Histidine-rich protein 2: a new pathogenic factor of Plasmodium falciparum malaria

Article activity feed

1. 

   Review Commons

   Feb 1, 2022

   Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   RESPONSE TO REVIEWER #1:

   We wish to express our appreciation to Reviewer #1 for his or her insightful comments, which will significantly improve this paper. We thank the reviewers for giving us the opportunity to improve the manuscript. We have responded to all the comments pointed out. The revised sections are highlighted in red characters and yellow backgrounds in the preliminary revised manuscript.

   Reviewer #1 (Evidence, reproducibility and clarity (Required)):

   This manuscript "Histidine-rich protein 2: a new pathogenic 1 factor of Plasmodium falciparum malaria" by Iwasaki, et al. reports effects of recombinant HRP2 protein on various mammalian cell …

   Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   RESPONSE TO REVIEWER #1:

   We wish to express our appreciation to Reviewer #1 for his or her insightful comments, which will significantly improve this paper. We thank the reviewers for giving us the opportunity to improve the manuscript. We have responded to all the comments pointed out. The revised sections are highlighted in red characters and yellow backgrounds in the preliminary revised manuscript.

   Reviewer #1 (Evidence, reproducibility and clarity (Required)):

   This manuscript "Histidine-rich protein 2: a new pathogenic 1 factor of Plasmodium falciparum malaria" by Iwasaki, et al. reports effects of recombinant HRP2 protein on various mammalian cell lines. The MS clearly demonstrates that recombinant HRP2 enters into HT1080 cells, causes inhibition of autolysosome fusion, increases lysosomal Ca ion concentration and reduces general autophagic degradation. The authors also show that the presence of FBS or metal chelators like EDTA and EGTA mitigate toxicity of HRP2, as the former traps HRP2 and the latter compete with HRP2 for Ca binding. The experiments are appropriately carried out with suitable controls in most of the cases. There are some concerns as listed below:

   **Major concerns:**

   1.HRP2 has been shown to be associated with virulence and causes vascular leakage, particularly cerebral malaria (references 37 and 38 ). Plasmodium falciparum histidine-rich protein II has been demonstrated to exacerbate experimental cerebral malaria in mice, which has been proposed to be associated with vascular leakage, activation of inflammasome and cytokine production (references 37, 38 and PMID: 31858717). This study complements the previous findings of the effect of HRP2 on mammalian cells. However, this study reveals another mechanism by which HRP2 might cause toxicity, which is inhibition of general autophagy and increase in lysosomal Ca concentration. However, whether these in vitro effects would translate in vivo needs to be shown.

   Response: We sincerely appreciate the reviewer's effort to evaluate our work. As the reviewer pointed out, this is an *in vitro *study, so further in vivo validation is essential in the future. However, it is also true that we discovered new findings that have been overlooked because we conducted an artificial and simple in vitro experiment. In the future, it is necessary to demonstrate the cytotoxicity, autophagy inhibition, and lysosomal calcium concentration variation of PfHRP2 by in vivo studies using model animals. Concretely, we need to confirm whether PfHRP2 behaves as a similar virulence factor in vivo by animal experiments using PfHRP2-administrated or PfHRP2-overexpressing/deficient P. falciparum-infected mouse models. These future tasks have been added to the Discussion (page 9, lines 294–297 and 309–310; page 10, lines 339–342). We have also added the study (PMID: 31858717) reporting PfHRP2 elicits pro-inflammatory effect and induces vascular permeability as reference 40.

   Furthermore, the title of the original paper was vague and gave the impression that it included in vivo experiments. Therefore, to avoid misunderstanding, we modified the paper's title to be more concrete, "Plasmodium falciparum histidine-rich protein II exhibits cell penetration and cytotoxicity with autophagy dysfunction".

   Reference

   P. Dinarvand, L. Yang, I. Biswas, H. Giri, A. R. Rezaie, Plasmodium falciparum histidine rich protein HRPII inhibits the anti-inflammatory function of antithrombin. J. Thromb. Haemost. 18, 1473–1483 (2020).

   2.All the experiments are done with recombinant HRP2 and BSA as a control. The authors should show if similar effects happen with infected parasites.

   Response: As the reviewer pointed out, it is required to perform *in vivo *experiments, i.e., to clarify whether the same phenomenon observed in the present study occurs in PfHRP2-administrated or P. falciparum-infected mouse models. However, in vivo studies are not possible immediately because we do not have the research facilities to carry out in vivo experiments. Therefore, we have added statements (page 9, lines 294–297 and 309–310; page 10, lines 339–342) to emphasize that the present findings are limited to in vitro and that further in vivo studies described above will be required in the future.

   3.HRP2 is released in circulation, making it accessible to endothelial cells and immune cells. How would it reach to the equivalents of these cells in the human body?

   Response: Since PfHRP2 induces vascular permeability as described in References 37–40, we propose that PfHRP2 can reach and contact cells in the human body after causing vascular leakage. I have added this possibility of contact between PfHRP2 and cells in the human body to Discussion (page 9, lines 287–290).

   **Minor concerns**

   1.p62 is an appropriate marker to assess autophagy cargo degradation. If possible, it would be good to support this with LC3 processing as well.

   Response: Following the reviewer's advice, we will use LC3 as an autophagy marker as well as p62 to evaluate the autophagy inhibition of PfHRP2. Concretely, we plan to treat HT1080 cells with PfHRP2 (1 μM) for 12–60 hours and quantify the amount of LC3 protein by Western blotting. The results of this experiment will be added to Fig. 5 in the main manuscript.

   2.HRP2 might affect general lysosomal degradation process. The authors can also check whether HPR2 affects degradation of a lysosomal substrate.

   Response: Following the reviewer's advice, we will determine the effect of PfHRP2 on lysosomal degradation activity using the plasmid-based lysosomal-METRIQ (MEasurement of protein Transporting integrity by RatIo Quantification) probe, reported in a previous study (https://doi.org/10.1038/s41598-019-48131-2), to quantify lysosomal activity. The results of this experiment will be added to Fig. 5 in the main manuscript.

   Reviewer #1 (Significance (Required)):

   This study compelements previous findings (references 37, 38 and PMID: 31858717). It identifies a new mechanism by which HRP2 might cause toxicity. However, it is completely an in vitro study, and the previous studies (references 37 and 38) have used in vivo models as well.

   Response: We wish to thank the reviewer for this comment. As the reviewer pointed out, this study is completely in vitro, and further in vivo studies are essential in the future. Therefore, we have added statements (page 9, lines 294–297 and 309–310; page 10, lines 339–342) to emphasize that the present findings are limited to in vitro and that further in vivo studies are required in the future. We have also added the study (PMID: 31858717) reporting PfHRP2 elicits pro-inflammatory effect and induces vascular permeability as reference 40.

   Furthermore, the title of the original paper was vague and gave the impression that it included in vivo experiments. Therefore, to avoid misunderstanding, we modified the paper's title to be more concrete.

   Reference

   P. Dinarvand, L. Yang, I. Biswas, H. Giri, A. R. Rezaie, Plasmodium falciparum histidine rich protein HRPII inhibits the anti-inflammatory function of antithrombin. J. Thromb. Haemost. 18, 1473–1483 (2020).

   We thank you again for giving us the opportunity to improve our paper, and we hope that the changes are satisfactory.

   RESPONSE TO REVIEWER #2:

   We wish to express our appreciation to Reviewer #2 for his or her insightful comments, which will significantly improve this paper. We thank the reviewers for giving us the opportunity to improve the manuscript. We have responded to all the comments pointed out. The revised sections are highlighted in red characters and yellow backgrounds in the preliminary revised manuscript.

   Reviewer #2 (Evidence, reproducibility and clarity (Required)):

   This paper showed that recombinant Plasmodium falciparum HRPII generated in E. coli is internalized by human tumor derived cells lines and at high concentrations, induces calcium-dependent cell death. The authors propose that HRPII inhibits autolysosome formation and autophagy.

   Of major concern is the use of E. coli generated HRP2 without addressing the inherent confounders of copurified bacterial components, namely endotoxin LPS. It is crucial for validation of their conclusions that the authors address steps taken to remove endotoxin which is known to bind poly-histidine and HRPII, the quantification of endotoxin bound to purified protein, and the LPS sensitivity of model cell lines. Even small quantities of LPS have been shown to potentially inhibit endosome maturation (https://doi.org/10.1074/jbc.M114.611442). Would recommend caution with conclusions regarding cytotoxicity and autophagy inhibition without addressing this issue.

   Response: We sincerely appreciate the reviewer's effort to evaluate our work. The reviewer points out that the endotoxin LPS may also affect the cytotoxicity and autophagy inhibition of PfHRP2 in this study. The reviewer's point is crucial, and we agree with the reviewer. In our study, recombinant PfHRP2 was captured by anti-FLAG antibody-immobilized affinity gel (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) and washed with 20-bed volumes of washing buffer (20 mM Tris-HCl pH7.4, 500 mM NaCl, 0.1% Triton X-100) to remove contaminants including endotoxin LPS according to the manufacturer's protocol (https://ruo.mbl.co.jp/bio/dtl/dtlfiles/3328R-ver4.0.pdf). After washing, affinity gel was equilibrated with 10-bed volumes of washing buffer without Triton X-100, and recombinant PfHRP2 was eluted by 10-bed volumes of elution buffer (20 mM Tris-HCl pH7.4, 500 mM NaCl, 0.1 mg/mL FLAG peptide: DYKDDDDK). However, we did not determine the residual endotoxin LPS bound to purified PfHRP2. To address the reviewer's concern, we will follow the reviewer's suggestion and quantify the residual endotoxin LPS in the purified PfHRP2 using the LAL Endotoxin Assay Kit. We also plan to test whether the same amount of endotoxin LPS alone as the residual endotoxin LPS affects cytotoxicity and autophagy inhibition. The results of additional experiments on endotoxin LPS will be added to Supplementary Information as Fig. S2. Furthermore, we have added additional information on the purification and washing of PfHRP2 to the Materials and methods section (page 11, lines 356–362).

   Additional concerns for specific experiments are as follows:

   Figure 2A. There is an increase in BSA penetration at lower pH as well which suggests nonspecific increased cell permeability.

   Response: As pointed out by the reviewer, the cell membrane permeability of BSA was enhanced at low pH (pH less than 5.8), and this result implies an increase in nonspecific cell permeability. Since we have reported in another study (https://doi.org/10.1093/bbb/zbab221) that BSA shows cell penetration to human gastric cancer cell lines at pH 5.0, the cell membrane permeability of BSA at low pH in this study is satisfactory. However, comparing pH 7.4 and pH 5.6, the net charge of BSA increased by 21.9 from -14.0 (pH7.4) to +7.9 (pH5.6), and the cell penetration increased by 34%. On the other hand, the net charge of PfHRP2 increased by 79.4 from -19.2 (pH7.4) to +60.2 (pH5.6), and the cell penetration increased by 246%. This suggests that the increase in cell membrane permeability of PfHRP2 under low pH conditions is due to the increase in net charge, not to the non-specific increase in cell permeability as seen in BSA. The above explanation has been added to lines 97–103.

   Figure 3A, 3B, and 4C. There is inconsistency between the cell viability data. For example, in panel A, 1 μM of HRPII for 24 h showed 84% cell viability whereas in panel B, the cell viability is 61% for 1 μM HRP2 by 24 hours. Figure 3A and 4C (full length) differ at cell viability for 5 μM HRP2.

   Response: We thank the reviewer for the critical remarks. There was an error in the time condition described in the graph of Fig. 3A. Correctly, Fig. 3A is the result of cell viability treated with 1 μM PfHRP2 for 3 hours, so we have corrected the time condition described in Fig. 3A. Namely, Fig. 3A and 3B show that a 3-hour treatment with 1 μM PfHRP2 results in 84% cell viability, but a 24-hour treatment with 1 μM PfHRP2 decreases cell viability to 61%. These results are correctly described in lines 119–120, highlighted in yellow.

   On the other hand, as the reviewer points out, in Fig. 3A and Fig. 4C (full-length PfHRP2), the cell viability treated with 5 μM PfHRP2 for 24 hours was 5% and 26%, respectively. We believe that the discrepancy in these values is an experimental error. However, both Fig. 3A and Fig. 4C (full-length PfHRP2) agree that 5 μM PfHRP2 is statistically and significantly cytotoxic, which should not affect the claims of this study.

   Figure 5C. It would be more informative if the cell viability data at 1 μM of HRP at timepoints beyond 60 hours and for bafilomycin treatment is also presented.

   Response: We thank the reviewer for their suggestions. However, the purpose of the experiment in Figure 5C is to prove that PfHRP2 induces autolysosomal dysfunction. Since we confirmed that treatment of cells with 1 µM PfHRP2 for 60 hours resulted in accumulation of p62 in the same amount as the positive control, Bafilomycin A1, we believe that no further additional experiments are necessary.

   Figure 3D. (Minor) Consider additional experimental detail regarding maintenance of cell cultures for 5 day. Are there interval media changes or supplement additions?

   Response: We apologize for the insufficient information in the description of the experimental procedure in Fig. 3D. In the experiment in Fig. 3D, cell culture was maintained for 5 days by changing a fresh medium containing each concentration of proteins every day. We have added this information to the legends of Figure 3 (page 23, lines 653–655) and Figure S2 (page 4, lines 28–29).

   Reviewer #2 (Significance (Required)):

   The authors present the novel finding of HRP2 permeability into human cells. The significance of these findings is limited given the major confounder with endotoxin and also since the experiments were conducted in tumor-derived cells lines with supraphysiologic concentrations of HRPII. Although the authors showed cell viability effects with lower concentrations over 3 and 5 days, the bulk of the experiments were at more than 10-fold mean physiological concentrations. Also, since these are early findings in tumor-derived cell lines, it is difficult to extrapolate the physiological relevance of these findings and use of calcium chelators as therapeutics.

   Several studies have proposed a pathogenic role for HRP2 including those cited in the paper regarding blood-brain barrier disruption (references 37 and 38), coagulation disruption (DOI: 10.1182/blood-2010-12-326876), and pro-inflammatory signaling (DOI: 10.1111/jth.14713). The challenge with all these studies is establishing the clinical relevance of the multitude of HRPII effects. If the issue of endotoxin is addressed, this paper could establish an interesting mechanism for further study in more clinically representative systems.

   Our lab has studied the many functions of HRPII including catalysis of heme polymerization, inhibition of antithrombin, brain endothelial disruption using tissue culture and mouse models.

   Response: As pointed out by the reviewer, this study must clear up the effect of endotoxin LPS. In this regard, as mentioned above, we plan to quantify the residual endotoxin LPS in the purified PfHRP2 using the LAL Endotoxin Assay Kit. We will also check the effect of the endotoxin LPS itself on cytotoxicity and autophagy inhibition.

   Furthermore, as the reviewer pointed out, this is an in vitro study using high concentrations of PfHRP2 and a tumor-derived cell line, so further in vivo validation is essential in the future. However, it is also true that we discovered new findings that have been overlooked because we conducted an artificial and simple in vitro experiment. In the future, it is necessary to demonstrate the cytotoxicity and autophagy inhibition of PfHRP2 by in vivo studies using model animals. Concretely, we need to confirm whether PfHRP2 behaves as a similar virulence factor in vivo by animal experiments using PfHRP2-administrated or PfHRP2-overexpressing/deficient P. falciparum-infected mouse models. We also need to demonstrate that calcium chelators such as EDTA have an *in vivo *therapeutic effect. These future tasks have been added to the Discussion (page 9, lines 294–297 and 309–310). We have also added the studies (DOI: 10.1182/blood-2010-12-326876, DOI: 10.1111/jth.14713) reporting PfHRP2 elicits pro-inflammatory effect and induces vascular permeability as reference 37 and 40.

   Furthermore, the title of the original paper was vague and gave the impression that it included in vivo experiments. Therefore, to avoid misunderstanding, we modified the paper's title to be more concrete, "Plasmodium falciparum histidine-rich protein II exhibits cell penetration and cytotoxicity with autophagy dysfunction".

   References

   M. Ndonwi, et al., Inhibition of antithrombin by Plasmodium falciparum histidine-rich protein II. Blood 117, 6347–6354 (2011). P. Dinarvand, L. Yang, I. Biswas, H. Giri, A. R. Rezaie, Plasmodium falciparum histidine rich protein HRPII inhibits the anti-inflammatory function of antithrombin. J. Thromb. Haemost. 18, 1473–1483 (2020).

   We thank you again for giving us the opportunity to improve our paper, and we hope that the changes are satisfactory.

   Read the original source
2. 

   Review Commons

   Feb 1, 2022

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

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   This paper showed that recombinant Plasmodium falciparum HRPII generated in E. coli is internalized by human tumor derived cells lines and at high concentrations, induces calcium-dependent cell death. The authors propose that HRPII inhibits autolysosome formation and autophagy.

   Of major concern is the use of E. coli generated HRP2 without addressing the inherent confounders of copurified bacterial components, namely endotoxin LPS. It is crucial for validation of their conclusions that the authors address steps taken to remove endotoxin which is known to bind poly-histidine and HRPII, the quantification of endotoxin bound to purified …

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

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   This paper showed that recombinant Plasmodium falciparum HRPII generated in E. coli is internalized by human tumor derived cells lines and at high concentrations, induces calcium-dependent cell death. The authors propose that HRPII inhibits autolysosome formation and autophagy.

   Of major concern is the use of E. coli generated HRP2 without addressing the inherent confounders of copurified bacterial components, namely endotoxin LPS. It is crucial for validation of their conclusions that the authors address steps taken to remove endotoxin which is known to bind poly-histidine and HRPII, the quantification of endotoxin bound to purified protein, and the LPS sensitivity of model cell lines. Even small quantities of LPS have been shown to potentially inhibit endosome maturation (https://doi.org/10.1074/jbc.M114.611442). Would recommend caution with conclusions regarding cytotoxicity and autophagy inhibition without addressing this issue.

   Additional concerns for specific experiments are as follows:

   Figure 2A. There is an increase in BSA penetration at lower pH as well which suggests nonspecific increased cell permeability.

   Figure 3A, 3B, and 4C. There is inconsistency between the cell viability data. For example, iIn panel A, 1 uM of HRPII for 24 h showed 84% cell viability whereas in panel B, the cell viability is 61% for 1 uM HRP2 by 24 hours. Figure 3A and 4C (full length) differ at cell viability for 5 uM HRP2.

   Figure 5C. It would be more informative if the cell viability data at 1 uM of HRP at timepoints beyond 60 hours and for bafilomycin treatment is also presented.

   Figure 3D. (Minor) Consider additional experimental detail regarding maintenance of cell cultures for 5 day. Are there interval media changes or supplement additions?

   Significance

   The authors present the novel finding of HRP2 permeability into human cells. The significance of these findings is limited given the major confounder with endotoxin and also since the experiments were conducted in tumor-derived cells lines with supraphysiologic concentrations of HRPII. Although the authors showed cell viability effects with lower concentrations over 3 and 5 days, the bulk of the experiments were at more than 10-fold mean physiological concentrations. Also, since these are early findings in tumor-derived cell lines, it is difficult to extrapolate the physiological relevance of these findings and use of calcium chelators as therapeutics.

   Several studies have proposed a pathogenic role for HRP2 including those cited in the paper regarding blood-brain barrier disruption (references 37 and 38), coagulation disruption (DOI: 10.1182/blood-2010-12-326876), and pro-inflammatory signaling (DOI: 10.1111/jth.14713). The challenge with all these studies is establishing the clinical relevance of the multitude of HRPII effects. If the issue of endotoxin is addressed, this paper could establish an interesting mechanism for further study in more clinically representative systems.

   Our lab has studied the many functions of HRPII including catalysis of heme polymerization, inhibition of antithrombin, brain endothelial disruption using tissue culture and mouse models.

   Read the original source
3. 

   Review Commons

   Feb 1, 2022

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

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   This manuscript "Histidine-rich protein 2: a new pathogenic 1 factor of Plasmodium falciparum malaria" by Iwasaki, et al. reports effects of recombinant HRP2 protein on various mammalian cell lines. The MS clearly demonstrates that recombinant HRP2 enters into HT1080 cells, causes inhibition of autolysosome fusion, increases lysosomal Ca ion concentration and reduces general autophagic degradation. The authors also show that the presence of FBS or metal chelators like EDTA and EGTA mitigate toxicity of HRP2, as the former traps HRP2 and the latter compete with HRP2 for Ca binding. The experiments are appropriately carried out with …

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

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   This manuscript "Histidine-rich protein 2: a new pathogenic 1 factor of Plasmodium falciparum malaria" by Iwasaki, et al. reports effects of recombinant HRP2 protein on various mammalian cell lines. The MS clearly demonstrates that recombinant HRP2 enters into HT1080 cells, causes inhibition of autolysosome fusion, increases lysosomal Ca ion concentration and reduces general autophagic degradation. The authors also show that the presence of FBS or metal chelators like EDTA and EGTA mitigate toxicity of HRP2, as the former traps HRP2 and the latter compete with HRP2 for Ca binding. The experiments are appropriately carried out with suitable controls in most of the cases. There are some concerns as listed below:

   Major concerns:

   1.HRP2 has been shown to be associated with virulence and causes vascular leakage, particularly cerebral malaria (references 37 and 38 ). Plasmodium falciparum histidine-rich protein II has been demonstrated to exacerbate experimental cerebral malaria in mice, which has been proposed to be associated with vascular leakage, activation ofinflamosome and cytokine production (references 37, 38 and PMID: 31858717). This study complements the previous findings of the effect of HRP2 on mammalian cells. However, this study reveals another mechanism by which HRP2 might cause toxicity, which is inhibition of general autophagy and increase in lysosomal Ca concentration. However, whether these in vitro effects would translate in vivo needs to be shown.

   2.All the experiments are done with recombinant HRP2 and BSA as a control. The authors should show if similar effects happen with infected parasites.

   3.HRP2 is released in circulation, making it accessibele to endothelial cells and immune cells. How would it reach to the equivalents of these cells in the human body?

   Minor concerns

   1.p62 is an appropriate marker to assess autophagy cargo degradation. If possible, it would be good to support this with LC3 processing as well.

   2.HRP2 might affect general lysosomal degradation process. The authors can also check whether HPR2 affects degradation of a lysosomal substrate.

   Significance

   This study compelements previous findings (references 37, 38 and PMID: 31858717). It identifies a new mechanism by which HRP2 might cause toxicity. However, it is completely an in vitro study, and the previous studies (references 37 and 38) have used in vivo models as well.

   Read the original source
4. 

   Version published to 10.1101/2021.11.19.469193v1 on bioRxiv

   Nov 19, 2021