A genetic toolkit for stable transgenesis in the anaerobic gut parasite Blastocystis ST7-B

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the reviewers' suggestions.]

Summary:

This paper presents a toolkit for the transformation of Blastocystis. The authors have screened a number of selectable agents, promoters and reporter genes and present their findings. This resource will be of immense use to those in Blastocystsis field, as well as those seeking to establish transformation tools in other species where such tools do not yet exist. Establishing new transformation tools is extremely challenging, and the authors have done an excellent job.

Strengths:

The authors have carried out a systematic screen of promoters, reporter genes and selectable agents. They have screened numerous for each, and all the data is presented. It is good to see when things did not work as well as when things did - so this data set is extremely useful indeed.

Comments on previous version.

The authors have revised their manuscript to clarify that molecular analyses have not yet occurred and have resolved the technical/publication issues with the figures. I look forward to seeing these tools used in future publications to answer important questions in Blastocystsis research.

Reviewer #3 (Public review):

Summary:

The primary objective of this study was to establish a practical and functional framework for propagation of stable transgenic cell lines of Blastocystis, a common animal gut microeukaryote. Although the work focused on Blastocystis ST7-B, a subtype with relatively low prevalence in humans, this choice is justified by its association with more frequent negative health effects. Beyond their relevance to the medical field, the methodological advances described here have the potential to also expand cell biology studies of this anaerobic organism, including its unusual mitochondria and redox metabolism.

Strengths:

Prior to this work, genetic tools for Blastocystis were very limited, relying on a single strong promoter-terminator combination. The authors successfully expanded the available promoter set across a range of expression strengths by testing two dozen variants in luciferase-based assays. Critically, they developed an integrated workflow from a modular transgenic construct design to an expanded inventory of molecular components (promoters, reporters), optimized DNA delivery, stepwise antibiotic resistance-mediated clonal selection and propagation, and to reporter validation. The evaluation of several anaerobiosis-compatible labeling strategies for live (and fixed) cell optical imaging will be particularly useful, with the SNAP-tag system appearing especially promising for Blastocystis.

Author response:  

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #1 (Public review):

Comments on revised version.

The authors have revised their manuscript to clarify that molecular analyses have not yet occurred and have resolved the technical/publication issues with the figures. I look forward to seeing these tools used in future publications to answer important questions in Blastocystis research.

We are grateful to Reviewer 1 for their careful and constructive assessment of our revised manuscript. We are pleased that the revisions have satisfactorily addressed their previous concerns, and we sincerely appreciate their recognition of the value of our work.

Reviewer #3 (Public review):

Comments on revised version.

The revised version provides sufficient clarity and appropriate visual presentation. Some confusion evidently arose due to my misunderstanding, so I thank the authors for their comprehensive clarifications and patience.

We are grateful to Reviewer 3 for their careful reading of the revised manuscript and for the additional constructive suggestions. We appreciate that these recommendations are aimed at improving clarity, accessibility, and presentation, and we have addressed them as detailed below.

Recommendations for the authors:

Reviewer #3 (Recommendations for the authors):

(1) Table 1

(1a) The description of the column "Promoter Lengths Tested" is a bit confusing because it actually shows acronyms of the used promoters. Suggestion for clarity: either keep the name and mention just the lengths, or rename to "Promoter abbreviation" or "Promoter acronym".

We thank the reviewer for pointing this out. To avoid confusion, we have renamed this column “Promoters Tested”, which better captures the information presented. The column contains both promoter identifiers and the corresponding promoter lengths, which are further clarified in the table notes.

(1b) It is not entirely clear based on the formatting of the table, which columns are relevant for "Blastocystis ST4-WR1" and which for "Blastocystis ST7-B".

We thank the reviewer for this helpful suggestion. We have revised the formatting of Table 1 to more clearly distinguish the data corresponding to Blastocystis ST7-B and Blastocystis ST4-WR1. Specifically, we have added a vertical divider between the relevant column groups to improve readability and make the species-specific information easier to follow.

(2) Figure 2:

If the authors wish to preserve the decorative colouring in the parts B and C, it would be better to at least keep the datapoint and boxplot hues consistent for individual conditions (or plot the datapoints in the same, neutral hue throughout, e.g., black or grey). Currently, this is only the case in the part C, but not in the part B.

We thank the reviewer for this useful suggestion. We have revised Figure 2B to improve visual consistency between the data points and boxplots while preserving the overall colour scheme of the figure. This adjustment improves readability without changing the data or its interpretation.

(3) Figure 3: With the modifications everything is clear. However, two issues are now apparent.

(3a) After the part A was modified, the arrow colours changed (had been yellow and red, now are cyan and magenta), but the description in the legend remained (yellow and red), so the text should be corrected. [By the way, the original colours worked well.]

We apologise for overlooking this inconsistency. The Figure 3 legend has now been corrected to match the revised arrow colours in panel A.

(3b) The care taken by the authors to make the images more accessible is really greatly appreciated, but being a person on the colourblind spectrum, I can attest that the issue is not always just about red and green discrimination: the greyscale and blueish green used in the part A photo in particular are discernible only at huge magnification (to some people). If the signal in the part A were of the same hue as in the part B (yellowish green instead of blueish green), it would readily pop out. For that matter, what is the reason for the use of a different colour profile of the UnaG fluorescence in these two images?

We sincerely thank the reviewer for this important accessibility-related comment. The difference in colour profiles between panels A and B was intentional because the images were acquired using different imaging modalities, as indicated in the figure legend. We wanted to avoid implying that the two panels were generated under identical imaging conditions. However, we appreciate the reviewer’s point that this distinction should not come at the expense of readability. We have therefore adjusted the display of the UnaG signal in panel A to improve contrast and visibility. 

(4) Conclusion:

The expression "bringing endogenous regulatory part discovery, namely the identification of native promoter and terminator elements" feels a bit clunky. What the part "endogenous regulatory part discovery" alludes to is now clearer, but consider reformulating it to "discovery of endogenous regulatory elements". This would make it clear without the need to add the explanation "namely the identification of native promoter and terminator elements". [It is now clear that the accumulation of noun adjectives and the significance of the word "part" was what originally blurred the overall meaning.

We thank the reviewer for this valuable suggestion. We agree that the original phrasing was unnecessarily clunky and have revised the sentence for clarity and flow. Lines 682–684 now read:

“By integrating endogenous promoter and terminator discovery, DNA delivery, selection, clonal recovery, and reporter validation into a single pipeline, we provide a flexible foundation for routine transgene-based studies.”