Microscopic Toxoplasma gondii parasite with glowing sugar molecules, CRISPR/Cas9 scissors, and interconnected pathways

Unlocking Toxoplasma's Secrets: How New Glycomics Tools Could Revolutionize Parasite Research

Elliot Brynn in Science & Nature June 2025 • 4 min read.

"Scientists develop innovative CRISPR/Cas9 and glycomics techniques to investigate Toxoplasma gondii, paving the way for novel therapies and a deeper understanding of parasitic infections."

Toxoplasma gondii, a single-celled parasitic protozoan, infects a significant portion of the human population worldwide. While many infections are asymptomatic, Toxoplasma can cause severe health problems, including birth defects, blindness, and encephalitis, particularly in individuals with weakened immune systems. The parasite's ability to persist within its host and reactivate makes developing effective treatments and preventive strategies a major challenge.

Glycosylation, the process of adding sugar molecules (glycans) to proteins and lipids, plays a crucial role in various biological processes, including cell-cell interactions, immune responses, and pathogen virulence. Toxoplasma relies on glycosylation to assemble complex structures on its cell surface and internal components. These structures are essential for the parasite's survival and interaction with host cells. However, the unique characteristics of Toxoplasma glycans have hindered in-depth research into their functions and potential as therapeutic targets.

Now, a team of researchers has developed a suite of innovative tools combining CRISPR/Cas9 gene editing and glycomics analysis to dissect the complex world of Toxoplasma glycobiology. This breakthrough promises to accelerate our understanding of the parasite's glycosylation pathways and identify new vulnerabilities that can be exploited for drug development.

CRISPR/Cas9 and Glycomics: A Powerful Combination

Microscopic Toxoplasma gondii parasite with glowing sugar molecules, CRISPR/Cas9 scissors, and interconnected pathways

The Toxoplasma genome encodes a vast array of genes, called glycogenes, which are thought to assemble a variety of glycans, including N-glycans, O-glycans, and polysaccharides. To investigate the roles of specific glycans in Toxoplasma, the researchers combined genetic and glycomic techniques to map the connections between 67 glycogenes, their enzyme products, the glycans to which they contribute, and cellular functions. To analyze the structure, the team adopted mass spectrometry.

The researchers applied a double-CRISPR/Cas9 strategy, in which two guide RNAs promote replacement of a candidate gene with a resistance gene, to edit the parasite's genome. They also adapted mass spectrometry-based glycomics workflows to test for effects on glycan formation and infected fibroblast monolayers to assess cellular effects. In short, the team created mutants to study the result of specific proteins being missing during the glycomics process.

Identified novel Glc0-2-Man6-GlcNAc2-type N-glycans.
Discovered a novel HexNAc-GalNAc-mucin-type O-glycan and Tn-antigen.
Identified the glycosyltransferases for assembling a novel nuclear O-Fuc-type and cell surface Glc-Fuc-type O-glycans.
Demonstrated the importance of these glycans for in vitro growth.

By editing 17 glycogenes, the scientists uncovered novel glycans, identified key enzymes involved in their assembly, and demonstrated their importance for parasite growth. The guide sequences, editing constructs, and mutant strains are freely available to researchers to investigate the roles of glycans in their favorite biological processes. This collaborative approach is sure to speed up the efforts to produce a drug.

Implications and Future Directions

These new tools and findings pave the way for a deeper understanding of Toxoplasma glycobiology and the development of novel therapeutic strategies to combat this widespread parasitic infection. By manipulating specific glycans, researchers may be able to disrupt the parasite's ability to invade host cells, evade the immune system, or establish chronic infections. The availability of these resources to the scientific community will undoubtedly accelerate progress in this field and ultimately lead to new and improved treatments for toxoplasmosis.

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This article is based on research published under:

DOI-LINK: 10.1074/jbc.ra118.006072, Alternate LINK

Title: Crispr/Cas9 And Glycomics Tools For Toxoplasma Glycobiology

Subject: Cell Biology

Journal: Journal of Biological Chemistry

Publisher: Elsevier BV

Authors: Elisabet Gas-Pascual, Hiroshi Travis Ichikawa, Mohammed Osman Sheikh, Mariam Isabella Serji, Bowen Deng, Msano Mandalasi, Giulia Bandini, John Samuelson, Lance Wells, Christopher M. West

Published: 2019-01-01

Everything You Need To Know

How does Toxoplasma gondii use glycosylation, and what are the therapeutic implications of targeting this process?

Toxoplasma gondii utilizes glycosylation, the process of adding sugar molecules (glycans) to proteins and lipids, to construct complex structures essential for its survival and interaction with host cells. These structures are present on its cell surface and internal components. By manipulating these glycans, it may be possible to disrupt the parasite's ability to invade host cells, evade the immune system, or establish chronic infections. Further research is needed to fully understand the intricacies of Toxoplasma glycosylation and its potential as a therapeutic target.

What specific methods did the scientists use to investigate Toxoplasma glycobiology, and what are the next steps in this research?

The scientists employed a combination of CRISPR/Cas9 gene editing and glycomics analysis. Specifically, they used a double-CRISPR/Cas9 strategy to edit the parasite's genome and adapted mass spectrometry-based glycomics workflows to analyze the effects on glycan formation and cellular processes. This involved creating mutants to study the impact of specific protein deficiencies during glycosylation. Further steps are needed to translate the insights gained from these techniques into effective drug therapies.

What novel glycans and glycosyltransferases were identified in Toxoplasma gondii, and how significant are these discoveries?

The research identified novel Glc0-2-Man6-GlcNAc2-type N-glycans, a novel HexNAc-GalNAc-mucin-type O-glycan and Tn-antigen, and the glycosyltransferases responsible for assembling novel nuclear O-Fuc-type and cell surface Glc-Fuc-type O-glycans. Furthermore, the importance of these glycans for in vitro growth was demonstrated. These findings provide a foundation for understanding the structure and function of glycans in Toxoplasma gondii.

What are glycogenes, and how did researchers investigate their roles in Toxoplasma gondii?

Glycogenes are a vast array of genes encoded within the Toxoplasma genome, believed to be responsible for assembling a variety of glycans, including N-glycans, O-glycans, and polysaccharides. Researchers investigated 67 glycogenes, mapping the connections between them, their enzyme products, the glycans they contribute to, and their cellular functions, to understand the roles of specific glycans in Toxoplasma. The exact mechanisms by which these glycogenes influence the parasite's virulence and survival remain areas for further investigation.

What resources are available to researchers for further study of Toxoplasma glycobiology, and how could these resources accelerate drug development?

The team made the guide sequences, editing constructs, and mutant strains freely available to researchers. This collaborative approach aims to speed up the development of new treatments for toxoplasmosis. Further studies are needed to validate these resources and translate them into clinical applications.