A novel glideosome-associated protein S14 coordinates sporozoite gliding motility and infectivity in mosquito and mammalian hosts
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Abstract
Plasmodium sporozoites are the infective forms of the malaria parasite in the vertebrate host. Gliding motility allows sporozoites to migrate and invade the salivary gland and hepatocytes. Invasion is powered by an actin-myosin motor complex linked to glideosome. However, the gliding complex and the role of several glideosome-associated proteins (GAPs) are poorly understood. In silico analysis of a novel protein, S14, which is uniquely upregulated in salivary gland sporozoites, suggested its association with glideosome-associated proteins. We confirmed S14 expression in sporozoites using real-time PCR. Further, the S14 gene was endogenously tagged with 3XHA-mCherry to study expression and localization. We found its expression and localization on the inner membrane of sporozoites. By targeted gene deletion, we demonstrate that S14 is essential for sporozoite gliding motility, salivary gland, and hepatocyte invasion. The gliding and invasion-deficient S14 KO sporozoites showed normal expression and organization of IMC and surface proteins. Using in silico and the yeast two-hybrid system, we showed the interaction of S14 with the glideosome-associated proteins GAP45 and MTIP. Together, our data show that S14 is a glideosome-associated protein and plays an essential role in sporozoite gliding motility, which is critical for the invasion of the salivary gland, hepatocyte, and malaria transmission.
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Reply to the reviewers
Manuscript number: RC-2023-02157
Corresponding author(s): Satish, Mishra
1. General Statements [optional]
We thank the editor and reviewers for their helpful comments. We have successfully addressed most of the comments. We are performing some additional experiments as suggested by the reviewers and will be included if considered further. We attempted to pulldown the S14 interacting partner using anti-mCherry antibody from S14-3XHA-mCherry transgenic sporozoites and then further tried to identify interactome using mass spectrometry but failed. So, accordingly, we have toned down the conclusion.
The point-by-point response to the reviewer’s comments is …
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Manuscript number: RC-2023-02157
Corresponding author(s): Satish, Mishra
1. General Statements [optional]
We thank the editor and reviewers for their helpful comments. We have successfully addressed most of the comments. We are performing some additional experiments as suggested by the reviewers and will be included if considered further. We attempted to pulldown the S14 interacting partner using anti-mCherry antibody from S14-3XHA-mCherry transgenic sporozoites and then further tried to identify interactome using mass spectrometry but failed. So, accordingly, we have toned down the conclusion.
The point-by-point response to the reviewer’s comments is given as follows.
2. Description of the planned revisions
Reviewer #1:
Figure 1F You have not formally shown that this signal corresponds to palmitoylated S14. Could be heavy chain. Response: The possibility of a heavy chain is negligible because we have used sporozoite samples and probed it with an anti-rabbit antibody conjugated to HRP. Also, the size of the S14 bands does not correspond to heavy chain. However, we have toned down the conclusion. Currently, we are performing the depalmitoylation experiment, and data will be included in the next round of revision.
Reviewer #2
Line 149: To definitively state S14 is a membrane protein, biochemical assays proving such should be performed. (or perhaps genetic mutation of the predicted palmitoylation site?) Otherwise, this should be rephrased. Response: We are performing the depalmitoylation assay, and the data will be included during the second round of revision. However, we have rephrased the sentence in the current version of the manuscript.
Lines 257-258: for yeast 2-hybrid, the controls of expressing S14, GAP45 and MTIP together with control proteins where no interaction would be predicted are absent. Response: We are performing experiments with additional controls, and data will be included in the next round of revision.
Reviewer #3
Conclusions that S14 knockout does not impact the expression and organization of two surface proteins, CSP and TRAP, and two IMC rely on a qualitative analysis only. However, quantitative analysis to support their observations is missing. Response: We are quantifying the IFA images and data will be included in the next round of revision.
3. Description of the revisions that have already been incorporated in the transferred manuscript
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Summary: The authors have identified a sporozoite gliding motility protein through bioinformatic analysis. From the main text I do not know how, or what bioinformatic analysis was performed, in order to focus on this protein which is called S14. The authors then go on to tag the protein, produce a KO and show its involvement in gliding motility. The KO shows that parasites lacking S14 fail to invade the mosquito salivary glands. This is due to a motility defect. Y2H and docking studies are used to define an interaction with MTIP and GAP45, two known components of the glideosome. Response: We identified this gene from the Kaiser et al., 2004 paper (DOI: 10.1046/j.1365-2958.2003.03909.x). The S14 was found to be highly upregulated in salivary gland sporozoites but lacked signal sequence and transmembrane domain. Next, we looked into other sporozoite proteins lacking signal sequence and transmembrane domain and found several gliding-associated proteins with similar properties. By using the guilt-by-association principle (DOI: 10.1186/gb-2009-10-4-104), we studied the following properties of existing glideosome components along with S14: (1) Classical pathway secretion using the signal peptide (SignalP, https://services.healthtech.dtu.dk/services/SignalP-5.0) (http://dx.doi.org/10.1016/j.jmb.2004.05.028). (2) Nonclassical pathway secretion (SecretomeP , https://services.healthtech.dtu.dk/services/SecretomeP-1.0/) (10.1093/protein/gzh037). (3) Presence of transmembrane domains (TMHMM , https://services.healthtech.dtu.dk/services/TMHMM-2.0/) (10.1006/jmbi.2000.4315). (4) Presence of a potential palmitoylation site (CSS-Palm, http://bioinformatics.lcd-ustc.org/css_palm) (Ren et al, 2008). This is a similar association prediction method as employed by the STRING database. However, mentioning that we identified a gliding motility protein by bioinformatic analysis was wrong, and we modified the sentence.
Major comments: The paper is sometimes hard to follow and lacks clarity. The reason: important information is omitted, or explained at the end of a section rather than at first mention; experimental details that are of essence need to be mentioned or explained in the main text; there is ample use of the word 'bioinformatic' without explaining what kind of analysis was performed in the main text. I cite from the abstract: 'In silico analysis of a novel protein, S14, which is uniquely upregulated in salivary gland sporozoites, suggested its association with glideosome-associated proteins.' I cite from the introduction: 'A study comparing transcriptome differences between sporozoites and merozoites using suppressive subtraction hybridization found several genes highly upregulated in sporozoites and named them 'S' genes (Kaiser et al, 2004). We narrowed it down to a candidate named S14, which lacked signal peptide and transmembrane domains.' From reading the main text, I do not know why Plasmodium berghei S14 was chosen in this manuscript. S14 is one of 25 transcripts identified by Kappe et al in Plasmodium yoelii (https://doi.org/10.1046/j.1365-2958.2003.03909.x) to be upregulated in sporozoites. The material and methods section does not explain either why S14 was chosen. Perhaps the authors could update Figure 2 from Kappe et al with the most recent annotations from plasmodb. Response: We have edited the manuscript for clarity and mentioned the name of the bioinformatic analysis performed. We chose S14 from Kaiser et al., 2004 (https://doi.org/10.1046/j.1365-2958.2003.03909.x) that identified transcripts in P. yoelii. We work on the rodent malaria parasite P .berghei and validated S14 transcripts by qPCR which showed its upregulation in sporozoites.
Rodent malaria parasites P. berghei and P. yoelii have been used extensively as models of human malaria. Both species have been widely used in studies on the biology of Plasmodium sporozoites and liver stages due to the availability of efficient reverse genetics technologies, and the ability to analyze these parasites throughout the life cycle stages have made these two species the preferred models for the analysis of Plasmodium gene function. A genetic screen and phenotype analysis were performed in P. berghei (DOI: 10.1016/j.cell.2017.06.030 and DOI: 10.1016/j.cell.2019.10.030) that makes in-depth characterization easier due to the availability of reagents and preliminary gene-phenotype like its dispensability in the blood. As suggested by this reviewer, we have updated the most recent annotations from PlasmoDB.
Reproducibility: None of the main Figures or Figure legends define ' N = '. For example I cite: 'The S14 KO clonal lines were first analyzed for asexual blood-stage propagation, and for this, 200 µl of iRBCs with 0.2% parasitemia was intravenously injected into a group of mice.' There are 2 mentions of 'N=' in the supplementary figures. I have not found any others.
I'm not sure what the convention is. Should unpublished data for this gene (PBANKA_0605900) found in pberghei.eu (a database for mutant berghei parasites) be cited? After all it confirms their findings.
The authors need to use more recent references for some of their statements; see some comments below. __Response: __We have mentioned N in the figures legends of the revised manuscript and also mentioned the unpublished data of RMGM. We have also added recent references in the revised manuscript.
Minor comments:
line 1-2 Add the Plasmodium species of this study.
Response: Added.
abstract Which species do you work with?
Response: We have mentioned P. berghei in the abstract of the revised manuscript.
29 mosquito salivary glands and human host hepatocytes
Response: Corrected.
30 to the glideosome, a protein complex containing [...]
Response: Corrected.
32-33 What kind of in silico analysis suggested S14 is part of the glideosome? S14 is not uniquely upregulated; there are other S-type genes identified by Kappe and Matuschewski. 25 I believe.
Response: Mentioning that in silico analysis suggested S14 is part of the glideosome was a wrong statement, and we have modified the sentence for clarity in the revised manuscript.
32 Please point out he species were S genes were identified. SGS of which species?
Response: The S genes were identified in the transcriptomic study of Plasmodium yoelii.
34 expression: change to transcription
Response: Changed.
39 What kind of in silico analysis was used here? and therefore malaria transmission
__Response: __In silico, protein-protein docking interaction analysis was used.
55 A single zygote transforms into a single ookinete, which establishes a single oocyst, which in turn can produce thousands of midgut sporozoites. Please correct the life cycle passage.
Response: Corrected. located or anchored in the IMC? And located between the IMC and plasma membrane?
Response: Glideosome is located between the plasma membrane and IMC, and the same has been corrected in the revised manuscript.
61-63 Refer to Table S1 and its contents here 64 Name the known GAPs. Response: Done.
65-67 Which transmembrane domain proteins? Please add more recent references than King 1988.
Response: We have added TRAP as a transmembrane domain protein and updated the reference.
71-72 TRAP was the first protein found to be ...
Response: Corrected.
74-76 Add additional, more recent references: for example search Frischknecht and TRAP
Response: Added.
76 S6 (TREP) is also [...]
Response: Done.
88 Some of these proteins are also expressed in ookinetes.
Response: Corrected.
89-91 The sentence needs a verb.
Response: Added.
88-96 Please add some more recent glideosome papers. After 2013.
Response: Added.
91 Why do you call it a peripheral protein?
Response: Because the GAP45 was detected at the periphery of the merozoite in P. falciparum. As there are no such reports in sporozoites hence we have removed peripheral in the revised manuscript.
91-93 There are more recent citations for GAP45 and GAP50. Response: We have added recent citations.
96 Insert a reference here.
Response: Added.
99 Please define the gliding-associated proteins. What are they? Aren't there papers on GAP40, 45 and 50? DOI: 10.1016/j.chom.2010.09.002
Response: Done.
99 .... What prompted you to identify a novel GAP? And why is S14 classified as a GAP?
Response: This was a wrong statement, which we removed in the revised manuscript.
99-102 What kind of bioinformatic study? Why was S14 chosen? Please outline how you ended up with S14. Any other proteins that came out of the bioinformatic screen from the list of S genes?
Response: We identified S14 from the Kaiser et al., 2004 paper and analyzed its properties using the “guilt-by-association” principle. The analysis showed that S14 had properties similar to GAP45 and MTIP. The S14 upregulation in sporozoites and its properties similar to known GAPs, we were prompted to study this gene's function.
How many proteins were identified in the screen for sporozoite upregulated proteins by Kappe and Matuschewski?
Response: 25 genes were identified in that paper, including the two characterized genes CSP and TRAP during that study.
102-103 Define the nonclassical secretion pathway. Please reference GAP45 and GAP50 data for the nonclassical pathway.
Response: When proteins are secreted out of the cytosol without predictable or known signal sequences or secretory motifs are classified as non-classically secreted proteins, and the pathway is called a non-classical protein secretory pathway. References: https://doi.org/10.1371/journal.pone.0125191; https://doi.org/10.1016/S0171-9335(99)80097-1; doi: 10.3389/fmicb.2016.00194
105 Please add P. berghei to the title, the abstract, the introduction.
Response: Added.
111 The results section does not outline what bioinformatic analysis was used
Response: The guilt-by-association principle was used, and it is outlined in the revised manuscript.
112-114 Please specify the exact number of upregulated in sporozoites genes. I think it was 25. And add the species the study was performed in. Why did you choose the Kappe study but not the uis genes from berghei?
Response: It was 25, and the species was P. yoellli. The domains of all 25 proteins are shown in Figure 2 of Kappe study. It intrigued us after having a glance at it. Later, we confirmed the upregulation of S14 transcripts in P. berghei sporozoites and chose to study the function of this gene.
114-115 How did you narrow it down to S14? The Kappe paper lists 25 S-type genes from P. yoelii.
Response: The domains of all 25 proteins are shown in the Kappe study. Two proteins, S14 and S15, lack signal sequence and transmembrane domain, which intrigued us after glancing at them. We chose S14 because its microarray induction is higher compared to S15.
118 Plasmodia is not the plural for a group of different Plasmodium species. Use: [...] conserved among Plasmodium spp.
Response: Corrected.
118-119 Which proteins did you analyze? And how did you analyze them? Where is the data for this analysis? Outline the amino acids that predict palmitoylation? The nonclassical pathway?
Response: The proteins we analyzed are given in Table S1. We analyzed them by the guilt-by-association principle. The data for this analysis is shown in Table S1. The amino acids predicted to be palmitoylated are C59 and C228 (S14), C5 (GAP45), C8 and C5 (MTIP). Non-classical pathway secretion was predicted by SecretomeP ( 10.1093/protein/gzh037).
119-122 Here: do you mean S14 has similar properties as GAP 45 and GAP50? Define the nonclassical pathway? How do you know S14 is in the IMC?
Response: The similar properties of S14 and GAP45 are Signal Peptide Prediction, Prediction of Non-classical pathway secretion, number of predicted transmembrane domains and prediction of Palmitoylation signal. GAP50 was wrongly mentioned here and has been removed from the revised manuscript.
When proteins are secreted out of the cytosol without predictable or known signal sequences or secretory motifs are classified as non-classically secreted proteins. The pathway is called a non-classical protein secretory pathway.
Our colocalization data of S14 with GAP45 and MTIP indicated that S14 is in the IMC.
122-123 Please reference the bioinformatic analysis plus URL that allows targeting to the IMC to be analyzed.
Response: All the URLs with references are given in the method section, lines 348-358 in the revised manuscript.
123-124 Please reference the URLs for TM, palmitoylation, and interactions analyses.
Response: All URLs with references are given in the method section, lines 348-358 in the revised manuscript.
125-127 How did you predict that S14 is secreted via the nonclassical pathway?
Response: We predicted non-classical pathway secretion of S14 using - SecretomeP (https://services.healthtech.dtu.dk/services/SecretomeP-1.0/) (10.1093/protein/gzh037).
128-130 Define the nonclassical pathway when it first appears in your manuscript.
The citation Moskes 2004 is not in the reference list
Response: The nonclassical pathway is defined in lines 105-107. The citation Moskes 2004 has been included in the revised manuscript.
132 Which membrane?
Response: Live S14-mCherry localization on the membrane does not differentiate between the outer membrane or IMC. Hence, only membrane is mentioned. Next, in Figure 4A, we confirmed S14 localization on IMC by treating sporozoites with Triton X-100 and colocalizing with IMC proteins GAP45 and MTIP.
134-135 In which species?
Response: We have mentioned P. berghei in the text in the revised manuscript.
141-142 Please include images of blood stage and liver stage parasites.
Response: Blood and liver stage images are included in the revised manuscript as Figure S2.
142-143 Which membrane?
Response: Live S14-mCherry localization on the membrane does not differentiate between the outer membrane or IMC. Hence, only membrane is mentioned. Next, in Figure 4A, we confirmed S14 localization on IMC by treating sporozoites with Triton X-100 and colocalizing with IMC proteins GAP45 and MTIP.
148-149 I cannot find the specific figure you refer to; I checked the online version of the Frenal 2010 paper.
Response: Electromobility shifts of GAP45 due to the palmitoylation have been reported in (Rees-Channer et al, 2006; DOI: 10.1016/j.molbiopara.2006.04.008). Frenal 2010 paper has stated about two bands but experimentally, it was shown in Rees-Channer et al, 2006 in Figures 1 and 2B.
175 gland, we counted [...]
Response: Corrected.
177 Compared to the
Response: Corrected.
177-179 Failed to invade (absolutely)? Or invaded in highly reduced numbers?
Response: Corrected.
182-186 Please be precise: I think you mean you let all types of mosquitoes take a blood meal; s14 knockout-infected mosquitoes did not infect mice.
Response: Corrected.
181-202 Perhaps use paragraphs to indicate the different types of experiments performed here.
Response: Done.
204 Please introduce paragraphs to identify the different experiments in this section
Response: Done.
208 Outer or inner membrane of what? IMC, the plasma membrane?
Response: We treated sporozoites with Triton X-100 to analyze whether S14 is present on the outer membrane (plasma membrane) or IMC.
228 onwards Structural models were obtained from whom? Which species did you use for the docking study? Could you use in one approach 3 berghei proteins, and confirm your docking studies with the falciparum proteins? That would strengthen your model. Should you include a negative control protein in the approach? Response: The structural models were obtained using the trROSETTA server. We used P. berghei for the docking study. In the old annotation and RMGM, the ortholog of P. berghei (PBANKA_0605900) in P.falciparum (PF3D7_1207400) was indicated. However, the updated PlasmodDB does not show PBANKA_0605900 ortholog in P. falciparum. We did try to generate structure models of P. falciparum MTIP, GAP45 and S14 using the trROSETTA server. We successfully reproduced the structure of MTIP, and GAP45 but the quality of S14 structure was unsuitable for the interaction studies. The negative control cannot be included in this kind of study because it gives a false interface, and none of the previous studies have used negative control.
250-251 Was all of the gene cloned? Please define amino acid range. discussion
Response: Full-length gene of S14, MTIP and GAP45 was cloned and the same has been mentioned in materials and methods in the revised manuscript.
Please discuss data from https://elifesciences.org/articles/77447 in relation to your protein Response: Discussed.
298-300 More recent glideosome papers exist. For example https://doi.org/10.1038/s42003-020-01283-8
Response: Included.
340 List the proteins you analysed. Add URL (websites) to the analyses tools.
Response: They are listed in Table S1. The method section gives all the URLs with references, lines 348-358 in the revised manuscript.
343 Known association from the literature: how was this done?
Response: The interactions demonstrated by different groups have been summarized in the review by Boucher & Bosch, 2015 (doi: 10.1016/j.jsb.2015.02.008).
346-349 A few glideosome components? On what basis were they selected and which are they? Response: The analysis showed that S14 had properties similar to GAP45 and MTIP. Additionally, S14 localized with GAP45 and MTIP, hence selected for interaction studies.
471 Can AlphaFold Structure Predictions be used in the docking studies?
Response: Even the Alphafold AI is trained on existing sequence/structure information despite being advertised as a de novo prediction system. That's why it can't produce good quality structures of evolutionarily unique proteins such as S14. We initially started our protein model generation by alphafold2, but the quality of the structure was very low; then we further used the trRosetta server (https://yanglab.nankai.edu.cn/trRosetta/), which shows the quality of all three protein structures above 95 after validation by using UCLA-DOE LAB-SAVES V6.0 (https://saves.mbi.ucla.edu/).
tr-Rosetta includes inter-residue distance, orientation distribution by a deep-neural network, and homologous template to improve the accuracy of models (DOI: 10.1038/s41596-021-00628-9).
We have given the model structure generated using alphafold2 for your reference.
__Model generated by using AlphaFold2.ipynb __(https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb#scrollTo=kOblAo-xetgx).
Structure quality assessment by http://saves.mbi.ucla.edu/.
GAP 45
__S14 __
MTIP
487 What parts of theses genes was cloned? Define the amino acid range.
Response: The full-length protein-encoding gene was cloned.
714 Please split the table into A Mosquito bite and B haemolymph Sporozoites Response: Done.
Figure 1 For clarity, maybe write S14::mCherry
Response: Done.
Figure 1 It would be useful to show blood stage parasite images.
Response: Blood stage parasite image is included in the revised manuscript as Figure S2.
Figure 2G Haemolymph sporozoites ?
Response: Done.
Figure 8 You argued that S14 is a membrane-bound protein through palmitoylation. Here the protein is shown to be cytoplasmic. Please update our model with more recent ones. Response: We have shown that S14 colocalizes with GAP45 and MTIP, suggesting its IMC localization. We have updated our model in Figure 8.
Figure S2B It would be good to include a positive control for these PCRs.
Response: We have replaced the figure's new gel with a positive control.
Figure S3 It would be good to include a positive control for these PCRs. Response: We already have positive controls in Figure S3C and S3F for all the primer pairs used.
Tabel S1 Table S1 is only mentioned twice in the text: lines 124 and 128. There is no mention that the table contains all (??) known gliding motility proteins.
Response: The table does not contain all the gliding proteins; however, most of the proteins mentioned in the Boucher & Bosch, 2015 paper (doi: 10.1016/j.jsb.2015.02.008) were included.
Table S1 The algorithms / websites used for bioinformatic prediction need to be listed here.
Response: Included.
Table S2 Add the plasmodb gene identifiers here. The table does not show all Plasmodium spp. but a selection. Response: All the orthologs mentioned in Figure S1 and Table S2 are not shown in the updated PlasmoDB. Accordingly, we have removed the Figure S1 and Table S2 in the revised manuscript__.__
Reviewer #1 (Significance (Required)):
General assessment: The authors provide an in-depth analyses of the Plasmodium berghei protein S14 and its involvement in gliding motility. Response: Thank you.
Advance: This paper is the first analysis of the S14 protein. The authors suggest a bridging function for the protein between MTIP and GAP45. Response: Thank you.
Audience: Gliding motility is of interest to the apicomplexan field. I think this particular proteins is specific to Plasmodium spp. Response: Thank you.
Reviewer #2
Summary:
The authors tag the sporozoite protein S14 in P. berghei and show localization near the sporozoite plasma membrane. They also convincingly show, through the generation of S14 knockout lines, that S14 is required for sporozoite motility and thereby also salivary gland and hepatocyte invasion. Their bioinformatic results support possible interactions between S14 and the inner membrane complex proteins MTIP and GAP45. These analyses were performed with these specific candidate proteins rather than being unbiased searches for potential interaction partners. The yeast 2-hybrid data to support these possible protein interactions need further controls.
Lines 143-144: Unless the sporozoites were not permeablized prior to staining, it is not clear if the protein is "on" the plasma membrane or just under the plasma membrane. Furthermore, this statement anyway seems contradictory to the authors' interpretation of Figure 4A. Response: Live S14-mCherry localization on the membrane does not differentiate between the outer membrane or IMC. Next, in Figure 4A, we confirmed S14 localization on IMC by treating sporozoites with Triton X-100 and colocalizing with IMC proteins GAP45 and MTIP. Further, we ensured that mCherrey signals were bleached post-fixation and performed IFA with and without permeabilization. We revealed the mCherry and CSP signals using Alexa 488 and Alexa 594, respectively. We observed the mCherrey signal with permeablized sporozoites, not without permeabilization.
Line 218: "This result indicates that S14 is present within the inner membrane of sporozoites." While this data shows that S14 is not in the plasma membrane of the parasite, how can the authors be sure it is at the IMC? Response: S14 colocalization with MTIP and GAP45 suggested its localization on IMC.
Line 225-226: This sentence overreaches in its conclusion. There is no indication that this protein provides the power or force behind the sporozoites forward movement. Several proteins are known to be required for gliding motility, but they are not all force-providing factors. Response: We have modified the sentence, and now it states, ‘These data demonstrate that S14 is an IMC protein, essential for the sporozoite's gliding motility.
Minor comments:
Line 99: "the role of gliding-associated proteins is unexplored" There are several publications on GAP40, GAP45 and GAP50 (some of which are referenced in the previous paragraph). Response: We have included the reference for studied proteins and modified the sentence for clarity.
Line 114: "We narrowed it down to a candidate" Narrowed down how? Or rephrase. Response: We identified the S14 gene from the Kaiser et al., 2004 paper (DOI: 10.1046/j.1365-2958.2003.03909.x) and rephrased the sentence in the revised manuscript.
Lines 120-123 are strangely written, and I don't follow the logic. What "similar properties" do GAP45 and GAP50 have with S14 and are they really indicative of function? Also if palmitoylation and myristylation and nonclassical secretion are present in most eukaryotes, why would they necessarily be evidence of IMC targeting? Response: It was wrongly written, we have modified the sentence for clarity.
Line 148-149. I did not see examples of this electromobility shift of GAP45 in this publication (although I may have overlooked it).
Response: Electromobility shifts of GAP45 due to the palmitoylation have been reported in (Rees-Channer et al, 2006; DOI: 10.1016/j.molbiopara.2006.04.008). Frenal 2010 paper has stated about two bands, but experimentally it was shown in Rees-Channer et al, 2006 in Figure 1 and 2B.
Table 1 legend should preferably specify that hemolymph sporozoites were used for IV infections. Response: Done.
Line 228: Should be rephrased for accuracy. "revealed the" should be replaced with "suggests" Response: Replaced.
Lines 305-307: I don't entirely understand the logic laid out here.
Response: This was written about GAP45 and MTIP coordination; however, it has been removed in the revised manuscript.
Lines 320-322: "We hypothesize that S14 possibly plays a structural role and maintains the stability of IMC required for the activity of motors during gliding and invasion." The data about the IMC structure shown is fluorescence microscopy - and there no change is observed in the IMC in the knockout line. I suggest removing or rephrasing this point if no extra data is provided to show this. Response: We have removed this sentence in the revised manuscript.
Reviewer #2 (Significance (Required)):
The work gives insights into an unstudied, conserved Plasmodium protein, S14, which the authors show is critical for Plasmodium transmission from mosquitoes. The parasite genetics and phenotyping demonstrating this are strong. The conclusions about interactions with glideosome/inner membrane complex components need further experimental support. The work is of interest to the Plasmodium field and may be also of interest to people interested in other protozoan parasites or in cellular motility.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
The manuscript by Gosh and colleagues demonstrates that S14 is a glideosome-associated protein in sporozoites. S14 knockout sporozoites fail to infect mosquito salivary glands and liver cells in the mammalian host. These sporozoites are also defective in gliding motility as S14 localizes to the inner membrane. S14 was shown to interact with the glideosome-associated proteins GAP45 and MTIP using the yeast two-hybrid system. The authors also provide an in-silico prediction on the S14, GAP45 and MTIP interaction.
Major issues:
Overall, there is information lacking in the manuscript, including on the figure legends, regarding experiments replication and n analyzed.
For complementation, the authors engineered an independent S14 knockout line. For this line is clear that parasites failed to infect salivary glands contrarily to the knockout line. Despite not showing it, did the authors confirm that this knockout line has no defects in infecting mosquito midguts and producing sporozoites? Response: We analyzed the midgut for sporozoite formation, which was comparable to the original KO line, and included the data (Figure 2D) in the revised manuscript.
Did the authors conduct IV injections in mice with a higher number of sporozoites? Hemolymph sporozoites are less infectious than sporozoites collected from the salivary glands and I was wondering whether patent infections with S14 ko sporozoites can be obtained by injecting a higher inoculum. The same applies to the infectivity experiments with HepG2cells. Response: We increased the sporozoites dose and infected mice with 10,000 hemolymph sporozoites, but no infection was observed (Table 1). No EEFs were observed in HepG2 cells infected with 10,000 S14 KO hemolymph sporozoites.
Please provide information on the number of sporozoites that were analyzed in the trails experiment. Response: We analyzed 210, 225, and 212 sporozoites for WT GFP, S14 KO c1, and S14 KO c2, respectively.
Minor issues:
In Figure 1. F) WB on S14-3xHA-mCherry tagged sporozoites showing two bands on the WB. The Palm-band is only inferred thus I suggest correcting the figure to S14-3xHA-mcherry. On 1D all the mcherry signal is detected on the membrane but then on WB, a smaller fraction is palm? What is the explanation for the ratio between the two bands? Why so distinct CSP intensity bands between wt and tagged line? Were very distinct amounts of protein loaded?
Response: We have corrected the Palm-S14-3xHA-mcherry to S14-3xHA-mcherry.
This reviewer raises a valid point regarding the discrepancy between IFA and Western blot. The non-palmitoylated S14-mCherrey signal was possibly corrected after deconvolution in image 1D and mainly the membrane signal was prominent. In Figure 1C, many sporozoites show some cytosolic signal, perhaps representing non-palmitoylated S14. Western blot concentrates the protein of interest as a single band, allowing more accurate visualization of protein.
The distinct CSP intensity bands between wt and the tagged line are due to the loading of a higher amount of parasite lysate in WT lane. To ensure that the western blot signal is specific to S14, we loaded a higher amount of protein in WT.
Figure 1. A) Statistical analysis is missing. Not clear if the bars represent mean values +/- standard deviation. No information on the material and methods of how the relative expression was calculated. Response: No error bars are shown in Figure 1 because it was performed once.
In the introduction lines 54 and 58 I suggest replacing humans with mammalian host. Response: Replaced.
Line 58. Not clear why the ref Ripp et al., 2021 is used for a general sentence to introduce the Plasmodium life cycle. Response: Removed.
Line 72: I suggest replacing "TRAP mutant" with "TRAP knockouts" (Sultan et al., 1997). More recently there are TRAP mutants with impaired motility and normal invasion of mosquito salivary glands (Klug et al., 2020) Response: Replaced.
Lines 78 to 86: In this paragraph, authors refer to several proteins involved in sporozoite gliding motility and host cell invasion, however for most of the studies this conclusion comes from the characterization of knockouts defective phenotype and actually a direct role for some of these molecules in the process awaits clear demonstration. Response: We have replaced involved with implicated.
Line 78: Authors do not consider that maebl knockout sporozoites display reduced adhesion, including to cultured hepatocytes, which could contribute to the defects in multiple biological processes, such as in gliding motility, hepatocyte wounding, and invasion. Response: We have corrected maebl role in the revised manuscript.
Line 80: I suggest authors reconcile the contradictory reports in the literature on the role of TRSP in sporozoites invasion. Response: We have removed this reference in the revised manuscript.
Line 82-83: Please revise it. Response: Revised.
Table 1. Correct table as when sporozoites were transmitted by mosquito bite the term "number of sporozoites injected" does not apply. Please give more details on the bite experiments. Is this the number of mosquitoes for all four animals? For how long the mosquitoes were allowed to bite? Response: For clarity, we have split the table into A Mosquito bite and B haemolymph Sporozoites. We used ten mosquitoes/mice in the bite experiment. Mosquitos were allowed to probe for blood meal for 20 minutes, and the feeding was ensured by observing mosquitoes post-blood meal; approximately 70% of mosquitoes received the blood meal in all the cages.
Line 288 and 289. There are several publications showing that maebl knockout sporozoites are impaired at invading the mosquito salivary glands and at infecting the vertebrate host contradicting Kariu et al., 2002 findings in the vertebrate host. Response: We have removed maebl from this line.
Line 290. I suggest "was most likely due to" instead of " due to" as sporozoite adhesion to cells was not evaluated. Response: Corrected.
Line 291: "Cellular transmigration and host cell invasion are prerequisites for gliding motility" please revise. Response: Revised.
Line 437: indicate which clone was used.
Response: Indicated (3D11).
Line: 463: indicate the % of the gel in the SDS-PAGE Response: We have used 10% SDS-PAGE gel and it is indicated in the revised manuscript.
Line 499: indicate the version of the GraphPad Prism software. Response: GraphPad Prism version 9.
Figure S3 legend needs to be corrected. Panels in the figure are from A to F while in legend G and H are included. Response: Corrected.
4. Description of analyses that authors prefer not to carry out
Reviewer #2
Line 39-41: "Using in silico and the yeast two-hybrid system, we showed the interaction of S14 with the glideosome-associated proteins GAP45 and MTIP. Together, our data show that S14 is a glideosome-associated protein" Although these interactions can be speculated based on the results shown, these interactions were not confirmed in this study. Response: We attempted to pulldown the S14 interacting partner using anti-mCherry antibody from S14-3XHA-mCherry transgenic sporozoites and then further tried to identify interactome using mass spectrometry but failed. Hence, we selected two known IMC localized gliding proteins MTIP and GAP45. Performing pull-down from sporozoites is challenging, so we checked this interaction using yeast 2-hybrid assay and bioinformatic analysis for protein-protein interaction.
In order to claim interaction between S14 and IMC proteins, interaction needs to be shown experimentally. Well-controlled yeast 2-hybrid would be a start - then interaction would be more than just speculative. But immunoprecipitation from sporozoites or other biochemical interactions would give more support to this idea. Response: We attempted to pulldown the S14 interacting partner using an anti-mCherry antibody from S14-3XHA-mCherry transgenic sporozoites and then further tried to identify interactome using mass spectrometry but failed. Hence, we selected two known IMC localized gliding proteins MTIP and GAP45. Performing pull-down from sporozoites is challenging, so we checked this interaction using yeast 2-hybrid assay and bioinformatic analysis for protein-protein interaction.
Reviewer #3
The authors provide convincing data on the S14 localization in the inner membrane of sporozoites and interaction with GAP45 and MTIP using the yeast model. Did the authors consider conducting co-IP followed by MS analysis to pull down S14 in the complex with GAP45 and MTIP? Response: We attempted to pulldown the S14 interacting partner using an anti-mCherry antibody from S14-3XHA-mCherry transgenic sporozoites and then further tried to identify the interactome using mass spectrometry but failed. Hence, we selected two known IMC localized gliding proteins, MTIP and GAP45. Performing pull-down from sporozoites is challenging, so we checked this interaction using yeast 2-hybrid assay and bioinformatic analysis for protein-protein interaction.
__Reviewer #3 (Significance (Required)):____ __ Sporozoite gliding motility is a critical feature of parasite infectivity. Impairment of this important feature has been described for several mutant/knockout parasite lines. This study goes beyond the phenotypic analysis of mutant parasites to infer the role of S14 by providing more mechanistic evidence to show S14 interaction with other glideosome-associated proteins. However, this interaction was investigated using the two-hybrid system in yeast. Still, in sporozoites, no experiments were conducted to evaluate the interaction between these proteins.
Response: We attempted to pulldown the S14 interacting partner using an anti-mCherry antibody from S14-3XHA-mCherry transgenic sporozoites and then further tried to identify interactome using mass spectrometry but failed. Hence, we selected two known IMC localized gliding proteins, MTIP and GAP45. Performing pull-down from sporozoites is challenging, so we checked this interaction using yeast 2-hybrid assay and bioinformatic analysis for protein-protein interaction.
Please consider I'm not an expert on the in-silico interaction studies.
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Referee #3
Evidence, reproducibility and clarity
The manuscript by Gosh and colleagues demonstrates that S14 is a glideosome-associated protein in sporozoites. S14 knockout sporozoites fail to infect mosquito salivary glands and liver cells in the mammalian host. These sporozoites are also defective in gliding motility as S14 localizes to the inner membrane. S14 was shown to interact with the glideosome-associated proteins GAP45 and MTIP using the yeast two-hybrid system. The authors also provide an in-silico prediction on the S14, GAP45 and MTIP interaction.
Major issues:
Overall, there is information lacking in the manuscript, including on the figure legends, regarding …
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Referee #3
Evidence, reproducibility and clarity
The manuscript by Gosh and colleagues demonstrates that S14 is a glideosome-associated protein in sporozoites. S14 knockout sporozoites fail to infect mosquito salivary glands and liver cells in the mammalian host. These sporozoites are also defective in gliding motility as S14 localizes to the inner membrane. S14 was shown to interact with the glideosome-associated proteins GAP45 and MTIP using the yeast two-hybrid system. The authors also provide an in-silico prediction on the S14, GAP45 and MTIP interaction.
Major issues:
Overall, there is information lacking in the manuscript, including on the figure legends, regarding experiments replication and n analyzed.
For complementation, the authors engineered an independent S14 knockout line. For this line is clear that parasites failed to infect salivary glands contrarily to the knockout line. Despite not showing it, did the authors confirm that this knockout line has no defects in infecting mosquito midguts and producing sporozoites?
Did the authors conduct IV injections in mice with a higher number of sporozoites? Hemolymph sporozoites are less infectious than sporozoites collected from the salivary glands and I was wondering whether patent infections with S14 ko sporozoites can be obtained by injecting a higher inoculum. The same applies to the infectivity experiments with HepG2cells.
The authors provide convincing data on the S14 localization in the inner membrane of sporozoites and interaction with GAP45 and MTIP using the yeast model. Did the authors consider conducting co-IP followed by MS analysis to pull down S14 in the complex with GAP45 and MTIP?
To characterize the gliding defective phenotype authors based their assessment on trails quantifications. This analysis is very limited as the percentage of attached, waving, floating sporozoites cannot be determined. Due to their conclusion on the role of S14 for sporozoite gliding motility, I recommend a more detailed investigation using live microscopy imaging. Please provide information on the number of sporozoites that were analyzed in the trails experiment.
Conclusions that S14 knockout does not impact the expression and organization of two surface proteins, CSP and TRAP, and two IMC rely on a qualitative analysis only. However, quantitative analysis to support their observations is missing.
Minor issues:
In Figure 1. F) WB on S14-3xHA-mCherry tagged sporozoites showing two bands on the WB. The Palm-band is only inferred thus I suggest correcting the figure to S14-3xHA-mcherry. On 1D all the mcherry signal is detected on the membrane but then on WB, a smaller fraction is palm? What is the explanation for the ratio between the two bands? Why so distinct CSP intensity bands between wt and tagged line? Were very distinct amounts of protein loaded?
Figure 1. A) Statistical analysis is missing. Not clear if the bars represent mean values +/- standard deviation. No information on the material and methods of how the relative expression was calculated.
In the introduction lines 54 and 58 I suggest replacing humans with mammalian host.
Line 58. Not clear why the ref Ripp et al., 2021 is used for a general sentence to introduce the Plasmodium life cycle.
Line 72: I suggest replacing "TRAP mutant" with "TRAP knockouts" (Sultan et al., 1997). More recently there are TRAP mutants with impaired motility and normal invasion of mosquito salivary glands (Klug et al., 2020)
Lines 78 to 86: In this paragraph, authors refer to several proteins involved in sporozoite gliding motility and host cell invasion, however for most of the studies this conclusion comes from the characterization of knockouts defective phenotype and actually a direct role for some of these molecules in the process awaits clear demonstration.
Line 78: Authors do not consider that maebl knockout sporozoites display reduced adhesion, including to cultured hepatocytes, which could contribute to the defects in multiple biological processes, such as in gliding motility, hepatocyte wounding, and invasion.
Line 80: I suggest authors reconcile the contradictory reports in the literature on the role of TRSP in sporozoites invasion.
Line 82-83: Please revise it.
Table 1. Correct table as when sporozoites were transmitted by mosquito bite the term "number of sporozoites injected" does not apply. Please give more details on the bite experiments. Is this the number of mosquitoes for all four animals? For how long the mosquitoes were allowed to bite?
Line 288 and 289. There are several publications showing that maebl knockout sporozoites are impaired at invading the mosquito salivary glands and at infecting the vertebrate host contradicting Kariu et al., 2002 findings in the vertebrate host.
Line 290. I suggest "was most likely due to" instead of " due to" as sporozoite adhesion to cells was not evaluated.
Line 291: "Cellular transmigration and host cell invasion are prerequisites for gliding motility" please revise.
Line 437: indicate which clone was used.
Line: 463: indicate the % of the gel in the SDS-PAGE
Line 499: indicate the version of the GraphPad Prism software.
Figure S3 legend needs to be corrected. Panels in the figure are from A to F while in legend G and H are included.
Significance
Sporozoite gliding motility is a critical feature of parasite infectivity. Impairment of this important feature has been described for several mutant/knockout parasite lines. This study goes beyond the phenotypic analysis of mutant parasites to infer the role of S14 by providing more mechanistic evidence to show S14 interaction with other glideosome-associated proteins. However, this interaction was investigated using the two-hybrid system in yeast. Still, in sporozoites, no experiments were conducted to evaluate the interaction between these proteins.
Please consider I'm not an expert on the in-silico interaction studies.
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Referee #2
Evidence, reproducibility and clarity
Summary:
The authors tag the sporozoite protein S14 in P. berghei and show localization near the sporozoite plasma membrane. They also convincingly show, through the generation of S14 knockout lines, that S14 is required for sporozoite motility and thereby also salivary gland and hepatocyte invasion. Their bioinformatic results support possible interactions between S14 and the inner membrane complex proteins MTIP and GAP45. These analyses were performed with these specific candidate proteins rather than being unbiased searches for potential interaction partners. The yeast 2-hybrid data to support these possible protein interactions …
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Referee #2
Evidence, reproducibility and clarity
Summary:
The authors tag the sporozoite protein S14 in P. berghei and show localization near the sporozoite plasma membrane. They also convincingly show, through the generation of S14 knockout lines, that S14 is required for sporozoite motility and thereby also salivary gland and hepatocyte invasion. Their bioinformatic results support possible interactions between S14 and the inner membrane complex proteins MTIP and GAP45. These analyses were performed with these specific candidate proteins rather than being unbiased searches for potential interaction partners. The yeast 2-hybrid data to support these possible protein interactions need further controls.
Major comments:
Line 39-41: "Using in silico and the yeast two-hybrid system, we showed the interaction of S14 with the glideosome-associated proteins GAP45 and MTIP. Together, our data show that S14 is a glideosome-associated protein" Although these interactions can be speculated based on the results shown, these interactions were not confirmed in this study.
Lines 143-144: Unless the sporozoites were not permeablized prior to staining, it is not clear if the protein is "on" the plasma membrane or just under the plasma membrane. Furthermore, this statement anyway seems contradictory to the authors' interpretation of Figure 4A.
Line 218: "This result indicates that S14 is present within the inner membrane of sporozoites." While this data shows that S14 is not in the plasma membrane of the parasite, how can the authors be sure it is at the IMC?
Line 149: To definitively state S14 is a membrane protein, biochemical assays proving such should be performed. (or perhaps genetic mutation of the predicted palmitoylation site?) Otherwise, this should be rephrased.
Line 225-226: This sentence overreaches in its conclusion. There is no indication that this protein provides the power or force behind the sporozoites forward movement. Several proteins are known to be required for gliding motility, but they are not all force-providing factors.
Lines 257-258: for yeast 2-hybrid, the controls of expressing S14, GAP45 and MTIP together with control proteins where no interaction would be predicted are absent.
In order to claim interaction between S14 and IMC proteins, interaction needs to be shown experimentally. Well-controlled yeast 2-hybrid would be a start - then interaction would be more than just speculative. But immunoprecipitation from sporozoites or other biochemical interactions would give more support to this idea.
Minor comments:
Line 99: "the role of gliding-associated proteins is unexplored" There are several publications on GAP40, GAP45 and GAP50 (some of which are referenced in the previous paragraph).
Line 114: "We narrowed it down to a candidate" Narrowed down how? Or rephrase.
Lines 120-123 are strangely written, and I don't follow the logic. What "similar properties" do GAP45 and GAP50 have with S14 and are they really indicative of function? Also if palmitoylation and myristylation and nonclassical secretion are present in most eukaryotes, why would they necessarily be evidence of IMC targeting?
Line 148-149. I did not see examples of this electromobility shift of GAP45 in this publication (although I may have overlooked it).
Table 1 legend should preferably specify that hemolymph sporozoites were used for IV infections.
Line 228: Should be rephrased for accuracy. "revealed the" should be replaced with "suggests"
Lines 305-307: I don't entirely understand the logic laid out here.
Lines 320-322: "We hypothesize that S14 possibly plays a structural role and maintains the stability of IMC required for the activity of motors during gliding and invasion." The data about the IMC structure shown is fluorescence microscopy - and there no change is observed in the IMC in the knockout line. I suggest removing or rephrasing this point if no extra data is provided to show this.
Significance
The work gives insights into an unstudied, conserved Plasmodium protein, S14, which the authors show is critical for Plasmodium transmission from mosquitoes. The parasite genetics and phenotyping demonstrating this are strong. The conclusions about interactions with glideosome/inner membrane complex components need further experimental support. The work is of interest to the Plasmodium field and may be also of interest to people interested in other protozoan parasites or in cellular motility.
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Referee #1
Evidence, reproducibility and clarity
Summary: The authors have identified a sporozoite gliding motility protein through bioinformatic analysis. From the main text I do not know how, or what bioinformatic analysis was performed, in order to focus on this protein which is called S14. The authors then go on to tag the protein, produce a KO and show its involvement in gliding motility. The KO shows that parasites lacking S14 fail to invade the mosquito salivary glands. This is due to a motility defect. Y2H and docking studies are used to define an interaction with MTIP and GAP45, two known components of the glideosome.
Major comments: The paper is sometimes hard to follow …
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Referee #1
Evidence, reproducibility and clarity
Summary: The authors have identified a sporozoite gliding motility protein through bioinformatic analysis. From the main text I do not know how, or what bioinformatic analysis was performed, in order to focus on this protein which is called S14. The authors then go on to tag the protein, produce a KO and show its involvement in gliding motility. The KO shows that parasites lacking S14 fail to invade the mosquito salivary glands. This is due to a motility defect. Y2H and docking studies are used to define an interaction with MTIP and GAP45, two known components of the glideosome.
Major comments: The paper is sometimes hard to follow and lacks clarity. The reason: important information is omitted, or explained at the end of a section rather than at first mention; experimental details that are of essence need to be mentioned or explained in the main text; there is ample use of the word 'bioinformatic' without explaining what kind of analysis was performed in the main text. I cite from the abstract: 'In silico analysis of a novel protein, S14, which is uniquely upregulated in salivary gland sporozoites, suggested its association with glideosome-associated proteins.' I cite from the introduction: 'A study comparing transcriptome differences between sporozoites and merozoites using suppressive subtraction hybridization found several genes highly upregulated in sporozoites and named them 'S' genes (Kaiser et al, 2004). We narrowed it down to a candidate named S14, which lacked signal peptide and transmembrane domains.' From reading the main text, I do not know why Plasmodium berghei S14 was chosen in this manuscript. S14 is one of 25 transcripts identified by Kappe et al in Plasmodium yoelii (https://doi.org/10.1046/j.1365-2958.2003.03909.x) to be upregulated in sporozoites. The material and methods section does not explain either why S14 was chosen. Perhaps the authors could update Figure 2 from Kappe et al with the most recent annotations from plasmodb.
Reproducibility: None of the main Figures or Figure legends define ' N = '. For example I cite: 'The S14 KO clonal lines were first analyzed for asexual blood-stage propagation, and for this, 200 µl of iRBCs with 0.2% parasitemia was intravenously injected into a group of mice.' There are 2 mentions of 'N=' in the supplementary figures. I have not found any others.
I'm not sure what the convention is. Should unpublished data for this gene (PBANKA_0605900) found in pberghei.eu (a database for mutant berghei parasites) be cited? After all it confirms their findings.
The authors need to use more recent references for some of their statements; see some comments below.
Minor comments:
line
1-2 Add the Plasmodium species of this study. abstract Which species do you work with? 29 mosquito salivary glands and human host hepatocytes 30 to the glideosome, a protein complex containing [...] 32-33 What kind of in silico analysis suggested S14 is part of the glideosome? S14 is not uniquely upregulated; there are other S-type genes identified by Kappe and Matuschewski. 25 I believe. 32 Please point out he species were S genes were identified. SGS of which species? 34 expression: change to transcription 39 What kind of in silico analysis was used here? and therefore malaria transmission 55 A single zygote transforms into a single ookinete, which establishes a single oocyst, which in turn can produce thousands of midgut sporozoites. Please correct the life cycle passage. located or anchored in the IMC? And located between the IMC and plasma membrane? 61-63 Refer to Table S1 and its contents here 64 Name the known GAPs.
65-67 Which transmembrane domain proteins? Please add more recent references than King 1988. 71-72 TRAP was the first protein found to be ... 74-76 Add additional, more recent references: for example search Frischknecht and TRAP 76 S6 (TREP) is also [...] 88 Some of these proteins are also expressed in ookinetes. 89-91 The sentence needs a verb. 88-96 Please add some more recent glideosome papers. After 2013. 91 Why do you call it a peripheral protein? 91-93 There are more recent citations for GAP45 andGAP50. 96 Insert a reference here. 99 Please define the gliding-associated proteins. What are they? Aren't there papers on GAP40, 45 and 50? DOI: 10.1016/j.chom.2010.09.002 99 .... What prompted you to identify a novel GAP? And why is S14 classified as a GAP? 99-102 What kind of bioinformatic study? Why was S14 chosen? Please outline how you ended up with S14. Any other proteins that came out of the bioinformatic screen from the list of S genes? How many proteins were identified in the screen for sporozoite upregulated proteins by Kappe and Matuschewski? 102-103 Define the nonclassical secretion pathway. Please reference GAP45 and GAP50 data for the nonclassical pathway. 105 Please add P. berghei to the title, the abstract, the introduction. 111 The results section does not outline what bioinformatic analysis was used 112-114 Please specify the exact number of upregulated in sporozoites genes. I think it was 25. And add the species the study was performed in. Why did you choose the Kappe study but not the uis genes from berghei? 114-115 How did you narrow it down to S14? The Kappe paper lists 25 S-type genes from P. yoelii. 118 Plasmodia is not the plural for a group of different Plasmodium species. Use: [...] conserved among Plasmodium spp. 118-119 Which proteins did you analyze? And how did you analyze them? Where is the data for this analysis? Outline the amino acids that predict palmitoylation? The nonclassical pathway? 119-122 Here: do you mean S14 has similar properties as GAP 45 and GAP50? Define the nonclassical pathway? How do you know S14 is in the IMC? 122-123 Please reference the bioinformatic analysis plus URL that allows targeting to the IMC to be analyzed. 123-124 Please reference the URLs for TM, palmitoylation, and interactions analyses. 125-127 How did you predict that S14 is secreted via the nonclassical pathway? 128-130 Define the nonclassical pathway when it first appears in your manuscript. The citation Moskes 2004 is not in the reference list 132 Which membrane? 134-135 In which species? 141-142 Please include images of blood stage and liver stage parasites. 142-143 Which membrane? 148-149 I cannot find the specific figure you refer to; I checked the online version of the Frenal 2010 paper. 175 gland, we counted [...] 177 Compared to the 177-179 Failed to invade (absolutely)? Or invaded in highly reduced numbers? 182-186 Please be precise: I think you mean you let all types of mosquitoes take a blood meal; s14 knockout-infected mosquitoes did not infect mice. 181-202 Perhaps use paragraphs to indicate the different types of experiments performed here. 204 Please introduce paragraphs to identify the different experiments in this section 208 Outer or inner membrane of what? IMC, the plasma membrane? 228 onwards Structural models were obtained from whom? Which species did you use for the docking study? Could you use in one approach 3 berghei proteins, and confirm your docking studies with the falciparum proteins? That would strengthen your model. Should you include a negative control protein in the approach? 250-251 Was all of the gene cloned? Please define amino acid range. discussion Please discuss data from https://elifesciences.org/articles/77447 in relation to your protein
298-300 More recent glideosome papers exist. For example https://doi.org/10.1038/s42003-020-01283-8 340 List the proteins you analysed. Add URL (websites) to the analyses tools. 343 Known association from the literature: how was this done? 346-349 A few glideosome components? On what basis were they selected and which are they?
471 Can AlphaFold Structure Predictions be used in the docking studies? 487 What parts of theses genes was cloned? Define the amino acid range. 714 Please split the table into A Mosquito bite and B haemolymph Sporozoites Figure 1 For clarity, maybe write S14::mCherry Figure 1 It would be useful to show blood stage parasite images. Figure 1F You have not formally shown that this signal corresponds to palmitoylated S14. Could be heavy chain. Figure 2G Haemolymph sporozoites ? Figure 8 You argued that S14 is a membrane-bound protein through palmitoylation. Here the protein is shown to be cytoplasmic. Please update our model with more recent ones.
Figure S2B It would be good to include a positive control for these PCRs. Figure S3 It would be good to include a positive control for these PCRs.
Tabel S1 Table S1 is only mentioned twice in the text: lines 124 and 128. There is no mention that the table contains all (??) known gliding motility proteins. Table S1 The algorithms / websites used for bioinformatic prediction need to be listed here. Table S2 Add the plasmodb gene identifiers here. The table does not show all Plasmodium spp. but a selection.
Significance
General assessment: The authors provide an in-depth analyses of the Plasmodium berghei protein S14 and its involvement in gliding motility.
Advance: This paper is the first analysis of the S14 protein. The authors suggest a bridging function for the protein between MTIP and GAP45.
Audience: Gliding motility is of interest to the apicomplexan field. I think this particular proteins is specific to Plasmodium spp.
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