An Atypical F-Actin Capping Protein Modulates Cytoskeleton Behaviors Crucial for Trichomonas vaginalis Colonization

ABSTRACT Cytoadherence and migration are crucial for pathogens to establish colonization in the host. In contrast to a nonadherent isolate of Trichomonas vaginalis, an adherent one expresses more actin-related machinery proteins with more active flagellate-amoeboid morphogenesis, amoeba migration, and cytoadherence, activities that were abrogated by an actin assembly blocker. By immunoprecipitation coupled with label-free quantitative proteomics, an F-actin capping protein (T. vaginalis F-actin capping protein subunit α [TvFACPα]) was identified from the actin-centric interactome. His-TvFACPα was detected at the barbed end of a growing F-actin filament, which inhibited elongation and possessed atypical activity in binding G-actin in in vitro assays. TvFACPα partially colocalized with F-actin at the parasite pseudopod protrusion and formed a protein complex with α-actin through its C-terminal domain. Meanwhile, TvFACPα overexpression suppressed F-actin polymerization, amoeboid morphogenesis, and cytoadherence in this parasite. Ser2 phosphorylation of TvFACPα enriched in the amoeboid stage of adhered trophozoites was reduced by a casein kinase II (CKII) inhibitor. Site-directed mutagenesis and CKII inhibitor treatment revealed that Ser2 phosphorylation acts as a switching signal to alter TvFACPα actin-binding activity and the consequent actin cytoskeleton behaviors. Through CKII signaling, TvFACPα also controls the conversion of adherent trophozoites from amoeboid migration to the flagellate form with axonemal motility. Together, CKII-dependent Ser2 phosphorylation regulates TvFACPα binding to actin to fine-tune cytoskeleton dynamics and drive crucial behaviors underlying host colonization by T. vaginalis. IMPORTANCE Trichomoniasis is one of the most prevalent nonviral sexually transmitted diseases. T. vaginalis cytoadherence to urogenital epithelium cells is the first step in the colonization of the host. However, studies on the mechanisms of cytoadherence have focused mainly on the role of adhesion molecules, and their effects are limited when analyzed by loss- or gain-of-function assays. This study proposes an extra pathway in which the actin cytoskeleton mediated by a capping protein α-subunit may play roles in parasite morphogenesis, cytoadherence, and motility, which are crucial for colonization. Once the origin of the cytoskeleton dynamics could be manipulated, the consequent activities would be controlled as well. This mechanism may provide new potential therapeutic targets to impair this parasite infection and relieve the increasing impact of drug resistance on clinical and public health.

The authors must recraft the figures to optimize viewing. All text should be the same size and font, the images and graphs should be easily visible without having to zoom in and out. The color of labels should be considered in terms of contrast. Also, some figure panels (such as Figure 1C) are not adjusted to optimize contrast and are difficult to see.
An example of how the figures could be revised is suggested here for figures 1 and 2. This is just a suggestion, but the concepts may help the authors. The impact of this work is lessened if it is hard to see and interpret the data. Figure 1: Panel A: by showing both CFSE and the merge with the phase image of the host cells, the 6 panels are displayed at a small size, along with the quantification. I suggest just showing the merged panels and putting an inset I or B for input or binding so that the size of the images can be increased. The scale bar does not have to include the text of its size -this can go in the legend. The height of the quantifying graph should correspond to the height of the image panels and if the text reads percent bound, it will fit on one line as the Y axis label. Panel B scale bar size can be put in the legend and omitted from the figure to improve legibility. Again, the height of the quantifying graph should correspond to the height of the image panels. The Y axis label should be simplified to fit in a legible font size. How about percent amoeba. Panel C should be adjusted to improve contrast and the labels for time can simply be numbers with the legend describing the timing. Again, the scale bar size can go in the legend. The graphs in panels A and C are not legible at the same scale as the blots. The labeling of the blots could be streamlined so that the graphs could be displayed at a larger size. One way to simplify everything is to make panels A and C one merged panel. This means that the labels only get shown once. The panels should be enlarged to occupy the full size of the figure -maximize your information! The quantification of colocalization (plot profile analysi) shown in D is illegible, particularly the inset. Consider if the graph should be a supplemental figure. Quantification in panel E is nearly illegible. Again, parts of panels (and all panels) should be presented at a size that can be viewed legibly. Again, for all cytoadherence assays, I would show the merge only so that the panels can be larger.
Reviewer #2 (Comments for the Author): The authors of this paper identified a role for a parasite protein, TvFACPα, in parasite colonization via its effects on the parasite's cytoskeleton. This was accomplished by comparing a more adherent strain of the parasite with a less adherent one in a number of assays that looked at parasite morphology, adherence, motility and finally comparative proteomics. They then go on to characterize the ability of TvFACPα on actin assembly in an in vitro assay and in the parasite using both the WT and a mutated version that lacks a putative actin binding domain. Finally, the authors demonstrate that TvFACPα activity is regulated by CKII phosphorylation using chemical inhibitors as well as hypo-and hyperphosphorylation mutants of TvFACPα. The experimental data in this paper is largely described well and experimentally rigorous but could use some minor revisions to strengthen their argument.
Strengths of the Paper: Experiments are largely well described and thorough.
Comparison a more adherent and less adherent strain of the parasites is a clever way to identify novel parasite colonization factors.
Multiple transgenic parasites are used which strengthen the argument and model the authors propose.
Major comments and concerns: Figure 1. In panel B the authors are measuring amoeboid forms of the parasite. The criteria by which parasites are scored as amoeboid is not clear in the methods and materials section. While most readers can probably appreciate the difference by eye (see figure B images) it is still unclear how an edge case would be assessed. If this is a purely qualitative assessment it should be stated as such in the methods. If there is a quantitative assessment to define amoeboid status (such as area or roundness) this should be stated in the methods.  . While it may be unlikely that the inhibitor, LatB, is killing or adversely affecting the parasites given the short time frame (2-hour pretreatment) the authors should still demonstrate that LatB is not cytotoxic to the parasites as dead or dying parasite could affect the outcome of the results. The results would be strengthened with a parasite viability assay to ensure any cytotoxic effects LatB might have on the parasite are not affecting the outcome of the results in figure 3.   Staff Comments:

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Kai-Hsuan Wang, Jing-Yang Chang1, Fu-An Li, Kuan-Yi Wu, Shu-Hao Hsu, Yen-Ju Chen, Tse-Ling Chu, Jessica Lin, and Hong-Ming Hsu, "An atypical F-actin capping protein modulates cytoskeleton behaviors crucial to Trichomonas vaginalis colonization" Summary: This manuscript investigates the role of TvFACPα, the Trichomonas homolog of the actin binding protein CAPZα (capping protein) in T. vaginalis cytoadherence, which is critical to pathogenesis. Actin, TvFACPα and adhesion were characterized in adherent and non-adherent isolates of T. vaginalis. Upon co-culture of T. vaginalis with a vaginal epithelial cell monolayer, trophozoites of the adherent isolate but not those of the nonadherent isolate convert from a flagellated form into an amoeboid form within 10 minutes. The amoeboid form moves by a crawling motility and latrunculin treatment reduces cytoadherence. These data suggest a role for actin in adhesion and amoeboid motility. However, since α-actin and α-actinin protein levels do not change between the flagellated and amoeboid forms within the adherent isolate, it is likely that changes in cytoskeletal organization are modulated by post translational modifications. Actin associated proteins were identified by co-immunoprecipitation and mass spectrometry. One of the hits was TvFACPα. In vitro biochemical assays indicate that TvFACPα inhibits pyrene actin polymerization, like CPs in metazoan organisms. However, TvFACPα has an atypical G-actin binding activity, which reduces actin assembly by sequestering subunits independent of barbed end binding/filament extension. Parasite based assays employed quantification of pelleted (polymerized) actin under conditions of over-expression and/or treatment with the CKII inhibitor TBB to investigate the predicted CKII phosphorylation site at TvFACPα S2. Overexpression of wild type or S2A (non-phosphorylated) TvFACPα reduces pelleted (F) actin but increases the co-sedimentation (association) of TvFACPα with F-actin. Expression of a phosphomimetic S2D TvFACPα does not change pelleted actin relative to the untransfected line but does show lower co-sedimentation (association). These and other experiments are consistent with the conclusion that CKII-dependent Ser2 phosphorylation causes TvFACPα and α-actin to dissociate. Both release of actin subunits and uncapping of the barbed ends of F-actin stimulate actin polymerization. Overexpression of wild type or S2A (nonphosphorylated) TvFACPα or TBB treatment reduces amoeboid morphogenesis, suggesting a role for this protein in the transition to a cytoadherent form.
Overall feedback: This MS is a data dense characterization of capping protein in T. vaginalis which shows that it plays key roles in morphological transitions and cytoadherence. The data and conclusions are exciting but many of the key findings get lost in the density and display of the data. Specifically, the figures contain panels that do not work well together because the labels, size and formatting are inefficient with text that is too small or large and patches of unused blank space due to the special placement of panels. While the authors do not need to do additional experiments, they should think carefully about what data is included so that the conclusions are evident to readers.
The authors must recraft the figures to optimize viewing. All text should be the same size and font, the images and graphs should be easily visible without having to zoom in and out. The color of labels should be considered in terms of contrast. Also, some figure panels (such as Figure 1C) are not adjusted to optimize contrast and are difficult to see.
An example of how the figures could be revised is suggested here for figures 1 and 2. This is just a suggestion, but the concepts may help the authors. The impact of this work is lessened if it is hard to see and interpret the data.

Figure 1:
Panel A: by showing both CFSE and the merge with the phase image of the host cells, the 6 panels are displayed at a small size, along with the quantification. I suggest just showing the merged panels and putting an inset I or B for input or binding so that the size of the images can be increased. The scale bar does not have to include the text of its size -this can go in the legend. The height of the quantifying graph should correspond to the height of the image panels and if the text reads percent bound, it will fit on one line as the Y axis label.
Panel B scale bar size can be put in the legend and omitted from the figure to improve legibility. Again, the height of the quantifying graph should correspond to the height of the image panels. The Y axis label should be simplified to fit in a legible font size. How about percent amoeba.
Panel C should be adjusted to improve contrast and the labels for time can simply be numbers with the legend describing the timing. Again, the scale bar size can go in the legend. The graphs in panels A and C are not legible at the same scale as the blots. The labeling of the blots could be streamlined so that the graphs could be displayed at a larger size. One way to simplify everything is to make panels A and C one merged panel. This means that the labels only get shown once. The panels should be enlarged to occupy the full size of the figuremaximize your information! The quantification of colocalization (plot profile analysi) shown in D is illegible, particularly the inset. Consider if the graph should be a supplemental figure. Quantification in panel E is nearly illegible. Again, parts of panels (and all panels) should be presented at a size that can be viewed legibly. Again, for all cytoadherence assays, I would show the merge only so that the panels can be larger.
The authors of this paper identified a role for a parasite protein, TvFACPα, in parasite colonization via its effects on the parasite's cytoskeleton. This was accomplished by comparing a more adherent strain of the parasite with a less adherent one in a number of assays that looked at parasite morphology, adherence, motility and finally comparative proteomics. They then go on to characterize the ability of TvFACPα on actin assembly in an in vitro assay and in the parasite using both the WT and a mutated version that lacks a putative actin binding domain. Finally, the authors demonstrate that TvFACPα activity is regulated by CKII phosphorylation through the use of chemical inhibitors as well as hypo-and hyperphosphorylation mutants of TvFACPα. The experimental data in this paper is largely described well and experimentally rigorous, but could use some minor revisions to strengthen their argument. However, the discussion section is very difficult to read and disjointed.

Strengths of the Paper:
Experiments are largely well described and thorough.
Comparison a more adherent and less adherent strain of the parasites is a clever way to identify novel parasite colonization factors Multiple transgenic parasites are used which strengthen the argument and model the authors propose.
Major comments and concerns:

Dear reviewers:
We are grateful for the valuable comments to make this manuscript more readable and convincing. The manuscript has been modified as per the reviewers' suggestions, and the related comments are responded to point by point as described below. Due to the line number shifting after PDF conversion by the submission system, you may track the issues in our attached Marked-Up manuscript PDF file.

Reviewer #1 (Comments for the Author):
This MS is a data dense characterization of capping protein in T. vaginalis which shows that it plays key roles in morphological transitions and cytoadherence. The data and conclusions are exciting but many of the key findings get lost in the density and display of the data. Specifically, the figures contain panels that do not work well together because the labels, size and formatting are inefficient with text that is too small or large and patches of unused blank space due to the special placement of panels. While the authors do not need to do additional experiments, they should think carefully about what data is included so that the conclusions are evident to readers.
The authors must recraft the figures to optimize viewing. All text should be the same size and font, the images and graphs should be easily visible without having to zoom in and out. The color of labels should be considered in terms of contrast. Also, some figure panels (such as Figure 1C) are not adjusted to optimize contrast and are difficult to see.

Re:
We are grateful for the valuable comments. In this revised manuscript, we rearranged the figure position and optimized label size and font for better readability. Also, the image contrast of Figure   1C is adjusted to be clearly visible.
An example of how the figures could be revised is suggested here for figures 1 and 2. This is just a suggestion, but the concepts may help the authors. The impact of this work is lessened if it is hard to see and interpret the data. Re: Cytoadherence assay only shows the merged images, magnified for better readability. The size texts are removed from the image scale bars and stated in the figure legends. Also, we adjust the height of quantifying graphs corresponding to that of the image panel and simplify the label text on the Y axis.
Panel B scale bar size can be put in the legend and omitted from the figure to improve legibility.
Again, the height of the quantifying graph should correspond to the height of the image panels. The Y axis label should be simplified to fit in a legible font size. How about percent amoeba.

Re:
The size text is removed from the image scale bar and described in the figure legend. The quantifying graph is adjusted to the height corresponding to the image panel. The Y-axis label is simplified with a legible font size.
Panel C should be adjusted to improve contrast and the labels for time can simply be numbers with the legend describing the timing. Again, the scale bar size can go in the legend.

Re:
The image contrast of panel C is adjusted to be clearly visible ( Figure 1C).

Figure 2:
The graphs in panels A and C are not legible at the same scale as the blots. The labeling of the blots could be streamlined so that the graphs could be displayed at a larger size. One way to simplify everything is to make panels A and C one merged panel. This means that the labels only get shown once. The panels should be enlarged to occupy the full size of the figure -maximize your information! The quantification of colocalization (plot profile analysi) shown in D is illegible, particularly the inset. Consider if the graph should be a supplemental figure. Quantification in panel E is nearly illegible. Again, parts of panels (and all panels) should be presented at a size that can be viewed legibly. Figure  Again, for all cytoadherence assays, I would show the merge only so that the panels can be larger.

Re:
The merged images were used for all cytoadherence assays of this revised manuscript ( Figures   1A, 3D, 8C, and 8D).
Reviewer #2 (Comments for the Author): The authors of this paper identified a role for a parasite protein, TvFACPα, in parasite colonization via its effects on the parasite's cytoskeleton. This was accomplished by comparing a more adherent strain of the parasite with a less adherent one in a number of assays that looked at parasite morphology, adherence, motility and finally comparative proteomics. They then go on to characterize the ability of TvFACPα on actin assembly in an in vitro assay and in the parasite using both the WT and a mutated version that lacks a putative actin binding domain. Finally, the authors demonstrate that TvFACPα activity is regulated by CKII phosphorylation using chemical inhibitors as well as hypoand hyperphosphorylation mutants of TvFACPα. The experimental data in this paper is largely described well and experimentally rigorous but could use some minor revisions to strengthen their argument.
Strengths of the Paper: Experiments are largely well described and thorough.
Comparison a more adherent and less adherent strain of the parasites is a clever way to identify novel parasite colonization factors.
Multiple transgenic parasites are used which strengthen the argument and model the authors propose.
Major comments and concerns:   The results would be strengthened with a parasite viability assay to ensure any cytotoxic effects LatB might have on the parasite are not affecting the outcome of the results in figure 3.

Re:
The cell viability assay was performed to demonstrate the little effects of LatB or TBB on the parasite's vitality. The procedure was stated in the revised Materials and Methods section (line 535), the data were shown in Supplementary Figures 2A and 11C, and the results were described in lines 159 and 278 of Marked-Up Manuscript PDF file.  The cell viability assay was tested to demonstrate the little effect of LatB or TBB on the parasite's viability. The procedure was described in the revised Materials and Methods section (line 535 of Marked-Up Manuscript.docx), the data were shown in Supplementary Figures 2A and 11C, and the results were written in lines 159 and 278 of Marked-Up Manuscript PDF file.  Staff Comments:

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.
• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.