The pathogen-encoded signaling receptor Tir exploits host-like intrinsic disorder to assist infection

  1. Marta F. M. Vieira
  2. Guillem Hernandez
  3. Tiago Veloso
  4. Hugo Monteiro
  5. Miguel Arbesú
  6. Andreas Zanzoni
  7. Tiago N. Cordeiro

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Abstract

The translocated intimin receptor (Tir) is a central effector of Attaching and Effacing (A/E) pathogens responsible for worldwide foodborne disease cases. Tir acts as a cell-surface receptor in host cells, rewiring intracellular processes to assist infection by targeting multiple host proteins. We sought to understand the basis for Tir binding diversity in signaling. Here, we establish that Tir is a disordered protein with host-like binding motifs. A trait we find prevalent in several other effectors secreted by A/E bacteria. We disclose that Tir has a disordered C-terminal intracellular tail (C-Tir) with non-random structural preferences at phosphorylation sites, including host-like tyrosine-based motifs, with versatile lipid- and SH2 domain binding capability pre-phosphorylation. We show that multi-site tyrosine phosphorylation enables C-Tir to engage SH2 domains in a multivalent manner, consistent with Tir’s scaffold/hub role for host proteins. Last, we uncover Tir’s ability to dimerizes via its partially disordered N-terminal intracellular domain. Collectively, our findings provide an updated picture of Tir’s intracellular side, highlighting its ability to mimic host disordered membrane receptors’ versatility as a molecular strategy for host evasion.

Summary

Tir is a cell-surface receptor secreted by life-threatening pathogens. Upon delivery into host cells, Tir inserts the host plasma membrane providing a means for these extracellular pathogens to control host intracellular processes. To prevent pathogens from relying on Tir, it is essential to understand its intracellular mechanics. This paper provides a coherent picture of the intracellular side of Tir, highlighting its ability to copycat the interactions of disordered intracellular domains of host immune receptors. This copycatting allows the bacterial pathogens to modulate critical host processes, allowing infection to spread further without triggering the immune system response. This work proposes that other bacterial secreted pathogenic proteins exploit intrinsic disorder to hijack human cells, suggesting a widespread host subversion mechanism.

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    1. General Statements

    We want to thank all three reviewers for their positive feedback, constructive comments, and suggestions for clarity and improvement. We are delighted to find their consensus that the manuscript represents a contribution to the field.

    Accordingly, we made changes in the text (all highlighted in blue in the revised manuscript) and added a new figure as detailed in the point-by-point response.

    2. Point-by-point description of the revisions

    __Reviewer #1 (Evidence, reproducibility and clarity (Required)): __

    The authors describe results of the comprehensive analysis of the prevalence and functionality of intrinsically disordered regions of the pathogen-encoded signaling receptor Tir, which serves as an illustrative example of the bacterial effector proteins secreted by Attaching and Effacing (A/E) pathogens. This is an interesting and important study that represents an impressive amount of data generated computationally and using a broad spectrum of biophysical techniques. The work serves as a model of the well-designed and perfectly conducted study, where intriguing conclusions are based on the results of the comprehensive experiments. The manuscript is well-written and concise, and I have a real pleasure reading it. The text and figures are clear and accurate.

    We thank the Reviewer for these positive comments on our work.

    Although, in general, prior studies are referenced appropriately, the authors should mention that the pre-formed structural elements they found in Tir are in line with the concept of "PreSMos" (pre-structured motifs) previously introduced and described in several important studies from the laboratory of Kyou-Hoon Han.

    We thank the Reviewer for this suggestion. We have added a sentence to acknowledge the presence of “PreSMos” in the target-free state of Tir as putative signatures for target-binding, referring to a review article summarizing several local structural elements in unbound IDPs:

    “This supports the presence of pre-structured motifs (PreSMos) as pre-existing signatures for target binding and function within target-free Tir (72).”

    Please, note that we decided to keep this discussion to a minimum, as we cannot rule out the contribution of the induced fit model to the binding mechanism (i.e., disorder-to-order transition upon binding).

    __Reviewer #1 (Significance (Required)): __

    Solid evidence is provided that structural disorder and short linear motifs represent common features of A/E pathogen effectors. In fact, using a set of bioinformatics tools, the authors first show that although prokaryotic proteins typically contain significantly less intrinsic disorder than eukaryotic proteins, A/E pathogen effectors are as disordered as eukaryotic proteins. Using the translocated intimin receptor (Tir) as a subject of focused study, the authors then utilized a number of biophysical techniques to draw an impressive picture of disorder-based functionality. This study clearly represents a major advancement in the field of functional intrinsic disorder in general and in disorder-based functionality of proteins expressed by pathogenic bacteria. This was adds significantly to the field and will have a noticeable impact.

    Again, reading this manuscript was a real joy. Finally, this work perfectly fits in the area of my expertise, since for the past 25 years or so I am working on the different aspects of intrinsically disordered proteins.

    Thank you for this encouraging assessment.

    **Referee Cross-commenting**

    I agree with the amended recommendation of reviewer #3 to add in the manuscript EPEC O127.

    According to the suggestion of Reviewer #3, we have now included EPEC O127:H6 in the manuscript.

    I completely agree with comments of reviewer #2 and partially agree with reviewer #3. In my view, comparison of various strains as references for EPEC represents an interesting but independent project. It can be recommended to the authors as one of the potential future developments of their work.

    Thanks for the suggestion. We are pursuing that line of research.

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

    The general impression is that this is an excellent study that establishes

    The C-terminal intracellular region of Tir called C-Tir spanning residues 338 to 550 is largely disordered, however, observe helical structural elements involved with lipid interactions; multi-phosphorylation. The intracellular N-terminal part of Tir called N-Tir spanning residues 1 to 233 is also partially disordered but include a folded domain that is shown to assemble into a dimer

    The only major concern is that no SDS-PAGE gels or size exclusion chromatograms have been included to verify purity and monodispersed of the various constructs worked on. In particular, the SAXS and CD measurement is highly sensitive to purity, and the level of degradation as IDPs are notorious for being difficult to handle in solution. it would strengthen the arguments made based that

    We produced N-Tir and C-Tir as fusion proteins with a cleavable N-terminal thioredoxin tag (Trx-His6) and C-terminal Strep-tag. The latter allowed us to purify them via Strep-tag affinity chromatography as indicated by SDS-PAGE (please see Fig. S1).

    We agree with the Reviewer that even small amounts of impurities (i.e., higher oligomers/degradation) can interfere with the data analysis and make interpretation of the resulting data difficult and potentially misleading. __So, to avoid such problems, all samples were purified in monodispersed forms by size-exclusion chromatography (SEC) before any biophysical study. __

    Following the Reviewer's suggestion, we added a new supplementary figure (Fig. S5) showing the SEC-SAXS chromatogram profiles of C-Tir, N-Tir, and NS-Tir. Briefly, in the inline SEC-SAXS experiment, the sample eluates from an HPLC system directly and continuously into a BioSAXS flow cell for subsequent X-ray interrogation. Under our experimental conditions, C-Tir elutes as a single peak with Rg-values and mass compatible with a disordered monomeric protein, providing an excellent fit to the experimental SAXS curves. For N-Tir and NS-Tir, by SEC-SAXS, we separated the dimer from small amounts of high-order oligomers to yield the experimental SAXS curves of the pure dimers.

    “Fig. S5.* SEC-SAXS chromatograms of (A) C-Tir, (B) N-Tir, and (C) NS-Tir. Each plane shows normalized total scattering intensity I(s), over the entire s range, from each frame acquired along elution volume and respective Rg-value (black circles). The flat variation of Rg reflects a pure monodisperse sample. The column type for size exclusion chromatography and sample concentrations are on the top left of each panel. For reference, the retention volume for monomeric BSA (66.4 kDa) is displayed by red triangles.”*

    __**Minor Comments** __

    Read through the manuscript to remove passages with spoken language

    We thank the Reviewer for this suggestion. We went through the manuscript and improved the writing to reduce passages with spoken language.

    Line 263, "To do so", should be removed

    Line 290 "Our data thus" replaced with "this"

    We have amended the manuscript accordingly.

    Line 292 "lipid bilayers that might potentially fine-tune Tir's activity in the host cell." Weak sentence and the word fine-tune is slang. Rewrite the sentence. The interaction with lipids is fascinating!

    Thanks for the suggestion. The sentence has now been changed to “This shows that C-Tir can undergo multivalent and tunable electrostatic interaction with lipid bilayers via pre-structured elements, suggesting that membrane-protein interplay at the intracellular side might control the activity and interactions of Tir in host cells.”

    We also reinforce this fascinating message in the abstract by adding the sentence: “Membrane affinity is residue-specific and modulated by lipid composition, suggesting a previously unrecognized mechanism for interaction with the host.”

    Line 192 "In figure Fig. 3A," remove the Fig

    Fixed.

    Line 326, "In a similar fashion," is redundant. Rewrite the sentences below.

    We have modified the sentence as follows: “We evaluated whether the N-terminal cytosolic region of Tir (N-Tir; Fig S1) was also intrinsically disordered ...”

    Line 342 add spaces between digit and SI unit "52kDa" there are more cases of this.

    Thank you for pointing this out. This has now been corrected to 52 kDa.

    __Reviewer #2 (Significance (Required)): __

    I expect this study to have broad relevance to microbiologists working with the intimin and translocated intimin receptor, in particular the lipid interaction is likely to be followed up by the community.

    We thank the reviewer for this comment. Indeed, we believe that further studies on Tir's lipid-binding ability as a novel molecular strategy in host-pathogen interactions, will potentially provide new insights on virulence, transmembrane signaling in general, and disorder-mediated functions.

    __**Referee Cross-commenting** __

    What reviewer 3 suggested in the comments sounds like added value and should be included.

    I agree with reviewer 1, that the strain comparison potentially is beyond the scope presented in this manuscript.

    We have now included EPEC O127:H6 in the manuscript.

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

    __**Summary:** __

    This interesting manuscript look at the structure of the Nter and Cter of the effector Tir from enteropathogenic E. coli. The authors confirmed previous study highlighting the "disordered" part of the Cter. However, the extended experimental work (NMR, Small-angle X-ray scattering and CD spectroscopy) from this study also reveals the connection between different area of Tir and its implication during Tir phosphorylation and its interactions with SH2 domain.

    We thank the Reviewer for this positive remark. Indeed, in our work, we highlight the structural features of the SH2-mediated interaction between Tir and host SHP-1 protein, and we also show that C-Tir is capable of lipid interaction via pre-structured motifs and that N-Tir is disordered but assembled into a dimer. Overall, we provide an updated and wide picture of Tir's intracellular side that goes beyond the scrutiny of previously described disorder features.

    __**Major Comments:** __

    The authors used E2348/69 (O127:H7) strain as a reference for EPEC. However, this strain are the least effectors of all the EPEC sequences and may over estimated the PDR in EPEC. It would be wiser to use a strain like B171 as a reference for EPEC to be able to conclude "Disordered Proteins (PDR) with long disordered regions occur in EPEC effectors similar to the human proteome". I believe that the PDR in EPEC is similar to EHEC and CR. I do not have any major concern for the rest of the work.

    We thank the Reviewer for this comment. So, to clarify, we amended “EPEC” with “EPEC O127:H6” in text and figures.

    We also added a paragraph at the beginning of the Discussion section to acknowledge that our prediction analysis concerns EPEC O127:H6 and two additional representative A/E bacteria strains:

    “Among the enteropathogenic Escherichia coli strains EPEC O127:H6 (E2348/69) is commonly used as a prototype strain to study EPEC biology, genetics, and virulence (69). Here, we have determined the structural disorder propensity of EPEC O127:H6 sequences and two additional representatives of A/E bacteria: EHEC O157:H7 and CR ICC168.”

    Finally, the Reviewer suggests to include EPEC strain B171 (serotype O111:NM) in our analysis. We agree that considering additional strains would be of value, however we believe that this is beyond the scope of this manuscript, which mainly focuses on the characterization of the structural features of the E2348/69 Tir effector. We are currently working on a broader comparative analysis among different Escherichia coli pathogenic strains, including B171, and we hope to share our findings with the community in the near future.

    **Minor comments**

    Statistic problem: Mann Whitney U Test (Wilcoxon Rank Sum Test) is a comparison of two independent samples with the underlying assumption is normally distributed or that the samples were sufficiently large. It is not certain that any of this assumption is correct. In addition, the effector are part of the whole proteome. Can it be then considered that both groups are independent?

    We thank the Reviewer for this remark, which allows us to clarify the choice of this particular test. Indeed the Mann Whitney U-test is a non-parametric test to compare two samples with the alternative hypothesis being that one of the two samples is stochastically greater than the other. As it is a nonparametric test samples are not required to be normally distributed, as it is for the Student t-test.

    Regarding the independence of the samples, when comparing the effectors collections to their corresponding proteomes, we did exclude the effectors sequences from the latter. We have clarified this point in the Supplementary Material and Methods section.

    Line 120 and 442: O127 not H127

    Thank you for pointing this out. It has now been corrected to O127.

    Line 212: positions 409 or 405?

    Yes, it should be 405. Thank you.

    __Reviewer #3 (Significance (Required)): __

    **Nature and significance:**

    Tir plays a major role during EPEC infection. It is a signalling platform that has been reported to interact with multiple proteins. Whereas the extracellular part has been well characterised and crystallised, the intracellular part has been proven so far to be difficult to study. Over the last decade, no progress has been made to explain how Tir works. This manuscript provides interesting information that shade some light on how the protein could work.

    __**Existing literature:** __

    The last research manuscript trying to highlight the structural function of Tir dates from 2007 (PMC1896257). This study is far more extended and in depth than any other previous work done.

    __**Audience:** __

    the Audience may probably limited to researcher working on the field of cellular microbiology and the function associated with bacterial effector in the host. This study could be also a useful tool to identify new effectors base on their "disorder".

    We thank the Reviewer for recognizing the importance of this study. We agree that our work highlights the pivotal role of disordered regions in bacterial effectors, thus enabling a better understanding of the molecular mechanisms used by pathogens to subvert the host-cell processes. We indeed believe that our work can stimulate further research on the characterization of intrinsically disordered effectors, and also beyond the cellular microbiology field, in order to gain a broader knowledge on the molecular dialogue at the host-pathogen interface, which is essential to design better therapeutic strategies.

    __**Expertise:** __

    I have been working on A/E pathogens for the last 15 years with a particular interest in Tir signalling. My domain of expertise is more in relation to cell signalling than crystallography or structural study.

    __**Referee Cross-commenting** __

    I agree with both reviewers. My comment about EPEC is more about the conclusion for some of the figures. I don't think they should conclude for the whole EPEC. The Tir variation among EHEC O157:H7 is low, but it is far more diverse for EPEC. Simply adding in the manuscript EPEC O127 should be enough.

    We thank the Reviewer for this comment. As mentioned above, we now state in the manuscript, in both Results and Discussion sections, that we used E2348/69 as a representative strain for EPEC.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    This interesting manuscript look at the structure of the Nter and Cter of the effector Tir from enteropathogenic E. coli. The authors confirmed previous study highlighting the "disordered" part of the Cter. However, the extended experimental work (NMR, Small-angle X-ray scattering and CD spectroscopy) from this study also reveals the connection between different area of Tir and its implication during Tir phosphorylation and its interactions with SH2 domain.

    Major Comments:

    The authors used E2348/69 (O127:H7) strain as a reference for EPEC. However, this strain are the least effectors of all the EPEC sequences and may over estimated the PDR in EPEC. It would be wiser to use a strain like B171 as a reference for EPEC to be able to conclude "Disordered Proteins (PDR) with long disordered regions occur in EPEC effectors similar to the human proteome". I believe that the PDR in EPEC is similar to EHEC and CR. I do not have any major concern for the rest of the work.

    Minor comments

    Statistic problem: Mann Whitney U Test (Wilcoxon Rank Sum Test) is a comparison of two independent samples with the underlying assumption is normally distributed or that the samples were sufficiently large. It is not certain that any of this assumption is correct. In addition, the effector are part of the whole proteome. Can it be then considered that both groups are independent?

    Line 120 and 442: O127 not H127

    Line 212: positions 409 or 405?

    Significance

    Nature and significance:

    Tir plays a major role during EPEC infection. It is a signalling platform that has been reported to interact with multiple proteins. Whereas the extracellular part has been well characterised and crystallised, the intracellular part has been proven so far to be difficult to study. Over the last decade, no progress has been made to explain how Tir works. This manuscript provides interesting information that shade some light on how the protein could work.

    Existing literature:

    The last research manuscript trying to highlight the structural function of Tir dates from 2007 (PMC1896257). This study is far more extended and in depth than any other previous work done.

    Audience:

    the Audience may probably limited to researcher working on the field of cellular microbiology and the function associated with bacterial effector in the host. This study could be also a useful tool to identify new effectors base on their "disorder".

    Expertise:

    I have been working on A/E pathogens for the last 15 years with a particular interest in Tir signalling. My domain of expertise is more in relation to cell signalling than crystallography or structural study.

    Referee Cross-commenting

    I agree with both reviewers. My comment about EPEC is more about the conclusion for some of the figures. I don't think they should conclude for the whole EPEC. The Tir variation among EHEC O157:H7 is low, but it is far more diverse for EPEC. Simply adding in the manuscript EPEC O127 should be enough.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    The general impression is that this is an excellent study that establishes The C-terminal intracellular region of Tir called C-Tir spanning residues 338 to 550 is largely disordered, however, observe helical structural elements involved with lipid interactions; multi-phosphorylation. The intracellular N-terminal part of Tir called N-Tir spanning residues 1 to 233 is also partially disordered but include a folded domain that is shown to assemble into a dimer

    The only major concern is that no SDS-PAGE gels or size exclusion chromatograms have been included to verify purity and monodispersed of the various constructs worked on. In particular, the SAXS and CD measurement is highly sensitive to purity, and the level of degradation as IDPs are notorious for being difficult to handle in solution. it would strengthen the arguments made based that

    Minor Comments

    Read through the manuscript to remove passages with spoken language

    Line 263, "To do so", should be removed

    Line 290 "Our data thus" replaced with "this"

    Line 292 "lipid bilayers that might potentially fine-tune Tir's activity in the host cell." Weak sentence and the word fine-tune is slang. Rewrite the sentence. The interaction with lipids is fascinating!

    Line 192 "In figure Fig. 3A," remove the Fig

    Line 326, "In a similar fashion," is redundant. Rewrite the sentences below.

    Line 342 add spaces between digit and SI unit "52kDa" there are more cases of this.

    Significance

    I expect this study to have broad relevance to microbiologists working with the intimin and translocated intimin receptor, in particular the lipid interaction is likely to be followed up by the community.

    Referee Cross-commenting

    What reviewer 3 suggested in the comments sounds like added value and should be included.

    I agree with reviewer 1, that the strain comparison potentially is beyond the scope presented in this manuscript.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The authors describe results of the comprehensive analysis of the prevalence and functionality of intrinsically disordered regions of the pathogen-encoded signaling receptor Tir, which serves as an illustrative example of the bacterial effector proteins secreted by Attaching and Effacing (A/E) pathogens. This is an interesting and important study that represents impressive amount of data generated computationally and using a broad spectrum of biophysical techniques. The work serves as a model of the well-designed and perfectly conducted study, where intriguing conclusions are based on the results of the comprehensive experiments. The manuscript is well-written and concise, and I have a real pleasure reading it. The text and figures are clear and accurate.

    Although, in general, prior studies are referenced appropriately, the authors should mention that the pre-formed structural elements they found in Tir are in line with the concept of "PreSMos" (pre-structured motifs) previously introduced and described in several important studies from the laboratory of Kyou-Hoon Han.

    Significance

    Solid evidence is provided that structural disorder and short linear motifs represent common features of A/E pathogen effectors. In fact, using a set of bioinformatics tools, the authors first show that although prokaryotic proteins typically contain significantly less intrinsic disorder than eukaryotic proteins, A/E pathogen effectors are as disordered as eukaryotic proteins. Using the translocated intimin receptor (Tir) as a subject of focused study, the authors then utilized a number of biophysical techniques to draw an impressive picture of disorder-based functionality. This study clearly represents a major advancement in the field of functional intrinsic disorder in general and in disorder-based functionality of proteins expressed by pathogenic bacteria. This was adds significantly to the field and will have a noticeable impact.

    Again, reading this manuscript was a real joy. Finally, this work perfectly fits in the area of my expertise, since for the past 25 years or so I am working on the different aspects of intrinsically disordered proteins.

    Referee Cross-commenting

    I agree with the amended recommendation of reviewer #3 to add in the manuscript EPEC O127.

    I completely agree with comments of reviewer #2 and partially agree with reviewer #3. In my view, comparison of various strains as references for EPEC represents an interesting but independent project. It can be recommended to the authors as one of the potential future developments of their work.

  5. Version published to 10.1101/2021.04.22.440577v2 on bioRxiv
  6. Version published to 10.1101/2021.04.22.440577v1 on bioRxiv