation is also confusing - does the lipid bilayer not go all the way around the tube? This would be important to clarify, since a lipid bilayer is one of the major components of the tube.

    The following publications have shown cryo-ET of the polar tube, and should be referenced appropriately in the introduction, as well as during interpretation: 1) BioRxiv, https://doi.org/10.1101/2023.05.01.538940 Figure 1 and 2) PMID: 31332877. The second is referenced but should be mentioned around line 70 in the introduction

    In the introduction, it would be helpful if the authors mention something about their microsporidia species of interest, and reason for choosing to study this species.

    Fig. S1C - please show individual data points

    Line 100 - the data presented do not show deformation of the cargo, so please reword to reflect the data being discussed

    Line 140: could the authors please outline how they confirmed that the handedness of the reconstructed tomogram is correct?

    Line 250: re-word to ensure that appropriate credit is given to previous work in the field; the large-scale rearrangement of the polar tube has been observed for many decades

    Line 352: out of curiosity, why could resolution not be determined for PTcargo?

    Fig S2a: diagram of the polar tube within the spore shows the polar tube with opposite handedness to what has been previously determined

    Supporting table 1 - is missing frames per movie and which mode data were collected in

    Fig 2b: We did not follow the rationale for the 3 colors of ribosomes

    Sup Fig 3a: please specify in legend and/or workflow software packages used in panel (a)

    Fig 2e: it is unclear whether averages presented are from 1 tomogram, or all tomograms where that pattern is visible - Is the measurement coming from all 5 tomograms?

    Fig 3 c-e: it is unclear how many tomograms were used for these averages. Was 1 STA per tomogram performed, or 1 STA per type of PT?

    Significance

    Overall, this paper provides interesting new insights into knowledge of the microsporidian polar tube. We thank the authors for making this paper available to the community on BioRixiv, and we summarize a few main comments below, which we hope will be helpful in preparing a revised version of the manuscript. There is a substantial advance in applying cryo-ET to studying the polar tube of microsporidian parasites. The audience this will be interesting to are those studying microsporidian parasites.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary: Sharma, et al. report the characterization of the polar tube (PT) from the microsporidian species, Vairimorpha necatrix, using a combination of optical microscopy, cryo-ET, and proteomics. The polar tube is a fascinating invasion apparatus which mediates the translocation of the parasite into the inside of a host cell to initiate infection. Similar to results obtained previously in other species, the authors show that PT firing in Vairimorpha necatrix is extremely fast, occurring on the order of 1 sec, and that the extruded PT is over 100 microns long in this species. Using cryo-ET to image the PT at a high resolution, they find that it exists in two major states: both an empty state and a state filled with cargo, and that the thickness of the tube wall changes when cargo is present. Strikingly, the authors observed that one of the cargo components, the ribosomes, are organized ordered array that may have helical symmetry. Finally, the authors took advantage of a naturally occurring "His tag" on PTP3 to affinity purify PTP3-containing protein complexes and analyze the composition using proteomics.

    Major comments

    ln 139-140: The absolute handedness of something can be very tricky to determine by cryo-ET (but certainly is possible). Variable hardware configurations between microscopes and differing conventions between software packages (e.g., for what direction is a positive tilt angle) can lead to inversion of the apparent handedness in the final tomogram. How certain are the authors that the absolute handedness is indeed right handed, as this seems to vary between the various display items in the manuscript? For example, in Fig 1c, my impression is that ribosome helices are left handed, as they are also in the supplemental movie. If this isn't known with certainty, perhaps it would be sufficient to describe the apparent helical symmetry but state that the handedness is ambiguous.

    Minor comments

    ln 39-40: Perhaps also cite the E. cuniculi genome paper?

    ln 97-98: It is interesting that the PT shortens in V. necatrix as well, and while I can pick this out in some of the individual traces in Sup Fig. 1b, it seems to get washed out in the trend line and isn't super obvious. If it isn't to laborious, it could be nice to add a panel showing the quantification of this (e.g., plotting the final length of each PT as a percentage of the maximum length achieved).

    ln 98-100: Strictly speaking, I don't think the referenced figure shows the sporoplasm being transformed into an extended conformation, only that it is spherical upon exit. Simply reword this to make clear that the deformations are inferred to occur but not directly observed.

    Because PT firing is so fast, the probability of trapping a PT in the process of transporting cargo would be pretty low. So then why does the PT still contain cellular cargo like ribosomes inside in the tomograms? Should these not have emerged in the sporoplasm which would enter the host cell? Are these "defective" spores that have failed to complete sporoplasm transport? Perhaps this is worth discussing.

    ln 118: The authors note an apparent correlation between the phase of germination and the thickness of the tube wall but don't specify what this correlation is. Is it thicker in the early phase and thinner in later phase, or vice versa? One could imagine "empty" tubes existing before or after sporoplasm transport, for example, so I'm not sure I follow how the phase is being inferred from the tomograms.

    ln 119-120: What is the evidence that the outer layer is made of PTPs, or that it is even protein (for example, as opposed to cell wall-like carbohydrate polymers)? I think this seems like a very reasonable hypothesis, but I would suggest explaining the logic and ensuring the degree of uncertainty is conveyed clearly. In light of this, I would also suggest changing figure labels, etc, that refer to the PTP layer (e.g., Fig. 3, PTPc and PTPe labels).

    ln 121, 123: "sheathed by a thin layer" and "enveloped by a thick outer layer": is this an additional layer being described? Or is this referring to the putative PTP layer, and that its thickness is variable?

    ln 125-126: While I understand how some features, like ribosomes, proteasomes, and generic membrane compartments could be identified, it is unclear to me how one would recognize the nucleus when inside the PT, nor are any examples shown. If the data is clear, perhaps the authors could show it in a figure? Otherwise, I suggest removing the claim regarding the nucleus.

    The arrangement of the ribosomes in a subset of tubes is really fascinating! While the number of observations is relatively small (n=5), it seems like it should be possible to comment preliminarily on whether there is much variability in their helical arrangement. Do the helical parameters vary much between observations? Does the til, pitch, etc vary much, are the 5 occurrences very similar? Is there any sign that they are associated with a membrane? Also, since the ribosomes form a lattice-like arrangement, it seems like it would be possible to trace ribosome helices in both the left and right handed directions. How did the authors decide between the two possibilities? This doesn't seem to be discussed.

    Fig. 2e: Are the two different colors/orientations meant to represent the two protamers of the ribosome dimer? When refined subvolumes are mapped back onto the original tomogram do the authors observe a similar crystalline arrangement of particles as in their segmentation? Are the orientations of the ribosomes correlated, and do the provide any evidence for the dimeric arrangement mentioned? The PlaceObjects plugin for Chimera can be very helpful for visualizing this: https://www.biochem.mpg.de/7939908/Place-Object

    Supp figure 4(b-d): Perhaps these models could be colored by pLDDT scores (with a key indicating the color scheme), so the reader can assess the quality of the predictions?

    How were the measurements of the membrane thickness and putative PTP layer carried out? On the tomogram projections? STAs? How were the boundaries of the layers established (e.g., map threshholding if STA?)? This information appears to be missing from the methods.

    Some tubes that are labeled as 'PTempty' actually contain cargo and look dense (example supp. Fig 2c, left and middle panels). Is it fair to classify these as empty tubes?

    Fig. 3d: I am not entirely clear on what is being shown here. Are independent reconstructions of PTcargo and PTempty superposed (aligned on membrane)? The description in the figure legend doesn't clearly say what is being displayed. I think it might be more clear to show these side-by-side instead of superposed (i.e., 4 panels instead of 2).

    Sup Fig 1: Define S and SP in legend or just spell out on figure? Missing x-axis label on panel b.

    Fig. 4b and Sup Fig 2a: The depictions of the PT in the spore here are left-handed. In a few species, the coil of the PT was found to form a right-handed helix (Jaroenlak, et al.), and it seems plausible that this may be a general feature that would be conserved across microsporidia. I appreciate that it might not be actually known to be right-handed in V. necatrix, but if there is no strong data either way, perhaps it would make sense for these depictions of the PT to be right-handed.

    I think all three of us are more or less in consensus about this manuscript, and I largely agree with the other reviewers comments. I think after addressing reviewer suggestions, this will be a pretty nice story.

    Significance

    Overall, this manuscript from Sharma, et al. presents interesting new findings about the structure and cargo transport function of the microsporidian PT. Microsporidia infect a wide range of hosts, including humans, and how the PT mediates parasite entry into cells is poorly understood. The approaches used in this study are appropriate for tackling the questions at hand, and appear to be generally well executed and interpreted. The observation that ribosomes assemble into an array within the PT is very unexpected and quite fascinating, and may be of broader interest to researchers working on ribosome structure and function, in addition to researchers studying microsporidia. The approach to investigating proteins interacting with PTP3 was quite elegant, and yielded a list of potential interactors that appears to be of very high quality and is highly plausible based on the literature field. We think this work is a substantial advance in the field and provides important new insights into the organization of the PT.

    • Please define your field of expertise with a few keywords to help the authors contextualize your point of view:

    Structural biology, microsporidia

    • Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    We are not experts in proteomics/mass spectrometry

  6. Version published to 10.1101/2023.05.31.543061v1 on bioRxiv