Conformational change of the Plasmodium TRAP I domain is essential for sporozoite migration and transmission of malaria

  1. Friedrich Braumann
  2. Dennis Klug
  3. Jessica Kehrer
  4. Chafen Lu
  5. Timothy A. Springer
  6. Friedrich Frischknecht

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Abstract

Eukaryotic cell adhesion and migration relies on surface adhesins connecting extracellular ligands to the intracellular actin cytoskeleton. Plasmodium sporozoites are transmitted by mosquitoes and rely on adhesion and gliding motility to colonize the salivary glands and to reach the liver after transmission. During gliding the essential sporozoite adhesin TRAP engages actin filaments in the cytoplasm of the parasite., while binding ligands on the substrate through its inserted (I)-domain. Crystal structures of TRAP from different Plasmodium species revealed the I-domain in closed and open conformations. Here, we probe the importance of these two conformational states by generating parasites expressing versions of TRAP with the I-domain stabilized in either the open or closed state with disulfide bonds. Strikingly, both mutations impact sporozoite gliding, mosquito salivary gland entry and transmission. Absence of gliding in sporozoites expressing the open TRAP I-domain could be partly rescued by adding a reducing agent. This suggests that dynamic conformational change is required for ligand binding, gliding motility and organ invasion and hence sporozoite transmission from mosquito to mammal.

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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity):

    Summary:
    Plasmodium sporozoites rely on gliding motility to invade the salivary glands of the mosquito vector where they reside until being inoculated into the vertebrate host. Once in the vertebrate host, sporozoites again have to glide to pass through liver cells and reach the final host cell before exo-erythrocytic replication occurs. In sporozoites, thrombospondin-related anonymous protein (TRAP) is the key adhesin, linking the molecular motor to the substrate. TRAP and TRAP-like proteins contain on the extracellular side an inserted (I) domain that is responsible for substrate binding. The I domain can be in an open or closed state which can be fixed by mutating specific amino acids. Braumann et al. then assessed the effect of these states on gliding motility and infection. Whilst no effect was seen for both mutants with closed or open states until the production of haemolymph sporozoites, the number of salivary gland sporozoites were highly reduced in both mutants. The closed mutant was not able to be transmitted from mosquitoes to mice whilst the open mutant is severely impacted in transmission. Gliding of both mutant is highly impaired. Some of these phenotypes could be reverse by adding a reducing agent.

    Major comments:
    This is an elegant study with exhaustive experiments to address the research questions. One of the things I wondered about is the effect of exchanging amino acids. As far as I understood, structural information of integrins from other organisms informed the authors about positions that should be mutated and about the impact (or loss of it) on the 3D structure of the domain. Would it not be useful to run a structure prediction programme such as Alphafold on all the mutants to at least confirm stability of the domain structure upon mutation in silico?_

    A: Thanks for this suggestion, we added the crystal structure models of P. falciparum and P. vivax as well as the Alphafold models of the open and closed state of P. berghei TRAP as new panels in Figure 1, added some corresponding text to the results, materials and methods, and the figure legend.

    Some of the concentrations of reducing agents tested are very high. Can the authors be sure that the parasites are not dead?

    A: We used sporozoites expressing GFP in the cytosol and did not see loss of fluorescence. While this does not rule out that the parasites are not alive anymore, it shows that the plasma membrane was not compromised. Importantly, when using the more moderate concentrations that rescued motility, wild type parasites were also moving, showing that the parasites were alive.

    Minor comments:
    -Please make sure that punctuation is correct (e.g. missing commas) and that there are no other typing errors.

    A: We carefully read and edited the manuscript again and hopefully corrected all those errors.

    • Line 50: This sentence may imply mechanical rupture of oocysts rather than active egress of the sporozoite. Please re-phrase.

    A: rephrased, it now reads “egress” instead of “burst”. (now line 53)

    • Line 82: Do you mean a complexed Mg2+ ion?

    A: Yes, this sentence was revised. (now line 99)

    • The introduction stops rather abruptly. Maybe a sentence of the significance of this study could be added.

    A: we added the following: “Our results suggest that active change between the open and closed conformation of the TRAP I domain is essential for sporozoite invasion of salivary glands and transmission from mosquitoes to mammals.” (line 122-125).

    • Line 361: 20 million parasites each? Please re-phrase to make it clearer.

    A: done, it now reads: “…into each of two naïve mice…”.(now line 517)

    • Line 363: Field of view needs to defined: What is the magnification used to observe exflagellation?

    A: done, it now reads: “as observed with a Zeiss Axiostar using a 100x objective lens – revealing 300-400 red blood cells per field of view) – now line 519-521.”

    • Materials and Methods: Please write out reagent names for the first time.

    A: done, e.g. “phosphate buffered saline” for PBS; line 540; Roswell Park Memorial Institute medium 1640 for RPMI; line 420; as well as for DTT and TCEP (lines 560, 561), BSA (line 565), etc

    Figures:

    • Figure 1A is very small. The label font of the whole figure 1 is often too small.

    A: We intentionally kept Figure 1A small as most readers will be familiar with the life cycle but increased font size as much as possible throughout the figure including Figure 1B, which we needed to shrink in the new design to accommodate the Alphafold structures.

    • Figure 1D: Please list the components in the figure legend, e.g. blue: substrate etc.

    A: done, now lines 722-725

    • Figure 3: The figure title should be changed as there is no complete failure of sporozoites with fixed I-domains to invade salivary glands.

    A: done, it now reads: “…show strong reduction in salivary gland invasion”, line 790-791

    • Figure 5: for clarity, it would be easier if Figure C would not be squeezed into the legends of A and B.

    A: We agree this is a tricky arrangement, but when composing the figure, we tried many different ways, which were all unsatisfactory too, leading to either too awkward an arrangement or too much white between the panels. We hence, despite anticipating this critique, decided on this arrangement and would hence like to keep it. However, we changed the color of the box around C so that it is marked more differently.

    Reviewer #1 (Significance):

    This study addresses in detail the mechanism of Plasmodium TRAP I domain in gliding motility and transmission. It builds on work from many years and groups including their own and contributes to deepen our understanding of TRAP-family proteins, gliding motility in Plasmodium sporozoites and maybe even in other Plasmodium stages or other Apicomplexan parasites.

    This work is of interest to researchers in the field of Plasmodium mosquito stages and transmission as well as scientist who work on apicomplexan gliding motility and transmission.

    I have previously worked on Plasmodium mosquito stages, but currently work on Toxoplasma_ gondii.

    A: Thank you for your appreciation of our study, the mechanistic insights it gives into gliding and transmission, and the helpful critique.

    Reviewer #2 (Evidence, reproducibility and clarity):

    This work seeks to investigate the effects on sporozoite motility of hypothesized conformational changes in TRAP's integrin domain. Previous structural studies suggested that the Integrin-like domain of TRAP assumes 'open' and 'closed' conformations induced by ligand binding. Here the authors hypothesize that TRAP's ability to dynamically switch between these states is crucial for sporozoite motility. The hypothesis is interesting and the authors elected to test it by 'fixing' TRAP in constitutively 'open' and 'closed' conformations by introducing Cys residues that are presumed to form disulphide bonds resulting in these states.

    Major Comments
    The approach is creative. The data that are presented are of high quality with adequate reproducibility. However, as written the manuscript does not provide a rationale for the specific substitutions they chose, how these substitutions are expected to lead to 'open' and 'closed' states, and how they mimic the natural route of TRAP transitioning between the 'open' and 'closed' conformations._

    A: We now add a more detailed rationale at the very start of the results section, which now reads: “We used closed and open structures of TRAP from P. falciparum and P. vivax, respectively (Song et al., 2012), to design cysteine mutations in P. berghei (Fig. 1E-J). The TRAP I domain of P. berghei is 42 and 48% identical in sequence to those of P. vivax and P. falciparum, respectively; lower identity was used for successful introduction of a disulfide into integrin αI domains (Shimaoka et al., 2001; Shimaoka et al., 2003). Between the open and closed TRAP I domain conformations, the I domain α7-helix pistons 9 Å relative to the neighboring β6-strand; therefore, disulfides introduced between these two structural elements can stabilize one state over the other, as previously pioneered in integrin I domains. β6-strand and α7-helix residues with Cβ atoms within 5 Å of one another in structures of the two states were chosen for mutation to cysteine (Fig. 1G&H). P. falciparum TRAP-Fc fusions with homologous cysteine mutations were well expressed in mammalian cells (Koksal et al,, 2013). Using the TRAP sequence alignment (Fig. 1F), homologous residues in P. berghei TRAP were mutated to cysteine to stabilize the closed conformation (Ser-210 and Phe-224 in the S210C/F224C mutation) and the open conformation (Ser-210 and Gln-216 in the S210C/Q216C mutation). AlphaFold (Mirdita et al 2022) predicted that the P. berghei TRAP I domains were well folded and assumed the desired conformations with formation of the mutationally introduced disulfide bonds (Fig. 1I&J). The I domain of P. berghei is 42 and 48% identical in sequence to those of P. vivax and P. falciparum in which the open and closed conformations are defined, assuring highly confident modeling (Song et al., 2012). Cysteines were substituted in positions that were sufficiently close in one conformation to form a disulfide but not in the alternative conformation..” lines 130-202 and also provided a detailed view of the structures in Figure 1.

    Furthermore, there is no biochemical, biophysical or modeling data demonstrating that the introduced mutations impact the folding/unfolding of TRAP's I domain in the manner hypothesized. Therefore, it is difficult to interpret subsequent phenotypic data from the two mutant lines. While mutant parasites display defects in gliding motility, these defects are unexpected, perhaps pointing to alternative explanations - such as aberrant inter- or intra-molecular disulphide bonding in TRAP's extracellular domain.

    A: The ability of such mutations to give biologically meaningful results has already been demonstrated in integrin I domains, where ligand-binding affinity was measured. We now describe the rationale in more detail, and demonstrate that the cysteines are recognized by Alphafold to yield well-folded domains that are stabilized in the desired conformations. These are presented as new panels in Figure 1.

    About 10% of mutant sporozoites assumed to be in a constitutively 'closed' conformation display gliding motility and they move faster that WT. Yet this mutant did not cause patency in mice when introduced either via mosquito bite or IV injection. In contrast, the mutant assumed to be in an 'open' conformation displayed no motility in vitro but was able to infect mice (albeit at a significantly reduced rate compared to controls). Presumably, these data suggest that the in vitro gliding motility assays used are insufficient for testing the effect of these mutations on motility that is relevant in vivo. The model is that TRAP's interaction with extracellular ligands stabilizes its 'open' position. This suggests that motility assays conducted with extracellular matrix eg Matrigel are a more appropriate test of motility.

    A: The reviewer raises interesting points. There are several types of sporozoite motility assays available, 2D on glass, 2D on ligands (e.g. Matrigel), 3D in polyacrylamide gels or Matrigel, and 3D in the skin. All of these have a number of advantages and disadvantages. 2D on glass is clearly the easiest, 3D in skin, clearly the most complex one. The advantage of the 2D on glass assay is that it reveals even tiny defects on motility that cannot be revealed in 3D assays as the enclosure of the parasite in a 3D matrix compensates for several defects, especially those in adhesion that are most relevant for our study here (see also e.g. Bane et al. Plos Path 2016, Ripp et al., EMBO Mol Med 2021). Hence, we believe that for understanding the role of adhesion in motility the 2D assay is highly valuable because it represents a minimal system. In contrast, the complex 3D environments, are closer to the natural situation but are less useful in our specific case. The fact that the mutants are severely limited in vivo (in the mosquito where they are blocked at the 2D surface of the salivary gland) suggests that the assays are highly relevant. We take this comment as a motivation to write a review/opinion like paper in the near future but feel it would distract if added here to e.g. the discussion.

    Experiments with DTT are difficult to interpret since there is once again no biochemical evidence that this treatment leads to a change in conformation of TRAP. DTT's effect on motility of the mutant could be non-specific. Overall, conclusions that are presented need to be supported by more data.

    A: We are aware of this and hence already discussed these potential shortcomings of our study in the discussion. Importantly: there is no known specific ligand to the TRAP I domain. Hence, we believe that biochemical tests would not justify the effort it would take to express the different domains.

    Reviewer #2 (Significance):

    Sporozoite motility is a prerequisite for infection by Plasmodium of the liver. A better mechanistic understanding of this process is significant for our understanding of the first step of malaria infection. TRAP is the major adhesin on the sporozoite surface and its loss abrogates sporozoite motility. The authors are to be commended for undertaking a challenging study.

    A: We thank the reviewer for appreciating our efforts, the advance provided, and for the constructive critique.

    Reviewer #3 (Evidence, reproducibility and clarity):

    This paper builds on previous structural studies from the Springer lab, which show that the I-domain of the malaria sporozoite surface protein TRAP can switch between two conformations ("open" and "closed"), similar to what happens with the I domain of integrins. In the current study the authors explore whether the open and closed conformations of TRAP are important for sporozoite migration and infectivity by generating parasites expressing each form locked in place using introduced disulfides. Locking TRAP into either form inhibits sporozoite gliding, invasion of the mosquito salivary glands and transmission from mosquito to mouse. The addition of low levels of a reducing agent can partially rescue these effects with the open form, which the authors suggest points to a requirement for TRAP to undergo a switch between its open and closed states to support these key processes in the parasite's life cycle.

    The paper is well written and the data and methods are clearly presented.

    Major Comments:

    Lines 109-115: The entire paper relies on the premise that the introduced disulfides lock the I domain of TRAP in either the open or closed conformation. In the absence of experimental proof that this is the case, it would be helpful to the reader to have more detail on how this can be confidently inferred from the previous work on integrins -- perhaps as a supplementary figure._

    A: We now added a statement on how the positions for the cysteines were selected and also a figure on structures and Alphafold prediction as also suggested by reviewers 1 and 2 as panels in Figure 1.

    The partial rescue of motility in the S210C/Q216C parasites by 50-100mM DTT is a VERY indirect approach to testing whether the conformational switch between the open and closed states is functionally important. The authors are appropriately reserved in their conclusions, but wildtype parasites are ultimately a better (less confounded) comparator; the authors may want to stress this more._

    A: Thanks for appreciating our efforts to navigate the limitations of our experimental system. We have changed our wording (e.g. replaced “fixed state” with “stabilized state” in the discussion) and added cautionary statements, which now read “While DTT clearly does not only act on TRAP, these data nevertheless suggest that…” (lines 353-354); “While not providing final proof, this phenotype indicates…” (lines 371); “…antibodies that stabilize… could contribute to stopping parasite migration” (line 415)

    The implications of the current results for the authors' previous stick-and-slip model of sporozoite motility should be discussed.

    A: We added a few sentences including two references on this in the discussion: “Sporozoites glide in a stick-slip fashion…” (lines 417-422)

    Minor Comments:

    The authors are experienced in the use reflection interference contrast microscopy to visualize attachment of the parasite to the substrate. Did they do any RICM on the mutants? Although not necessary for the paper, this would be a good way to support the interpretation of some of their results (eg, lines 185-190)._

    A: We indeed hoped that the mutants would allow us to exploit these techniques as this would give much insight into the mode of migration. However, all our previous work on RICM was done with salivary gland derived sporozoites, which are robustly gliding. In contrast, those derived from the hemolymph or oocysts do not glide very well. While we can quantitatively image them at low magnification (large field of view) settings, we have consistently failed to get sufficient data on most of our advanced assays that require single sporozoite imaging such as RICM, TIRFM, and laser tweezer experiments. This, unfortunately, is a real limitation of the system as most interesting mutants (see also e.g. recent paper by Yee et al., Plos Pathogens 2022) are not entering the salivary glands.

    Line 127: Figure 2A does not show "no growth difference to wild type mice"

    A: we split the sentence in two with Figure 2A now only referring to mosquito infections (now line 212)

    Line 154: "higher, but not significantly higher" - if the data don't meet the significance threshold being used, they cannot be called higher

    A: Yes, the thing here is that we always got more from the open mutant and hence could do experiments but indeed the stats showed it not to be significant. We changed the sentence to read “…was not significantly higher…” (line 242)

    Line 186: "showed nearly no floating parasites compared to the controls S210C and cFluo" - cFluo and S210C/Q216C show the same amount of floaters in Fig 5B. Also, S210C/Q216C HLS show similar levels of floaters to both controls in 2A.

    A: Thanks for spotting this mixup, we changed it to: “Interestingly, hemolymph sporozoites expressing the closed I domain (S210CF224C) showed more floating parasites suggesting reduced adhesion. Intriguingly, the few moving sporozoites of this mutant were also significantly faster than the corresponding controls.” (lines 296-299). We also pick up on this observation in the discussion in the new paragraph on stick-slip motility, lines 414-422) which links back to the Munter et al paper cited in line 303. This is in line with observations in mammalian cells where reduced ligand density also leads to less adhesion and faster migration.

    Fig S2 defines unproductive gliding to include waving, but Figs. 5 and S3 scores waving as a separate category

    A: We define it as unproductive motility, which we now changed into “unproductive movement” in the hope to be less misleading, clearly “lazy gliding” is motility while waving is no motility but some type of movement.

    Fig S3B and Fig 5D,E appear to be the same data presented twice

    A: Correct, we now state this explicitly in the figure legend to Figure S3B (lines 958-960) where they are reproduced together with the matching controls

    Line 304: "the inability of sporozoites with the TRAP I domain to migrate" (?)

    A: “conformationally stabilized” was missing and has now been included (line 453)

    Line 226: please explain what the + > ++++ qualitative descriptors signify in the tables and how they were scored

    A: Now added in the legends to the table, lines 885-887 and 892, respectively.

    Lines 282, 312: the authors should mention here the extensive work that has been done on efficacy of viral vaccines directed against a particular conformational state of the immunogen

    A: We were naturally tempted to do this but feel that this could hype the study more than is justified by our data.

    Fig 2B: The labels above the lanes are incomplete and therefore confusing; suggest sticking to the same nomenclature as in 2A

    A: Modified as suggested

    Typographical errors on lines: 58 (space missing), 80-81 (parentheses), 155 (Figure misspelled), 188 (expressing the closed (S210C/F224C)), 306 (comma after both)

    A: Thanks, these were corrected.

    CROSS-CONSULTATION COMMENTS
    Since all three reviewers questioned whether the introduced disulfides would have the assumed effects on I domain structure, the authors should provide a stronger rationale for this assumption or -- as suggested by reviewer 2 -- some actual data to support it. Reviewer 2's comment about the extracellular ligands available for the parasite to bind to in the gliding assay and whether this could influence the outcome (and relevance to what occurs in vivo) also deserves consideration.

    A: We have now added Alphafold predictions of the mutants (Figure 1) and also described better how we selected the mutations based on extensive work by the Springer lab (see newly added lines 130-202). We appreciate the idea of using extracellular ligands, however, all our previous assays (e.g. Klug et al. eLife 2020 and Perschmann et al., Nano Letter 2011) suggest that the parasites move on a large variety of surfaces at the same rate.

    Reviewer #3 (Significance):

    This is an elegant study that contributes important new information to our understanding of apicomplexan parasite motility and the function of the TRAP protein. The results will be of significant interest to those who study parasite motility, and likely also to those who study the role of integrins in cellular adhesion and signaling. The data nicely connect what was previously known about conformational changes in integrins with parasite adhesion to the substrate and motility.

    I have expertise in the area of parasite motility._

    A: We thank the reviewer for the constructive comments and appreciation of our work

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    This paper builds on previous structural studies from the Springer lab, which show that the I-domain of the malaria sporozoite surface protein TRAP can switch between two conformations ("open" and "closed"), similar to what happens with the I domain of integrins. In the current study the authors explore whether the open and closed conformations of TRAP are important for sporozoite migration and infectivity by generating parasites expressing each form locked in place using introduced disulfides. Locking TRAP into either form inhibits sporozoite gliding, invasion of the mosquito salivary glands and transmission from mosquito to mouse. The addition of low levels of a reducing agent can partially rescue these effects with the open form, which the authors suggest points to a requirement for TRAP to undergo a switch between its open and closed states to support these key processes in the parasite's life cycle.

    The paper is well written and the data and methods are clearly presented.

    Major Comments:

    Lines 109-115: The entire paper relies on the premise that the introduced disulfides lock the I domain of TRAP in either the open or closed conformation. In the absence of experimental proof that this is the case, it would be helpful to the reader to have more detail on how this can be confidently inferred from the previous work on integrins -- perhaps as a supplementary figure.

    The partial rescue of motility in the S210C/Q216C parasites by 50-100mM DTT is a VERY indirect approach to testing whether the conformational switch between the open and closed states is functionally important. The authors are appropriately reserved in their conclusions, but wildtype parasites are ultimately a better (less confounded) comparator; the authors may want to stress this more.

    The implications of the current results for the authors' previous stick-and-slip model of sporozoite motility should be discussed.

    Minor Comments:

    The authors are experienced in the use reflection interference contrast microscopy to visualize attachment of the parasite to the substrate. Did they do any RICM on the mutants? Although not necessary for the paper, this would be a good way to support the interpretation of some of their results (eg, lines 185-190).

    Line 127: Figure 2A does not show "no growth difference to wild type mice"

    Line 154: "higher, but not significantly higher" - if the data don't meet the significance threshold being used, they cannot be called higher

    Line 186: "showed nearly no floating parasites compared to the controls S210C and cFluo" - cFluo and S210C/Q216C show the same amount of floaters in Fig 5B. Also, S210C/Q216C HLS show similar levels of floaters to both controls in 2A.

    Fig S2 defines unproductive gliding to include waving, but Figs. 5 and S3 scores waving as a separate category

    Fig S3B and Fig 5D,E appear to be the same data presented twice

    Line 304: "the inability of sporozoites with the TRAP I domain to migrate" (?)

    Line 226: please explain what the + > ++++ qualitative descriptors signify in the tables and how they were scored

    Lines 282, 312: the authors should mention here the extensive work that has been done on efficacy of viral vaccines directed against a particular conformational state of the immunogen

    Fig 2B: The labels above the lanes are incomplete and therefore confusing; suggest sticking to the same nomenclature as in 2A

    Typographical errors on lines: 58 (space missing), 80-81 (parentheses), 155 (Figure misspelled), 188 (expressing the closed (S210C/F224C)), 306 (comma after both)

    Referees cross-commenting

    Since all three reviewers questioned whether the introduced disulfides would have the assumed effects on I domain structure, the authors should provide a stronger rationale for this assumption or -- as suggested by reviewer 2 -- some actual data to support it. Reviewer 2's comment about the extracellular ligands available for the parasite to bind to in the gliding assay and whether this could influence the outcome (and relevance to what occurs in vivo) also deserves consideration.

    Significance

    This is an elegant study that contributes important new information to our understanding of apicomplexan parasite motility and the function of the TRAP protein. The results will be of significant interest to those who study parasite motility, and likely also to those who study the role of integrins in cellular adhesion and signaling. The data nicely connect what was previously known about conformational changes in integrins with parasite adhesion to the substrate and motility.

    I have expertise in the area of parasite motility.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    This work seeks to investigate the effects on sporozoite motility of hypothesized conformational changes in TRAP's integrin domain. Previous structural studies suggested that the Integrin-like domain of TRAP assumes 'open' and 'closed' conformations induced by ligand binding. Here the authors hypothesize that TRAP's ability to dynamically switch between these states is crucial for sporozoite motility. The hypothesis is interesting and the authors elected to test it by 'fixing' TRAP in constitutively 'open' and 'closed' conformations by introducing Cys residues that are presumed to form disulphide bonds resulting in these states.

    Major Comments

    The approach is creative. The data that are presented are of high quality with adequate reproducibility. However, as written the manuscript does not provide a rationale for the specific substitutions they chose, how these substitutions are expected to lead to 'open' and 'closed' states, and how they mimic the natural route of TRAP transitioning between the 'open' and 'closed' conformations.

    Furthermore, there is no biochemical, biophysical or modeling data demonstrating that the introduced mutations impact the folding/unfolding of TRAP's I domain in the manner hypothesized. Therefore, it is difficult to interpret subsequent phenotypic data from the two mutant lines. While mutant parasites display defects in gliding motility, these defects are unexpected, perhaps pointing to alternative explanations - such as aberrant inter- or intra-molecular disulphide bonding in TRAP's extracellular domain.

    About 10% of mutant sporozoites assumed to be in a constitutively 'closed' conformation display gliding motility and they move faster that WT. Yet this mutant did not cause patency in mice when introduced either via mosquito bite or IV injection. In contrast, the mutant assumed to be in an 'open' conformation displayed no motility in vitro but was able to infect mice (albeit at significantly reduced compared to controls). Presumably these data suggest that the in vitro gliding motility assays used are insufficient for testing the effect of these mutations on motility that is relevant in vivo. The model is that TRAP's interaction with extracellular ligands stabilizes its 'open' position. This suggests that motility assays conducted with extracellular matrix eg Matrigel are a more appropriate test of motility.

    Experiments with DTT are difficult to interpret since there is once again no biochemical evidence that this treatment leads to a change in conformation of TRAP. DTT's effect on motility of the mutant could be non-specific. Overall, conclusions that are presented need to be supported by more data.

    Significance

    Sporozoite motility is a prerequisite for infection by Plasmodium of the liver. A better mechanistic understanding of this process is significant for our understanding of the first step of malaria infection. TRAP is the major adhesin on the sporozoite surface and its loss abrogates sporozoite motility. The authors are to be commended for undertaking a challenging study.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Plasmodium sporozoites rely on gliding motility to invade the salivary glands of the mosquito vector where they reside until being inoculated into the vertebrate host. Once in the vertebrate host, sporozoites again have to glide to pass through liver cells and reach the final host cell before exo-erythrocytic replication occurs. In sporozoites, thrombospondin-related anonymous protein (TRAP) is the key adhesin, linking the molecular motor to the substrate. TRAP and TRAP-like proteins contain on the extracellular side an inserted (I) domain that is responsible for substrate binding. The I domain can be in an open or closed state which can be fixed by mutating specific amino acids. Baumann et al. then assessed the effect of these states on gliding motility and infection. Whilst no effect was seen for both mutants with closed or open states until the production of haemolymph sporozoites, the number of salivary gland sporozoites were highly reduced in both mutants. The closed mutant was not able to be transmitted from mosquitoes to mice whilst the open mutant is severely impacted in transmission. Gliding of both mutant is highly impaired. Some of these phenotypes could be reverse by adding a reducing agent.

    Major comments:

    This is an elegant study with exhaustive experiments to address the research questions. One of the things I wondered about is the effect of exchanging amino acids. As far as I understood, structural information of integrins from other organisms informed the authors about positions that should be mutated and about the impact (or loss of it) on the 3D structure of the domain. Would it not be useful to run a structure prediction programme such as Alphafold on all the mutants to at least confirm stability of the domain structure upon mutation in silico?

    Some of the concentrations of reducing agents tested are very high. Can the authors be sure that the parasites are not dead?

    Minor comments:

    • Please make sure that punctuation is correct (e.g. missing commas) and that there are no other typing errors.
    • Line 50: This sentence may imply mechanical rupture of oocysts rather than active egress of the sporozoite. Please re-phrase.
    • Line 82: Do you mean a complexed Mg2+ ion?
    • The introduction stops rather abruptly. Maybe a sentence of the significance of this study could be added.
    • Line 361: 20 million parasites each? Please re-phrase to make it clearer.
    • Line 363: Field of view needs to defined: What is the magnification used to observe exflagellation?
    • Materials and Methods: Please write out reagent names for the first time.

    Figures:

    • Figure 1A is very small. The label font of the whole figure 1 is often too small.
    • Figure 1D: Please list the components in the figure legend, e.g. blue: substrate etc.
    • Figure 3: The figure title should be changed as there is no complete failure of sporozoites with fixed I-domains to invade salivary glands.
    • Figure 5: for clarity, it would be easier if Figure C would not be squeezed into the legends of A and B.

    Significance

    This study addresses in detail the mechanism of Plasmodium TRAP I domain in gliding motility and transmission. It builds on work from many years and groups including their own and contributes to deepen our understanding of TRAP-family proteins, gliding motility in Plasmodium sporozoites and maybe even in other Plasmodium stages or other Apicomplexan parasites.

    This work is of interest to researchers in the field of Plasmodium mosquito stages and transmission as well as scientist who work on apicomplexan gliding motility and transmission.

    I have previously worked on Plasmodium mosquito stages, but currently work on Toxoplasma gondii.

  5. Version published to 10.1101/2022.08.24.505106v1 on bioRxiv