Characterization of Dense Granule Metalloproteinase INS-16 in Cryptosporidium parvum
, Yaoyu Feng 1,* and Na Li 1,*
Round 1
Reviewer 1 Report
Cryptosporidium parvum is an apicomplexan parasite that infects the intestinal epithelium of humans and livestock worldwide. The disease is particularly dangerous to infants and immunocompromised individuals for whom there is no effective treatment or vaccination. Nitazoxanide is approved for use in adult, immunocompetent patients, but it is not effective or approved for use in the most vulnerable populations: infants and immunocompromised patients.
Currently, there are no effective vaccines against C. parvum, and basic research on the biology of the parasite and host-pathogen interactions is needed to identify potential therapeutic targets and vaccines. These results fit well with targeted research and this topic.
The C. parvum genome contains an extended family of 22 M16 metalloproteases, most of which have unknown function. The results of the present studies have shown marked differences in the expression and subcellular localization of two INS members. This suggests that INS -16 likely exerts multiple functions in C. parvum invasion and growth. Of particular note is the comprehensive research from obtaining recombinant proteins, to analysis at the level of gene and protein expression, to inhibition assay in in vitro culture, which provides the basis for further use in in vivo assays or gut organoids and the introduction of a three-dimensional culture model.
The work is scientifically sound and worthy of consideration for publication. The experimental design is correct; the methods are correctly described.
Author Response
Many thanks for the constructive comments and suggestions. We have taken all suggestions by the reviewers into consideration, and in each case, made the suggested modifications. Below are the specifics of the revision (reviewers’ suggestion first followed by the response). The following line numbers correspond to those in the marked copy of the revised manuscript.
Reviewer #1:
Cryptosporidium parvum is an apicomplexan parasite that infects the intestinal epithelium of humans and livestock worldwide. The disease is particularly dangerous to infants and immunocompromised individuals for whom there is no effective treatment or vaccination. Nitazoxanide is approved for use in adult, immunocompetent patients, but it is not effective or approved for use in the most vulnerable populations: infants and immunocompromised patients. Currently, there are no effective vaccines against C. parvum, and basic research on the biology of the parasite and host-pathogen interactions is needed to identify potential therapeutic targets and vaccines. These results fit well with targeted research and this topic.
The C. parvum genome contains an extended family of 22 M16 metalloproteases, most of which have unknown function. The results of the present studies have shown marked differences in the expression and subcellular localization of two INS members. This suggests that INS -16 likely exerts multiple functions in C. parvum invasion and growth. Of particular note is the comprehensive research from obtaining recombinant proteins, to analysis at the level of gene and protein expression, to inhibition assay in in vitro culture, which provides the basis for further use in in vivo assays or gut organoids and the introduction of a three-dimensional culture model.
The work is scientifically sound and worthy of consideration for publication. The experimental design is correct; the methods are correctly described.
RESPONSE: Many thanks for the positive comments.
Reviewer 2 Report
The manuscript presented for review (ijms-1800222-peer-review-v1) concerns the insulinase-like metalloproteinase 16 (INS-16) from Cryptosporidium parvum. So far known INSs have impact on the host-pathogen interaction, therefore, the comparison of two INS which are paralogs and additionally taking the attempt to identify the function of this protein is very valuable.
The research conducted by the authors was very well planned, the methodology was chosen very accurately and the manuscript was written very clearly. Additionally, the added materials in the supplement indicate the Authors' high meticulousness and the use of good laboratory practices. My only minor comments are on the Materials and methods chapter, and it seems to me that some data should be supplemented. Below, I indicate exactly the places in the manuscript that, in my opinion, should contain more precise descriptions of the methodology.
l.291 First of all, there is no information about the conditions of running cultures of HCT-8 cells, I suppose, prior the addition of oocysts (because the description is this place is not clear). And also, with what kind of medium, these oocysts were suspended? I would be grateful to the authors if they supplemented this or provided a reference publication.
l. 308 Please complete the PCR amplification conditions for both full cgd3_4270 gene and domain I.
Author Response
Many thanks for the constructive comments and suggestions. We have taken all suggestions by the reviewers into consideration, and in each case, made the suggested modifications. Below are the specifics of the revision (reviewers’ suggestion first followed by the response). The following line numbers correspond to those in the marked copy of the revised manuscript.
Reviewer #2:
The manuscript presented for review (ijms-1800222-peer-review-v1) concerns the insulinase-like metalloproteinase 16 (INS-16) from Cryptosporidium parvum. So far known INSs have impact on the host-pathogen interaction, therefore, the comparison of two INS which are paralogs and additionally taking the attempt to identify the function of this protein is very valuable.
The research conducted by the authors was very well planned, the methodology was chosen very accurately and the manuscript was written very clearly. Additionally, the added materials in the supplement indicate the Authors' high meticulousness and the use of good laboratory practices. My only minor comments are on the Materials and methods chapter, and it seems to me that some data should be supplemented. Below, I indicate exactly the places in the manuscript that, in my opinion, should contain more precise descriptions of the methodology.
RESPONSE: Many thanks for the positive comments.
L291 First of all, there is no information about the conditions of running cultures of HCT-8 cells, I suppose, prior the addition of oocysts (because the description in this place is not clear). And also, with what kind of medium, these oocysts were suspended? I would be grateful to the authors if they supplemented this or provided a reference publication.
RESPONSE: Thanks for the suggestion. We have added the description in Lines 291-297.
L308 Please complete the PCR amplification conditions for both full cgd3_4270 gene and domain I.
RESPONSE: Thanks for the suggestion. We have added the description in Lines 310-315.
