BopN is a Gatekeeper of the Bordetella Type III Secretion System

ABSTRACT The classical Bordetella species infect the respiratory tract of mammals. While B. bronchiseptica causes rather chronic respiratory infections in a variety of mammals, the human-adapted species B. pertussis and B. parapertussisHU cause an acute respiratory disease known as whooping cough or pertussis. The virulence factors include a type III secretion system (T3SS) that translocates effectors BteA and BopN into host cells. However, the regulatory mechanisms underlying the secretion and translocation activity of T3SS in bordetellae are largely unknown. We have solved the crystal structure of BopN of B. pertussis and show that it is similar to the structures of gatekeepers that control access to the T3SS channel from the bacterial cytoplasm. We further found that BopN accumulates at the cell periphery at physiological concentrations of calcium ions (2 mM) that inhibit the secretion of BteA and BopN. Deletion of the bopN gene in B. bronchiseptica increased secretion of the BteA effector into calcium-rich medium but had no effect on secretion of the T3SS translocon components BopD and BopB. Moreover, the ΔbopN mutant secreted approximately 10-fold higher amounts of BteA into the medium of infected cells than the wild-type bacteria, but it translocated lower amounts of BteA into the host cell cytoplasm. These data demonstrate that BopN is a Bordetella T3SS gatekeeper required for regulated and targeted translocation of the BteA effector through the T3SS injectisome into host cells. IMPORTANCE The T3SS is utilized by many Gram-negative bacteria to deliver effector proteins from bacterial cytosol directly into infected host cell cytoplasm in a regulated and targeted manner. Pathogenic bordetellae use the T3SS to inject the BteA and BopN proteins into infected cells and upregulate the production of the anti-inflammatory cytokine interleukin-10 (IL-10) to evade host immunity. Previous studies proposed that BopN acted as an effector in host cells. In this study, we report that BopN is a T3SS gatekeeper that regulates the secretion and translocation activity of Bordetella T3SS.

1. A gatekeeper protein is a protein that switches the substrate secretion from the secretion of translocators to the secretion of effectors. In Fig. 2, the authors report the secretion of BopN, which is not a classical characteristic of a gatekeeper protein. Fig. 3 shows the secretion of the BteA effector, which is more relevant; therefore, the authors should change the order of the figures and present the results that demonstrate the effect of BopN on effector secretion first. 2. Figure 2C -The presentation of BopN amounts as a fraction of that expressed in a specific culture (BopNrep grown with 2 mM calcium) is not ideal. As it looks like the growing conditions affect the expression of BopNrep, it should be presented as a fraction of each sample (how much is secreted vs. intracellular). Also, please change the color of the black bars to a lighter color so that the error bars will be visible. 3. In Fig. 3B -the normalization of BteA secretion/expression is normalized according to the levels in the culture grown without calcium, while in Fig. 2B, it is normalized to a culture grown in 2 mM calcium. It is not clear why. Since this normalization method is not ideal, as discussed in my previous point, I think it should be presented as a ratio between each sample's secreted and intracellular levels. 4. Figure 2D-E -to have a complete picture of the localization BopN under high and low calcium concentrations, the authors should add the BscD localization for zero calcium and its merged image (so the two panels will become one). 5. Figure 3B-C -In Fig. 3B, the secretion/expression is measured under exponential conditions, while in Fig. 3C, it is examined for overnight cultures (stationary phase). To conclude the effect of BopN on effector/translocator secretion -the authors should present data from similar conditions or at least provide a scientific explanation for the difference. 6. Figure 4C -please add a positive control (WT strain or delta BopN strain) to show that NF-kB activity is enhanced under these conditions. 7. The hierarchy of type III secretion should be explained in the introduction and not in the discussion (lines 333-343), as it is critical for understanding the work described in the manuscript. 8. Can you detect BopN-StcV interaction in vitro? Is it calcium-dependent? 9. Usually, gatekeeper proteins are not secreted. Here the authors describe a unique situation where BopN functions as a gatekeeper but is also secreted. Since its role as a gatekeeper requires the protein to be folded, the authors should explain how it later/simultaneously gets secreted. As a folded protein? How is this possible based on the reported structure? 10. Lines 182-185: the authors cannot conclude that the secretion of BopN is T3-dependent because it is not secreted in the delta BscN mutant, as it is hardly expressed in this strain, as mentioned by the authors in lines 186-188. 11. The authors should include the statistical analysis in their figures and clearly mark significant differences. 12. While the structural data presented in this manuscript was BopN of B. pertussis, the functional experiments were carried out with B. bronchiseptica. Can the authors add sequence alignment of the BopN of the two strains to determine that these proteins are homologous? 13. The design of BopN includes a flag-tag, which allows a direct measurement of BopN expression and secretion using western blot. Therefore, the authors should add this method to figures 2 and 3. 14. The authors should demonstrate that the BopN-HiBit-3xFlag-SPOT is a functional protein (preferably by showing that the labeled protein can complement the infection activity of the delta BopN mutant strain). 15. According to the Dali algorithm, "strong matches" have a Z-score higher than n/100-4 (where n is the number of amino acids). When taking ~280 aa of the BopN, only a z-score above 24 is meaningful. However, the authors report Z-scores of 12.5-20.3 but suggest structural homology. This point should be discussed when concluding the similarity between BopN and other T3SS gatekeepers.
Minor comments: 1. "secretion-restrictive calcium conditions", "physiological concentration", "high calcium condition". Please use one term to avoid confusion. 2. The abbreviation T3E is used in many figures but is not explained. Please add the acronym "type III effector" to the figure legend of Figure 2. 3. Figure 3C -the authors should add the size marker of the blots. 4. Line 175, add the calcium concentration tested. 5. Line 155 -the authors present the calcium response as a hypothesis, although it was already reported. I would recommend rephrasing the sentence to "previously reported." 6. Bordetella should be italic throughout the manuscript (for example, lines 76, 148, etc.).
Reviewer #2 (Comments for the Author): In this study, Munoz Navarrete and co-workers report an extensive structure, biochemical and functional characterisation of the Bordetella T3SS protein BopN. Notably, they demonstrate that it possess a fold similar to that of the canonical T3SS gatekeeper protein SctW, that it regulates T3SS in a calcium-dependent manner, and that it regulates effector secretion. Notably, through an elegant reporter assay, they demonstrate that it is secreted (unlike the gatekeeper in many other T3SSs), but contrary to previous reports, it does not appear to impact the NF-kB signalling cascade, nor impacts the secretion of the translocon components. Collectively the work reported here is very compelling and performed thoroughly. While there are no major surprises, this study contributes to the characterisation of this critical component of the T3SS, whose role and mechanism of action remains poorly understood. Accordingly, I only have very minor comments.
-In the abstract, line 30: HU after parepetrussi is probably a typo -Abstract line 32, and also introduction line 77: why "so-called"? These are effectors, and have been shown to be secreted in the host.
-Line 96: While I commend the use to the standard T3SS nomenclature throughout the manuscript, it would probably help the reader to mention the name of the orthologue in other species here (SepD/L in EPEC, MxiC in Shigella...) -Line 145: What is the sequence identity between homologous proteins? They are indicated in table 2, which should be refereed to here -but is the sequence identity for the whole protein, or just the aligned residues? -In the discussion, with might be worth mentioning if BopN possesses a bonafide T3SS signal sequence. It has been shown to have one in some orthologues (SepL I think?), but still is not secreted in this species. Is there something different in the signal sequence of BopN?
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In this manuscript, the authors studied the BopN protein of Bordetella, which so far was considered a T3SS effector, and suggest that based on its structure and function, it is the unidentified gatekeeper protein of the Bordetella T3SS. While the results of this study are exciting and support the notion that BopN is involved in BteA secretion and its ability to translocate into the host cells' cytoplasm, they do not support the classical function of a gatekeeper. Therefore, I recommend reconsidering the presentation of BopN as the gatekeeper protein as well as addressing the following concerns: Main comments: 1. A gatekeeper protein is a protein that switches the substrate secretion from the secretion of translocators to the secretion of effectors. In Fig. 2, the authors report the secretion of BopN, which is not a classical characteristic of a gatekeeper protein. Fig. 3 shows the secretion of the BteA effector, which is more relevant; therefore, the authors should change the order of the figures and present the results that demonstrate the effect of BopN on effector secretion first. 2. Figure 2C -The presentation of BopN amounts as a fraction of that expressed in a specific culture (BopN rep grown with 2 mM calcium) is not ideal. As it looks like the growing conditions affect the expression of BopN rep , it should be presented as a fraction of each sample (how much is secreted vs. intracellular). Also, please change the color of the black bars to a lighter color so that the error bars will be visible. 3. In Fig. 3B -the normalization of BteA secretion/expression is normalized according to the levels in the culture grown without calcium, while in Fig. 2B, it is normalized to a culture grown in 2 mM calcium. It is not clear why. Since this normalization method is not ideal, as discussed in my previous point, I think it should be presented as a ratio between each sample's secreted and intracellular levels. 4. Figure 2D-E -to have a complete picture of the localization BopN under high and low calcium concentrations, the authors should add the BscD localization for zero calcium and its merged image (so the two panels will become one). 5. Figure  To conclude the effect of BopN on effector/translocator secretion -the authors should present data from similar conditions or at least provide a scientific explanation for the difference. 6. Figure 4C -please add a positive control (WT strain or delta BopN strain) to show that NF-kB activity is enhanced under these conditions. 7. The hierarchy of type III secretion should be explained in the introduction and not in the discussion (lines 333-343), as it is critical for understanding the work described in the manuscript. 8. Can you detect BopN-StcV interaction in vitro? Is it calcium-dependent? 9. Usually, gatekeeper proteins are not secreted. Here the authors describe a unique situation where BopN functions as a gatekeeper but is also secreted. Since its role as a gatekeeper requires the protein to be folded, the authors should explain how it later/simultaneously gets secreted. As a folded protein? How is this possible based on the reported structure? 10. Lines 182-185: the authors cannot conclude that the secretion of BopN is T3dependent because it is not secreted in the delta BscN mutant, as it is hardly expressed in this strain, as mentioned by the authors in lines 186-188.
11. The authors should include the statistical analysis in their figures and clearly mark significant differences. 12. While the structural data presented in this manuscript was BopN of B. pertussis, the functional experiments were carried out with B. bronchiseptica. Can the authors add sequence alignment of the BopN of the two strains to determine that these proteins are homologous? 13. The design of BopN includes a flag-tag, which allows a direct measurement of BopN expression and secretion using western blot. Therefore, the authors should add this method to figures 2 and 3. 14. The authors should demonstrate that the BopN-HiBit-3xFlag-SPOT is a functional protein (preferably by showing that the labeled protein can complement the infection activity of the delta BopN mutant strain). 15. According to the Dali algorithm, "strong matches" have a Z-score higher than n/100-4 (where n is the number of amino acids). When taking ~280 aa of the BopN, only a z-score above 24 is meaningful. However, the authors report Z-scores of 12.5-20.3 but suggest structural homology. This point should be discussed when concluding the similarity between BopN and other T3SS gatekeepers.
Minor comments: 1. "secretion-restrictive calcium conditions", "physiological concentration", "high calcium condition". Please use one term to avoid confusion. 2. The abbreviation T3E is used in many figures but is not explained. Please add the acronym "type III effector" to the figure legend of Figure 2. 3. Figure 3C -the authors should add the size marker of the blots. 4. Line 175, add the calcium concentration tested. 5. Line 155 -the authors present the calcium response as a hypothesis, although it was already reported. I would recommend rephrasing the sentence to "previously reported." 6. Bordetella should be italic throughout the manuscript (for example, lines 76, 148, etc.).

Response to reviewers of the manuscript Spectrum04112-22:
Reviewer #1: Recommendation: While the results of this study are exciting and support the notion that BopN is involved in BteA secretion and its ability to translocate into the host cells' cytoplasm, they do not support the classical function of a gatekeeper. Therefore, I recommend reconsidering the presentation of BopN as the gatekeeper protein as well as addressing the following concerns. 2. Figure 2C -The presentation of BopN amounts as a fraction of that expressed in a specific culture (BopNrep grown with 2 mM calcium) is not ideal. As it looks like the growing conditions affect the expression of BopNrep, it should be presented as a fraction of each sample (how much is secreted vs. intracellular). Also, please change the color of the black bars to a lighter color so that the error bars will be visible.

A2:
The reviewer is correct that normalizing to protein levels in the specific culture may simplify the presentation and the recommendation was followed. However, the price for this type of normalization is the loss of the information on protein levels under specific culture conditions and/or during T3SS activation status. Indeed, the data show a downregulation of BopN levels in the T3SS-deficient ΔbscN strain that is independent of 2 mM calcium, as shown now in Fig. 3B. Furthermore, an increase in total BteA levels under secretion-promoting conditions with no effect on intracellular BteA levels was observed, as now shown in Fig. 2C. Therefore, on top of the performed normalization, shown in Fig. 3C, also absolute BopN levels from a representative experiment are now shown as the new Fig. 3B. Further, we replaced the color of the black bars with a lighter color to visualize the error bars.
3. In Fig. 3B -the normalization of BteA secretion/expression is normalized according to the levels in the culture grown without calcium, while in Fig. 2B, it is normalized to a culture grown in 2 mM calcium. It is not clear why. Since this normalization method is not ideal, as discussed in my previous point, I think it should be presented as a ratio between each sample's secreted and intracellular levels.
A3: As outlined above, the new Fig. 2 (previous Fig. 3) was modified according to the suggestion of the reviewer and data from a representative experiment were included. Figure 2D-E -to have a complete picture of the localization BopN under high and low calcium concentrations, the authors should add the BscD localization for zero calcium and its merged image (so the two panels will become one).

4.
A4: The suggestion was followed and the image of BscD/BopN localization for zero calcium was included into new Fig. 4 along with the corresponding fields from previous Fig. 2E. In the new Fig. 3 (previous Fig. 2), we show the merged images without the BscD protein to document the localization of the BopN protein in respect to the bacterial envelope, which is better seen in these images. Figure Fig. 3C) was performed to examine whether ablation of BopN production affects the secretion of the Bsp22 and BopB/D structural subunits that are important for function of the injectisome. As shown, the secretion of the Bsp22 and BopB/D was not affected and we thus believe that use of higher amounts of these proteins accumulated in stationary culture for improved detection on the immunoblot is acceptable.

5.
6. Figure 4C -please add a positive control (WT strain or delta BopN strain) to show that NF-kB activity is enhanced under these conditions.

A6:
The suggestion of the reviewer could not be followed because under the conditions used, the BteA protein produced by wild-type and ΔbopN strains exerts such a potent cytotoxic action on A549 cells that the cells lyze before activation of the NF-kB pathway can be assessed by the used SEAP reporter system. In contrast, the ΔbteA strains are not cytotoxic and activation the NF-kB pathway in infected A549 cells could be assessed, showing that injection of BopN into cells does not inhibit NF-kB activation (now Fig. 5C). These data are now mentioned in the manuscript on lines 305-308.

The hierarchy of type III secretion should be explained in the introduction and not in
the discussion (lines 333-343), as it is critical for understanding the work described in the manuscript.
A7: The suggestion was followed and the description of the hierarchy of the type III secretion is now included in the Introduction. In fact, replacement of the T122 residue of B. bronciseptica BopN by a proline residue in BopN of B. pertussis may potentially be among the reasons of the reduced functionality of the T3SS in B. pertussis, which will be subject of further examination.
However, here we used the B. pertussis BopN structure only to reveal that Bordetella BopN has the characteristic three X-bundle domain architecture of T3SS gatekeepers, which prompted us to explore its gatekeeper function.
13. The design of BopN includes a flag-tag, which allows a direct measurement of BopN expression and secretion using western blot. Therefore, the authors should add this method to figures 2 and 3.

A13:
The suggestion of the reviewer was followed only in part, as we have used mouse polyclonal antibodies raised against BopN for immunoblot detection of BopN, now shown in the new Fig. 3B. This method of detection is specific, as no signal is detected in the ΔbopN strain. Indeed, BopN detection by immunoblot was already included in the previous version of the Fig. 3C, now Fig. 2E.
14. The authors should demonstrate that the BopN-HiBit-3xFlag-SPOT is a functional protein (preferably by showing that the labeled protein can complement the infection activity of the delta BopN mutant strain).

A14:
The data requested by the reviewer have been shown in another way in the initial manuscript by demonstrating that fusion of the HiBit-3xFlag-SPOT tag to BopN did not affect the BteA-dependent cytotoxicity of B. bronchiseptica, which would have been reduced upon loss of BopN function. This is now shown in Suppl. Fig. 3A, documenting that the bopN rep reporter producing strain elicits the same T3SS-dependent cytotoxicity as the wild-type B. bronchiseptica RB50 strain. The issue is now also clarified in the manuscript.
15. According to the Dali algorithm, "strong matches" have a Z-score higher than n/100-4 (where n is the number of amino acids). When taking ~280 aa of the BopN, only a z-score above 24 is meaningful. However, the authors report Z-scores of 12.5-20.3 but suggest structural homology. This point should be discussed when concluding the similarity between BopN and other T3SS gatekeepers.

A15:
The reviewer is correct that DALI defines a "strong match" between structures as an empirically determined Z-score higher than (n/10)-4, where n is the number of residues in the queried structure. Funnily enough, DALI also defines "significant similarities" to have a Z-score above 2. In any case, the Z-scores of BopN match well with the previously reported Z-scores within the SctW gatekeeper protein family. For example, DALI search of SepL of E. coli yields Z-scores between 11.9 and 7.6 for other gatekeeper proteins (Burkinshaw BJ et al. 2015, PMID: 26457522). The issue is now discussed on lines 161-168.

A1:
We apologize for the lack of consistency. The term has been unified to secretion restrictive conditions.
2. The abbreviation T3E is used in many figures but is not explained. Please add the acronym "type III effector" to the figure legend of Figure 2.
A2: The acronym has been added to the figure legend of Figure 2.
3. Figure 3C -the authors should add the size marker of the blots.

A3:
We politely disagree with the reviewer, as only a section of the immunoblots comprising the detected proteins are shown in Fig. 3C 1. In the abstract, line 30: HU after parepetrussi is probably a typo A1: We thank reviewer, the typo has been corrected.
2. Abstract line 32, and also introduction line 77: why "so-called"? These are effectors, and have been shown to be secreted in the host.

A2:
The words "so-called" have been removed.
3. Line 96: While I commend the use to the standard T3SS nomenclature throughout the manuscript, it would probably help the reader to mention the name of the orthologue in other species here (SepD/L in EPEC, MxiC in Shigella...)

A3:
The suggestion is appreciated, and the introduction has been revised to provide more information on gatekeeper function, including names of orthologues in other species. Specifically, we have highlighted information on Yersinia YopN, while also other orthologues are also briefly mentioned.
4. Line 145: What is the sequence identity between homologous proteins? They are indicated in table 2, which should be refereed to here -but is the sequence identity for the whole protein, or just the aligned residues?
A4: The sequence identity of other BopN and YopN protein is now included in the introduction. The sequence identity in Table 2 represents the identity of the aligned residues. The issue is now clarified in the manuscript. Thank you for submitting your revised manuscript to Microbiology Spectrum. It has been sent back to the two original referees. Both acknowledge that you properly answered their intiial comments and recommend publication. Reviewer #1 however suggests to add protein markers on the western-blots, and I agree with this remark. Per ASM style, it is required to have at least two weight markers on cropped blots. When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed instructions on submitting your revised paper are below.

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Response to reviewers of the manuscript Spectrum04112-22R1 (BopN is a gatekeeper of the Bordetella type III secretion system) Reviewer #1: Q: The only thing left is my request (as a minor comment) to add a size marker to the bolts (presented in figures 2 and 3). In their rebuttal letter, the authors explain that since they show only a small section of the blots, it makes little sense to present a size marker. I beg to differ and argue that this is an elementary standard to show that the bands correspond to the expected protein size. I don't expect them to include the entire Mw markers (and I'm sorry I wasn't clear about it), but only one or two markers close to the bands. If the authors worry that it will clutter their presentation, I suggest they add the complete bolts with the full Mw markers as supplementary material.
A: We thank the reviewer for the explanation, and we have followed the recommendation. However, due to technical problems, the Western blots previously shown were not saved with the size marker in the correct format (overlay of the chemiluminescence signal with the brightfield view of the pre-stained marker).
Therefore, we included a new set of Western blots in Figure 2 and Figure Figure S4 (previous Figure S3).

Reviewer #2:
Q: The authors have suitably addressed the comments from both reviewers, and I am happy to recommend publication of this revised manuscript in Spectrum.