Toleco, M. R. | Search bioRxiv

bioRxiv2026

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

Blastocystis is among the most prevalent microbial eukaryotes 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 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 benchmarked their activity using NanoLuc luciferase (Nluc), defining near-background, weak, moderate, and robust expression tiers. We optimised 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(R)). Results showed reporter-dependent performance consistent with constraints such as chromophore availability and substrate permeability. Together, this toolkit makes Blastocystis ST7-B markedly more amenable to genetic engineering.

molecular biology↗

bioRxiv2023

A mitochondrial carrier transports glycolytic intermediates to link cytosolic and mitochondrial glycolysis in the human gut parasite Blastocystis

Stramenopiles form a clade of diverse eukaryotic organisms, including multicellular algae, the fish and plant pathogenic oomycetes, such as the potato blight Phytophthora, and the human intestinal protozoan Blastocystis. In most eukaryotes, glycolysis is a strictly cytosolic metabolic pathway that converts glucose to pyruvate, resulting in the production of NADH and ATP. In contrast, stramenopiles have a branched glycolysis in which the enzymes of the pay-off phase are located in both the cytosol and the mitochondrial matrix. Here, we identify a mitochondrial carrier in Blastocystis that can transport glycolytic intermediates, such as dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, across the mitochondrial inner membrane, linking the cytosolic and mitochondrial branches of glycolysis. Comparative analyses with the phylogenetically-related human mitochondrial oxoglutarate carrier (SLC25A11) and dicarboxylate carrier (SLC25A10) show that the glycolytic intermediate carrier has lost its ability to transport the canonical substrates malate and oxoglutarate. Blastocystis lacks several key components of oxidative phosphorylation required for the generation of mitochondrial ATP, such as complexes III and IV, ATP synthase and ADP/ATP carriers. The presence of the glycolytic pay-off phase in the mitochondrial matrix generates ATP, which powers energy-requiring processes, such as macromolecular synthesis, as well as NADH, used by mitochondrial complex I to generate a proton motive force to drive the import of proteins and molecules. Given its unique substrate specificity and central role in carbon and energy metabolism, the carrier for glycolytic intermediates identified here represents a specific drug and pesticide target against stramenopile pathogens, which are of great economic importance.

biochemistry↗