Developmental Transitions Coordinate Assembly of the Coxiella burnetii Dot/Icm Type IV Secretion System

  1. Donghyun Park
  2. Samuel Steiner
  3. Meng Shao
  4. Craig R. Roy
  5. Jun Liu

  • Curated by eLife

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    Evaluation Summary:

    Park et al.'s work provides insight into the infection processes of the human pathogen Coxiella burnetii with unprecedented detail. Their time course of cellular infection reveals the timing of key events and detects a previously unrecognized membrane structure. This work will shed new insight into the infection process of this pathogen allowing new targets for the treatment of infection with Coxiella.

    (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

Coxiella burnetii is an obligate intracellular bacterial pathogen that has evolved a unique biphasic developmental cycle. The infectious form of C. burnetii is the dormant small cell variant (SCV), which transitions to a metabolically active large cell variant (LCV) that replicates inside the lysosome-derived host vacuole.

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  1. Version published to 10.1128/iai.00410-22
  2. eLife

    Evaluation Summary:

    Park et al.'s work provides insight into the infection processes of the human pathogen Coxiella burnetii with unprecedented detail. Their time course of cellular infection reveals the timing of key events and detects a previously unrecognized membrane structure. This work will shed new insight into the infection process of this pathogen allowing new targets for the treatment of infection with Coxiella.

    (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):

    The manuscript by Park et al. uses CLEM, cryo-milling, and cryo-ET to visualize the major morphological transitions that occur in the bacteria Coxiella burnetii as it infects mammalian cells. These bacteria move from a small cell variant (SCV) to a large cell variant (LCV) that can replicate in the host lysosome. The authors focus on the C. burnetii T4SS which is similar in composition to the legionella Dot/Icm T4SS. They generate a homology model from the sub-tomographic 3D reconstruction of a little over 7,000 T4SSs using alpha-fold and structure of the Lp T4SS. This is a very descriptive analysis of gross cellular changes that occur in this bacteria during infection. While the authors suggest models for the developmental steps they think are occurring, it doesn't appear that they have fully mined their data for a more quantitative analysis of what is happening during the transition - let alone a mechanistic model for T4SS assembly and regulation.

    Major Suggestions:

    1. While it seems a reasonable hypothesis that the Dot/Icm T4SS assembly is regulated during the SCV-to-LCV transition it is unclear from the data shown if this is actually the case? Can the authors quantify the number and assembly state of the T4SSs during the transition from SCV to LCV (like they quantified the number of ribosomes)? They should also show sub-tomographic averages of the various structures making clear what parts of the complexes are assembled early and late during this transition? This type of detailed analysis is essential since it is the main point of the paper.
    2. "The results indicate that 38.6% of the Dot/Icm machines identified from LCV images displayed an assembled inner membrane complex. By contrast, none of the bacteria in the transitional phase displayed a Dot/Icm-associated inner membrane complex, indicating that the complete core complex assembles upon transition to the LCV form (Fig. 5)."

    Related to the previous comment - Figure 5 is a cartoon model - the authors should actually show this quantification and show structures.

    3. A more detailed structural comparison between the Legionella and Coxiella burnetii T4SSs should be included as a major figure.

    4. The number of ribosomes is quantified between SCV and LCV cells - however, it is unclear whether the argument is that in the SCV they are poised to be transcribed or that there is no change in transcriptional activity of the ribosomes during the transition.

    5. The authors should further explain how folding of the inner membrane alone would allow for rapid expansion - shouldn't the outer membrane also have to rapidly expand? Do they see any sort of protein structures within the folded membrane that explains its topology?

    6. Can the authors explain more why the LCV cells appear to be further away from the lysosome membrane? I would have predicted the bacteria would be closer so that proteins translocated through the T4SS would be able to get through the lysosome membrane and into the host cell cytoplasm - otherwise wouldn't these bacterial effector proteins simply be degraded in the acidified lysosome?

    7. More information about the methods used needs to be included. How was phase shift correction done? How were different symmetry mismatches dealt with in the 3D reconstructions (in methods it simply states 13-fold symmetry was applied).

  4. eLife

    Reviewer #2 (Public Review):

    This study aims to use the latest cryo-electron tomography methods to understand key events in the process of cellular infection by Coxiella burnetii. The study focuses on the type IV secretion system, which is recognised as a critical feature of a successful infection. The study aims to image cells at different stages of infection to understand the order of events and potential roles of the secretion system.

    Key strengths of the study:

    The design of the study takes advantage of a detailed understanding of Coxiella biology to image cells in a very appropriate way. As a consequence, the study is successful in determining when the secretion system is present, and that the position of the bacterium in the Coxiella-containing vesicle is determined by a different structure.

    The cryo-ET has clearly succeeded in providing novel details of Coxiella. The authors report a previously unobserved membrane folding that likely has high importance for the infection process. Further study of this phenomenon may open new avenues of research in Coxiella.

    The structure of the type IV secretion system produced from cryo-ET is beautiful and adds new insight to the understanding of this structure.

    Key weaknesses:

    The analysis of ribosome numbers in the different stages of the Coxiella life-cycle is based on very few cells.

    This work would be more impactful if the images were submitted to a public database so that others can repeat the analysis,

    My opinion is that the authors have met their stated aims clearly, and their results are strong and support the conclusions.

    This work is most likely to be impactful from the insight that it provides into when the type IV secretion system is active, and from the discovery of a novel infection-related membrane structure. The methods used should be reproducible but are largely an elegant application of previously described methods to a well-defined problem. Providing the raw images to the community would increase the utility of the work by allowing comparison with future research on similar topics.

    Generally, I found this paper to be an elegant and interesting study that was written very clearly and succinctly. I congratulate the authors on their excellent work.

  5. Version published to 10.1101/2022.05.29.493899v1 on bioRxiv