Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review of Gonzalez-Chavez 2026 ; Genes

 

Summary

 

Gonzalez-Chavez et al. present a well-thoughtout manuscript that describes a strategy for examining the contribution of the P. falciparum CLAG3 helix 44 to functional nutrient uptake using a CRISPR screen. Using a double-transfection methodology, the authors use a mutagenesis library to infer which sections of helix 44 contribute most to nutrient uptake. The main findings highlight the importance of alpha H44 to nutrient uptake, particularly the region closest to the extracellular space and HVR region. These findings, particularly the mutagenesis screen, could be further used to interrogate the impact of clag3 on nutrient uptake.

 

Report

The manuscript is clear, relevant and well-written. Citations are accurate and fair. The manuscript is scientifically sound and well thought out with appropriate controls. The limitations of the study have been ackkowledged. I overall recommend this manuscript after minor revision (see below).

 

To be addressed:

 

1. In Figure 1C, what is the loading control? A clearer image is needed with more equal loading or quantitation
2. Line 263: “Thus, the C-terminal CLAG3 region, including the a-H44, is required for formation of the channel pore” is an overstatement, and cannot be concluded from the data due to the complications from compensation from other clags.
3. Figure 3B: provide a non cropped and soomed out image to show that what is observed is the prevailing phenotype. This could be included as a supplementary figure.
4. Figure 4A: it was mentioned that significance was not achived for the NNK1-A12 mutant, yet there is significance in figure 4B (which is derived from Figure 4A). Can you explain this better? Are you doing a 1 way ANOVA between all the samples, or a t-test between 2 samples. Which would be a better test?

 

Author Response

Please see attached pdf for itemized response to these questions.  We thank the reviewer for their comments, which have been fully addressed and have improved presentation of the manuscript results.  

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, the authors generated a library of mutants of the CLAG3 gene from Plasmodium falciparum. Among them, two mutants were selected for functional analyses, including protein expression and parasite growth. Future analyses using these mutants may further clarify the mechanism of nutrient uptake in malaria parasites and could contribute to the development of effective antimalarial drugs.

Although several comments are provided below, the manuscript contains findings that could be worthy in Genes if the issues are appropriately addressed.

 

Line 118:

The origin of Pronase E should be described. If it was purchased commercially, the manufacturer should be specified.

 

Line 129:

The source of the anti-CLAG3 antibody should be properly described.

 

Lines 139–140:

The incubation time is described as “overnight,” but the temperature at which the incubation was performed should also be specified.

 

Line 337:

What happened to the 123 kDa fragment (160 − 37 = 123 kDa)? Please clarify this point. Was it cleaved and therefore not included in the Western blot sample? Was it further degraded into smaller fragments? Or is it present in the sample but not detectable by immunoblotting (for example, because it is not recognized by the antibody)? Additional explanation would be helpful.

 

Line 378:

Here the fragment is described as 35 kDa. Do the authors consider this to be different from the 37 kDa fragment mentioned elsewhere? If so, please explain the difference. According to Figure 4A, the fragment appears to be 37 kDa in H44rep and 35 kDa in NNK4C8, suggesting that their lengths may differ.

 

Figure 3:

Neither CLAG3 nor RhopH3 appears to be expressed on the cell surface. Rather, the staining seems to be distributed throughout the entire cell, including the cytoplasm. If the authors obtained the images using a confocal microscope, expression on the cell surface should appear as a ring-like pattern.

 

NNK4C8 mutant:

In Figures 3 and 4, this mutant shows results similar to those of the wild type (H44rep) and appears to retain wild-type-like functions. However, in Figure 5 it shows properties that are completely different from those of the wild type. The authors should discuss possible interpretations that could explain this discrepancy.

Author Response

Please see attached pdf for itemized response to these questions.  We thank the reviewer for their comments, which have been fully addressed and have improved presentation of the manuscript results.

Author Response File: Author Response.pdf