A genetic toolkit for stable episomal transgenesis in the anaerobic gut parasite Blastocystis ST7-B
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eLife Assessment
This paper presents a valuable methodology for genetic manipulation of Blastocystis. Although some imaging data are compelling, higher-quality figures together with more rigorous biochemical assays would strengthen support for the authors' claims. With the experimental evidence and graphics improved, the study would be of interest both to researchers investigating mitochondrial evolution under anaerobic conditions and to medical biologists studying human pathogens.
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Abstract
Blastocystis is among the most prevalent microbial eukaryote in the human gut, yet it has remained largely inaccessible to functional genetics. Here, we report a combinatorial toolkit for Blastocystis ST7-B that enables stable episomal transgene maintenance under antibiotic selection and recovery of colony-derived transgenic lines. Guided by a proteomics-informed candidate screen, we identified endogenous promoter–terminator pairs and benchmark their activity using NanoLuc luciferase (Nluc), defining near-background, weak, intermediate, and robust expression tiers. We optimise square-wave electroporation and establish conditions that balance DNA delivery with culture viability, providing a practical operating regime for routine transfection. Using resazurin-based viability assays alongside culture outgrowth validation, we identified puromycin and trimethoprim as the most reliable selectable systems. A three-stage workflow combining liquid enrichment, solid-phase selection, and liquid culture expansion supports recovery of colony-derived transgenic lines that can be cryopreserved and revived with retained growth, antibiotic resistance, and reporter expression. Finally, bicistronic constructs incorporating a codon-optimised P2A peptide supported selection-linked expression of anaerobic-compatible reporters (UnaG, smURFP, and SNAP-tag). Results showed reporter-dependent performance consistent with constraints such as chromophore availability and substrate permeability. Together, these make Blastocystis ST7-B markedly more amenable to genetic engineering.
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eLife Assessment
This paper presents a valuable methodology for genetic manipulation of Blastocystis. Although some imaging data are compelling, higher-quality figures together with more rigorous biochemical assays would strengthen support for the authors' claims. With the experimental evidence and graphics improved, the study would be of interest both to researchers investigating mitochondrial evolution under anaerobic conditions and to medical biologists studying human pathogens.
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Reviewer #1 (Public review):
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 the 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.
Weaknesses:
The findings are …
Reviewer #1 (Public review):
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 the 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.
Weaknesses:
The findings are reported by reporter gene assay (microscopy). No evidence is given using genetics. The authors claim that the DNA is maintained episomally. However, could it be possible that there is integration? No PCRS/RT-PCRs are shown (although it can safely be assumed that the DNA/RNA is present where the transformation was successful), nor are any Western blots. These would have been useful to show that the P2A ribosomal skipping had occurred, and that proteins were expressed individually rather than as a polyprotein.
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Reviewer #2 (Public review):
This manuscript presents a substantial technical advance for the genetic manipulation of Blastocystis by establishing an integrated workflow for stable episomal transgenesis, antibiotic selection, clonal recovery, and reporter-based imaging in the ST7-B subtype. The study is particularly valuable because it combines multiple previously fragmented approaches into a coherent and practically applicable toolkit, including endogenous regulatory elements, optimized electroporation conditions, selectable markers, and anaerobic compatible fluorescent reporters. This methodological work greatly expands the molecular toolbox and future studies focused on both basic and infection biology can now build on the ability to express and localize proteins in fixed as well as live cells.
The microscopy data are convincing and …
Reviewer #2 (Public review):
This manuscript presents a substantial technical advance for the genetic manipulation of Blastocystis by establishing an integrated workflow for stable episomal transgenesis, antibiotic selection, clonal recovery, and reporter-based imaging in the ST7-B subtype. The study is particularly valuable because it combines multiple previously fragmented approaches into a coherent and practically applicable toolkit, including endogenous regulatory elements, optimized electroporation conditions, selectable markers, and anaerobic compatible fluorescent reporters. This methodological work greatly expands the molecular toolbox and future studies focused on both basic and infection biology can now build on the ability to express and localize proteins in fixed as well as live cells.
The microscopy data are convincing and clearly demonstrate functional reporter expression and successful recovery of stable transgenic lines. Nevertheless, because this is primarily a methodological paper, the study would be further strengthened by the inclusion of Western blot validation of reporter expression and bicistronic constructs. In particular, biochemical analysis of the P2A-containing constructs would help assess the efficiency of ribosomal skipping and exclude the possible presence of uncleaved fusion proteins, thereby providing stronger support for the interpretation of the imaging data and the functionality of the expression system.
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Reviewer #3 (Public review):
Summary:
The primary objective of this study was to establish a practical and functional framework for the 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 …
Reviewer #3 (Public review):
Summary:
The primary objective of this study was to establish a practical and functional framework for the 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.
Weaknesses:
The presented data generally provide solid support for the conclusions that the work reached, but clarification of reasoning and several inconsistencies, as well as amendments to the visual presentation of the data, would be highly beneficial, as detailed below.
(1) Episomal persistence of the construct:
The manuscript repeatedly assumes, including in its title, that constructs persist in Blastocystis in their episomal form, but no direct evidence is provided. Although this interpretation is plausible, it should be identified more clearly as provisional. Nuclear genomic integration (e.g., via NHEJ) remains a possible explanation unless supporting evidence or rationale is provided to exclude it. Testing whether the phenotype persists without drug-mediated selection in the generated transgenic cell lines would help strengthen the case for episomal maintenance.(2) Promoters and terminators:
2.1) There is a discrepancy between the claimed number of loci (14), from which promoters used to drive luciferase expression were derived, and those detailed as having been actually generated in Table 1 (11). This inconsistency should be corrected or explained, as it creates uncertainty around the accuracy of the dataset.
2.2) Based on the presented evidence, constructs benchmarked in bioluminescence assays differed only in their promoter composition. Although terminator selection is mentioned in the Methods section, no additional details are provided; for instance, Table 1 and Figure 2 only list 23 promoters in total. Figure 2A likewise shows only promoter-dependent variation. If the terminator was held constant (LeguP1?), this should be stated explicitly. The authors may then consider revising the wording of having tested "23 promoter-terminator pairs" to better reflect that only promoters varied.
2.3) Promoter benchmarking was done with a plasmid lacking a selection marker, so it is unclear how the maintenance of the luciferase construct was ensured. Without selection, the observed reporter intensity could reflect differential or stochastic plasmid retention rather than promoter strength alone. The luminescence assay was performed 16-18 hours after transfection, but the rationale for this particular timeframe should be explained. In this context, the authors should explicitly state whether the experiments shown in Fig.2A represent biological triplicates or technical triplicates from a single transfection.(3) Figure 2:
3.1) Several aspects of the current design may lead to ambiguity for the reader. The boxplots are colour-coded, but it is unclear whether the colours carry meaning or are purely decorative. Because the data are already spatially separated into bins, additional random colouring is redundant and may suggest distinctions that are not intended. In addition, part A of Figure 2 is split into two panels, with the scale for the left panel shown in the right panel and some of the boxplot colours falling in the range of the scale, but not in line with their counterparts in the left panel. Because the colour use is not consistent, it is difficult to tell whether the same scale should be applied to both panels or how it should be interpreted.
3.2) The left panel of part A uses a diverging blue-white-red colour scheme, which is most appropriate when the midpoint represents a meaningful central value such as zero. Because the values shown in this graph are only positive, a non-diverging 2-colour scale or a colour palette such as 'viridis' would make the plot easier to interpret.
3.3) A black background should be avoided: 'B' and 'C' labels are invisible, and it draws attention to a distracting design feature rather than the data themselves.(4) Figure 3:
4.1) Individual snapshots should be separated more clearly, either by using a white background or by adding visible borders to make the overall composition clearer. As currently displayed, some boundaries between fluorescent channels resemble image artifacts rather than intentional panel divisions.
4.2) In parts B-D, the legend should explain more clearly what each image shows, and the figure itself would benefit from annotations. There seem to be three sub-panels in each 'condition' of part B (as well as C and D): while the middle and rightmost panel can be easily inferred to represent the fluorescent protein and bright-field image, what the leftmost panels represent is not specified. If DAPI was used to dye DNA, an explanation why mostly multiple labelled regions are visible should be provided.
4.3) Cell morphology and appearance differ markedly between UnaG/smURFP and SNAP-tag images, which should be explained. A microscope issue is mentioned in the main text, but if that was the cause, the authors should consider replacing the images, as the current distortions complicate interpretation. -
