Distinct regions of H. pylori’s bactofilin CcmA regulate protein–protein interactions to control helical cell shape

  1. Sophie R Sichel
  2. Benjamin P Bratton
  3. Nina R Salama

  • Curated by eLife

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    Evaluation Summary:

    The helical shape of the bacterial pathogen Helicobacter pylori is important for its ability to colonize the human gut. Building on previous work identifying a complex of proteins required for generating helicity, this study focuses on the molecular mechanisms by which this complex modulates cell shape. Based on results from genetic, cytological, and pull-down experiments, the authors propose that one member of the complex, the bactofilin CcmA, interacts with two other complex members to generate helicity through a combination of cell wall synthesis and degradation. While data is supportive of this idea, the conclusions of the study require additional experimental support to rule out competing models.

    (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

The helical shape of Helicobacter pylori cells promotes robust stomach colonization; however, how the helical shape of H. pylori cells is determined is unresolved. Previous work identified helical-cell-shape-promoting protein complexes containing a peptidoglycan-hydrolase (Csd1), a peptidoglycan precursor synthesis enzyme (MurF), a non-enzymatic homolog of Csd1 (Csd2), non-enzymatic transmembrane proteins (Csd5 and Csd7), and a bactofilin (CcmA). Bactofilins are highly conserved, spontaneously polymerizing cytoskeletal bacterial proteins. We sought to understand CcmA’s function in generating the helical shape of H. pylori cells. Using CcmA deletion analysis, in vitro polymerization, and in vivo co-immunoprecipitation experiments, we identified that the bactofilin domain and N-terminal region of CcmA are required for helical cell shape and the bactofilin domain of CcmA is sufficient for polymerization and interactions with Csd5 and Csd7. We also found that CcmA’s N-terminal region inhibits interaction with Csd7. Deleting the N-terminal region of CcmA increases CcmA-Csd7 interactions and destabilizes the peptidoglycan-hydrolase Csd1. Using super-resolution microscopy, we found that Csd5 recruits CcmA to the cell envelope and promotes CcmA enrichment at the major helical axis. Thus, CcmA helps organize cell-shape-determining proteins and peptidoglycan synthesis machinery to coordinate cell wall modification and synthesis, promoting the curvature required to build a helical cell.

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  1. Version published to 10.7554/elife.80111 on eLife
  2. eLife

    Evaluation Summary:

    The helical shape of the bacterial pathogen Helicobacter pylori is important for its ability to colonize the human gut. Building on previous work identifying a complex of proteins required for generating helicity, this study focuses on the molecular mechanisms by which this complex modulates cell shape. Based on results from genetic, cytological, and pull-down experiments, the authors propose that one member of the complex, the bactofilin CcmA, interacts with two other complex members to generate helicity through a combination of cell wall synthesis and degradation. While data is supportive of this idea, the conclusions of the study require additional experimental support to rule out competing models.

    (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):

    In previous work, the Salama group identified a large complex of proteins required for the distinctive shape of Helicobacter pylori. Focusing on one member of this complex, CcmA, Sichel and colleagues use a combination of genetics, biochemistry, and quantitative image analysis to identify regions of CcmA involved in shape determination and illuminate the mechanism underlying H. pylori cell shape. They pinpoint the bactofilin domain and N terminal region of CcmA as important for both CcmA polymerization and interactions between CcmA and complex members Csd5 and Csd7 which in turn influences the activity of the peptidoglycan hydrolase Csd1. Super-resolution microscopy indicates that Csd5 recruits CcmA to the cell envelope and promotes CcmA localization to the major helical axis. Synthesizing these data, the authors propose a model in which CcmA coordinates interactions with the peptidoglycan synthesis machinery and associated hydrolases to promote helical shape. Experimentally the paper is extremely solid, containing data acquired through an extensive array of approaches. I was impressed by the development of quantitative approaches to assess how much CcmA was associated with the cell envelope versus cytoplasmic. Figure 7 is beautiful. At the same time, and despite the presentation of a beautiful model figure at the end, the sheer volume of data and the dense writing make it difficult for readers to make the connection between the results and the model in a meaningful way.

  4. eLife

    Reviewer #2 (Public Review):

    The authors' introduction is clearly written and sets the stage for the rest of the paper. They state what is unclear currently in the field: the mechanism for the transfer of the cytoplasmic regulation of bactofilins to the periplasmic space; and this is exactly what they set out to elucidate. This work is powered by the creation of truncated mutants of the bactofilin-like protein CcmA. From there, the authors showed that CcmA has a central role in integrating Csd1-Csd7 and Csd5-MurF sub-complexes. By disentangling the CmmA anatomy, the authors showed that CcmA lacks the first 17 amino acids to sequester Csd7, leaving Csd1 unprotected and likely exposed to proteolysis. However further studies will be necessary to connect other pathways to this observation, this is (to my knowledge) the first instance of a mechanism combining localization and proteolytic instability to balance synthesis and hydrolysis of the cell wall to achieve specific shapes.

    Strengths: This work is a masterclass on how to perform solid science with controls. The authors' narrative is smooth, making it easy for non-expert readers to understand how they unveil each segment of the story. It also makes it look as if their assays are easy to perform provided the number of parallel scenarios tested - disguising how challenging it is to master the cell biology of this tiny, skinny bacterium. Another highlight of this study is how almost every claim is supported by the data.

    Weaknesses: There is a disconnection between the CcmA polymerization evidence, the pulldowns showing its interactions with other factors, and the microscopy data displaying CcmA patterns under different genetic backgrounds. The fact that CcmA is capable of polymerization with the bactofilin domain alone is important and supported by the microscopy data, but the polymer width measurements shown in the TEM images look completely detached from the story. The authors should try to discuss if these different polymer architectures have any role in the localization and/or interactions with other proteins.

  5. eLife

    Reviewer #3 (Public Review):

    This work examines the interaction(s) between several cell shape determining proteins and a bactofilin protein (CcmA) in the spiral-shaped bacterium Helicobacter pylori. The study utilizes a nice mix of methodologies and an overall reductionist approach to examine how various domains of CcmA influence its ability to affect cell wall curvature, homo- and hetero- protein interactions, and the regulation of a cell wall hydrolase enzyme. The localization study along with an in-depth analysis of the Gaussian curvature intrinsic to the various mutant strains examined is particularly stunning, and this approach would be very beneficial to others in the field.

    Strengths:

    - The author's approach to investigating the contributions of various domains of CcmA to their previously identified phenotypes is excellent.
    - The TEM filament phenotypes are striking and convincing.
    - The use of co-IP was utilized well by the authors throughout the study and was beneficial in moving the study forward.
    - The cell curvature analysis and circular dichroism studies were well-conducted.
    - The computational rigor present in the CcmA localization study is commendable, and the data convincing.

    Weaknesses/Concerns:

    - In their model, the authors do not comment on how the N-terminal domain might effectively regulate the interactions of CcmA and Csd7 (assuming direct interactions exist).
    o Despite generating a nice assortment of N-terminal truncation mutants, the authors do not utilize them to demonstrate that the N-terminus is actually important for what they define it as (ie. MM, membrane-binding motif); this is a notable omission, given the authors' hypothetical model. Membrane association may potentially underlie the mechanism by which CcmA occludes the protein's association with Csd7.
    o Assuming that the N-terminal domain regulates the interaction between CcmA and Csd7, the authors do not address whether this regulation is transient or permanent.

    - Despite significantly contributing relevant data to the study, there is some concern that the authors rely too heavily on their co-IP studies when developing their model. The co-IPs appear to have been designed to exclusively utilize a CcmA polyclonal antibody. This effectively prevents the investigation of the interactions of hypothetical complex members (specifically Csd5-Csd7) in the absence of CcmA. Given that the N-terminal truncation mutant shares the cell shape characteristics of a ccmA deletion mutant (curved rod; Yang 2019) rather than that of the csd5 deletion mutant (straight rod), this is a potential concern.
    o Additional controls or alternative approaches are needed to support the authors' overall conclusions that the interactions they describe are occurring directly between CcmA and Csd7/Csd5 (specifically investigating potential interactions between Csd5 and Csd7 in the absence of CcmA).

    Are the authors' conclusions justified?
    The following conclusions appear to be well-supported by the authors' observations:

    1. Both the bactofilin domain and N-terminal region of CcmA are required for helical cell shape.
    2. The bactofilin domain of CcmA is sufficient for polymerization.
    3. Deleting the N-terminal region of CcmA destabilizes the peptidoglycan-hydrolase Csd1.
    4. Csd5 recruits CcmA to the cell envelope and promotes CcmA enrichment at the major helical axis.

    The following conclusions require clarification, modification, or additional experimental support:

    i) The bactofilin domain of CcmA is sufficient for interactions with Csd5 and Csd7.
    o The evidence presented indicates that the bactofilin domain is sufficient for all three proteins to associate together. It does not demonstrate that CcmA interacts directly with either Csd5 or Csd7.

    ii) CcmA's N-terminal region inhibits interaction with Csd7.
    iii) Deleting the N-terminal region of CcmA increases CcmA-Csd7 interactions.
    o The evidence presented indicates that the three proteins only associate together when the N-terminal portion of CcmA is removed.
    o The competing hypothesis, that CcmA only associates with Csd5 and that it effectively prevents the association of Csd5 with Csd7 (likely via its N-terminal domain) is not acknowledged or disproven.

  6. Version published to 10.1101/2022.04.05.487110v2 on bioRxiv
  7. Version published to 10.1101/2022.04.05.487110v1 on bioRxiv