The study represents the first comprehensive in silico drug-target prioritisation for T. foetus in roughly a decade. The previous analysis, published ten years ago, proposed 113 candidate targets based solely on homology to compounds catalogued in the ChEMBL database. Since then, both the genomic resources available for the parasite and the pharmacological landscape have changed considerably. The new work, led by Joanna Dąbrowska with colleagues Maciej Kochanowski and Jacek Sroka, set out to close that gap with a far more layered approach, drawing on multiple independent lines of evidence rather than a single homology filter.

The pipeline began with the predicted proteome of T. foetus strain K, which contains 25,030 proteins. From this starting point, the researchers applied a subtractive strategy designed to strip away anything unlikely to make a safe and effective drug target. Proteins with close counterparts in the feline host were filtered out, reducing the risk that an inhibitor would also attack the cat’s own biology. The remaining proteins were then cross-referenced against three pharmacological reference databases, which flag targets already known to be druggable, and against essential-gene resources drawn from three model eukaryotic organisms, which highlight genes that parasites of this kind cannot survive without.

A further layer of prioritisation drew on PHI-base, a curated virulence reference database that catalogues genes experimentally shown to contribute to pathogenicity in other organisms. Proteins appearing in this resource are attractive candidates because disrupting them could blunt the parasite’s ability to cause disease, not merely its survival in culture. When all of these filters were combined, the pipeline returned 433 priority candidates from strain K. To test whether the result was an artefact of one particular genome assembly, the team ran the identical pipeline on a chromosome-scale assembly of a second strain, KV-1, which yielded 436 candidates. Sixty-six percent of the candidates were recovered as reciprocal best hits between the two strains, a substantial cross-strain overlap that strengthens confidence in the robustness of the target list.

Expression data added a third dimension to the prioritisation. A candidate protein may look perfect on paper, but if the gene encoding it is silent in the disease-relevant life stage, it offers little therapeutic purchase. Using gene-level transcriptomic data from the feline isolate G10/1, the researchers found that 82.7 percent of the priority candidates were actively transcribed in that isolate, measured in transcripts per million. This high proportion of expressed targets suggests that the subtractive pipeline is not merely cataloguing genomic relics but is surfacing proteins the parasite genuinely deploys during infection of its feline host.

The most striking finding emerged when the candidates were ranked by expression level. Nine of the ten most highly expressed priority candidates belong to protein families for which clinically advanced small-molecule inhibitors are already available. These families include the molecular chaperones Hsp70 and Hsp90, the CDC48/p97 AAA+ ATPase, components of the ubiquitin-proteasome pathway, and serine/threonine kinases. Each of these protein classes has been the subject of intensive pharmaceutical development in human medicine, particularly in oncology, where inhibitors of Hsp90, p97, the proteasome, and various kinases have advanced through clinical trials or reached approval. The implication is direct: medicinal chemistry against these families is mature, and existing compounds or close analogues could be screened for activity against T. foetus without the need to invent inhibitor scaffolds from scratch.

The tenth member of that top-ten list stands out for a different reason. It is a Rab4B GTPase, a member of the broader Ras/GTPase superfamily, which is generally considered pharmacologically tractable because GTP-binding proteins have well-defined nucleotide pockets that small molecules can target. However, no Rab4B-specific inhibitor has been characterised to date. This makes the Rab4B candidate both an intriguing opportunity and an open challenge: it would require de novo inhibitor development rather than repurposing, but it sits within a protein superfamily with a long track record of successful drug discovery.

To demonstrate that the candidate list can support structure-based work, the team carried out a proof-of-concept molecular docking screen against three of the top-ranked candidates. Docking computationally fits small ligands into the three-dimensional structures of target proteins and scores the predicted binding affinity. Using a ligand library that included positive controls, negative control analogues, selective inhibitors, and natural ligand controls, each annotated with its clinical or pre-clinical development status, the researchers recovered internally consistent, structure-based affinity rankings across the three targets. The authors are careful to note that these computational results await experimental validation, but the internal consistency of the rankings suggests the protein structures and docking workflow are behaving as expected and can serve as a foundation for laboratory follow-up.

The practical significance of the work lies in the disease it addresses. Feline tritrichomonosis is a chronic large-bowel diarrhoea that persists in infected cats, and with no approved therapy, veterinarians are left using ronidazole off-label. The drug’s narrow therapeutic window means the margin between an effective dose and a toxic one is slim, and the documented spread of resistance across three continents makes the search for alternatives urgent. By identifying targets in protein families with existing clinical-grade inhibitors, the study offers what the authors describe as an immediate opportunity for drug-repurposing studies. Repurposing, in which drugs developed for one indication are tested against another, can compress the timeline and cost of drug development dramatically, because safety, pharmacokinetic, and manufacturing data for the original compounds may already exist.

The researchers have made their full candidate list and supporting data openly available as supplementary tables, including UniProt and RefSeq identifiers, locus tags, protein descriptions, Pfam and InterPro annotations, Gene Ontology terms, pathway counts, expression values, the complete docking ligand library, and the cross-strain reciprocal best hit pairs. The work was supported by the Internal Research Fund of the National Veterinary Research Institute under project F/123, and the authors acknowledge the Galaxy Europe community and the European Bioinformatics Institute for maintaining the public bioinformatic resources on which the analysis relied, including Ensembl Protists, InterProScan, and the AlphaFold Protein Structure Database. For a parasite that has received far less research attention than its importance to feline health would justify, the study provides both a starting point for experimental target validation and a template for how integrative omics can accelerate drug discovery in neglected veterinary pathogens.

Subject of Research: Integrative omics identification of drug-repurposing targets in the feline parasite Tritrichomonas foetus

Article Title: Integrative omics reveals candidate drug-repurposing targets in Tritrichomonas foetus

Article References: Dąbrowska, J., Kochanowski, M., & Sroka, J. (2026). Integrative omics reveals candidate drug-repurposing targets in Tritrichomonas foetus. BMC Genomics. https://doi.org/10.1186/s12864-026-13330-5

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13330-5

Keywords: Tritrichomonas foetus, feline tritrichomonosis, drug repurposing, drug-target prioritisation, subtractive proteomics, comparative genomics, molecular docking, Hsp90, ubiquitin-proteasome pathway, kinases, Rab4B GTPase, ronidazole resistance

The protein in question is NcDYRK2, a member of the dual-specificity tyrosine phosphorylation-regulated kinase family. DYRKs are ancient and highly conserved serine/threonine kinases found across eukaryotes, where they orchestrate processes as varied as cell cycle progression, cellular differentiation and signal transduction. While their functions in model organisms such as yeast, flies and mammals have been mapped in considerable detail, the roles of DYRK family members in apicomplexan parasites have remained largely unexplored. Given that neosporosis lacks effective chemotherapy, the researchers reasoned that identifying parasite kinases governing virulence and development could expose promising drug targets that would be absent, or at least very different, in the host animal.

To interrogate NcDYRK2’s function, the team turned to CRISPR-Cas9 gene editing. They generated a knockout strain lacking the dyrk2 gene, and in parallel constructed complemented parasites in which the gene was restored, allowing any observed defects to be attributed specifically to the deleted kinase rather than to off-target effects of editing. They also engineered parasites carrying an epitope-tagged version of the native protein, which enabled them to pinpoint where NcDYRK2 resides inside the cell by immunofluorescence microscopy. The answer was striking: the kinase localizes predominantly to the endoplasmic reticulum, the membrane network that in most eukaryotes serves as a major intracellular calcium store.

That ER localization proved to be a meaningful clue rather than a curiosity. When the researchers compared the knockout parasites with wild-type controls across a battery of standard fitness assays, a paradoxical picture emerged. Loss of NcDYRK2 significantly enhanced gliding motility, the distinctive substrate-crawling movement that apicomplexans use to traverse tissues and invade new cells, and it also boosted plaque formation, a composite readout of a parasite population’s ability to spread and destroy a host cell monolayer. Yet the more fundamental steps of the parasite’s life cycle inside the host cell were untouched: host cell invasion, intracellular replication and egress all proceeded normally in the knockout strain.

Because gliding motility in apicomplexans is tightly governed by intracellular calcium, the team next examined calcium dynamics directly using real-time fluorescence imaging. The Δdyrk2 parasites showed elevated basal concentrations of intracellular Ca²⁺, suggesting that in the absence of the kinase the parasite’s calcium homeostasis is chronically disturbed. Even more telling was the response to ionomycin, an ionophore that floods the cytosol with calcium: knockout parasites mounted a diminished Ca²⁺ response compared with wild type. Together these observations indicate that NcDYRK2 helps regulate both the resting calcium set point and the parasite’s capacity to mobilize calcium from internal stores, consistent with its position on the endoplasmic reticulum.

To understand how a single kinase could produce such broad phenotypic effects, the researchers performed RNA sequencing on knockout and wild-type parasites. The transcriptomic comparison revealed extensive gene expression alterations: 554 differentially expressed genes in total, of which 393 were downregulated and 161 upregulated. Principal component analysis cleanly separated the knockout group from the parental controls along the first principal component, which accounted for over 70 percent of the total variance, and biological replicates clustered tightly, with intra-group correlation coefficients above 0.986, underscoring the robustness of the dataset.

Among the downregulated genes were several with well-known roles in stage differentiation, a process of central importance to the parasite’s biology and transmission. Bradyzoite-associated genes including BPK1, CST1, MCP3, MCP4 and MAG2 were all transcriptionally reduced in the knockout strain, as was MIC17A, a gene associated with the merozoite stage. Bradyzoites are the slowly dividing, cyst-forming stage that persists chronically in infected animals, and the cyst wall proteins they produce are what allow the parasite to hide from the immune system and from drugs. The downregulation of this entire developmental program implicates NcDYRK2 as a regulator of stage differentiation, not merely a housekeeping enzyme.

The calcium signaling machinery itself also bore the fingerprint of NcDYRK2 loss. Calmodulin genes CAM1 and CAM3 were transcriptionally upregulated in the knockout parasites, a pattern that could reflect compensatory responses to the disrupted calcium balance, while PIPLCβ, encoding phosphoinositide phospholipase C beta, an enzyme that generates calcium-mobilizing second messengers, was downregulated. Quantitative reverse transcription PCR confirmed these transcriptional changes independently. The coordinated alteration of calmodulins and phospholipase signaling provides a mechanistic bridge between the kinase’s ER location, the observed calcium dysregulation and the hypermotile phenotype of the knockout parasites.

The most consequential result came from experiments in living animals. When mice were infected with the knockout parasites, virulence was markedly attenuated compared with wild-type infection, and quantitative PCR of brain tissue at 40 days post-infection revealed significantly reduced brain cyst burdens. This finding ties the in vitro molecular phenotypes directly to pathogenicity: a parasite that cannot properly regulate its calcium signaling and cannot fully express its bradyzoite developmental program is also a parasite that establishes fewer chronic infections in the brain. All animal work was conducted under the approval of the Institutional Animal Care and Use Committee of China Agricultural University, with measures taken to minimize animal suffering.

Taken together, the study establishes NcDYRK2 as a critical coordinator of calcium-dependent motility, stage-specific gene expression and virulence in N. caninum. What makes the kinase especially interesting from a drug discovery standpoint is its unusual profile of dispensability and necessity: the parasite can invade, replicate and exit host cells without it, yet it loses much of its disease-causing capacity in its absence. Inhibiting such a target would not need to kill the parasite outright in culture but could instead defang it in the host, blunting tissue migration and chronic cyst formation, the hallmarks of neosporosis. The work also adds to a growing appreciation that apicomplexan parasites have co-opted conserved eukaryotic kinase families for parasite-specific purposes, and that the endoplasmic reticulum, long studied as a calcium store, functions in these organisms as a signaling platform integrated with motility and differentiation. The research was supported by the National Natural Science Foundation of China, and the authors suggest that NcDYRK2 now merits further evaluation as a candidate target for future anti-neosporosis strategies, a prospect that could not come soon enough for an industry losing calves to this silent abortifacient.

Subject of Research: Role of the endoplasmic reticulum-associated kinase NcDYRK2 in regulating calcium dynamics, motility, development and virulence in Neospora caninum

Article Title: Endoplasmic reticulum-associated NcDYRK2 regulates Ca2⁺ dynamics, motility, and development gene expression in Neospora caninum

Article References: Yang, S., Wang, X., Yu, S., Fu, Y., Liu, Q., & Liu, J. (2026). Endoplasmic reticulum-associated NcDYRK2 regulates Ca2⁺ dynamics, motility, and development gene expression in Neospora caninum. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07681-x

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07681-x

Keywords: Neospora caninum, NcDYRK2, calcium signaling, endoplasmic reticulum, gliding motility, transcriptomics, virulence, bradyzoite differentiation, CRISPR-Cas9, apicomplexan parasites, kinases, neosporosis