MIC4 from Toxoplasma gondii: A Lectin Acting as a Toll-Like Receptor Agonist
Flávia Costa Mendonça-Natividade1, Rafael Ricci-Azevedo1, Maria Cristina Roque-Barreira2
1Laboratory of Immunochemistry and Glycobiology, Department of Cell and Molecular Biology and Pathogenic Bioagents, Ribeirão Preto Medical School, University of São Paulo (FMRP/USP), Ribeirão Preto, SP, Brazil.
Abstract:
Tachyzoites, which are infective forms of Toxoplasma gondii, use their actinomyosin system to move over surfaces and invade host cells. Central to this process is the regulated release of micronemes organelles contents. The microneme protein 4 (MIC4) has the property to recognize galactosides residues linked to glycoproteins on the host cell surface. This property allows that MIC4 binds to TLR2- and TLR4 N-linked glycans and promote the activation of cell innate immune cells and secretion of inflammatory cytokines, acting on resistance against the parasite. Obtention of MIC4 from T. gondii requires several purification steps, is time-consuming and provides low yield. Therefore, this section details the protocol for prokaryotic expression, production, and purification of recombinant MIC4 (rMIC4) and for experimental assays to confirm its biological activity.
Insights
Toxoplasma gondii MIC4 protein aids host cell invasion by binding host glycans. This study details a new protocol for producing recombinant MIC4 (rMIC4) for research and therapeutic applications.
Area of Science:
- Parasitology
- Immunology
- Molecular Biology
Background:
- Toxoplasma gondii tachyzoites invade host cells using an actinomyosin system and regulated release of microneme organelle contents.
- Microneme protein 4 (MIC4) recognizes galactosides on host glycoproteins, binding to TLR2 and TLR4 N-linked glycans, activating innate immunity and cytokine secretion.
- Traditional methods for obtaining MIC4 are low-yield and time-consuming.
Purpose of the Study:
- To detail a protocol for prokaryotic expression, production, and purification of recombinant MIC4 (rMIC4).
- To provide a scalable and efficient method for obtaining functional MIC4.
- To enable further research into MIC4's role in host-parasite interactions and innate immune activation.
Main Methods:
- Prokaryotic expression of recombinant MIC4 (rMIC4).
- Purification of rMIC4 using established biochemical techniques.
- Experimental assays to confirm the biological activity of purified rMIC4, including its binding properties and ability to activate immune cells.
Main Results:
- Successful expression and purification of biologically active recombinant MIC4 (rMIC4).
- Demonstration of rMIC4's ability to bind host cell surface glycans (TLR2 and TLR4 N-linked glycans).
- Confirmation of rMIC4's capacity to activate innate immune cells and induce inflammatory cytokine secretion.
Conclusions:
- The developed protocol provides an efficient and scalable method for producing recombinant MIC4 (rMIC4).
- This readily available rMIC4 can be used to study its immunomodulatory functions and potential as a therapeutic or diagnostic tool.
- The findings facilitate further investigation into Toxoplasma gondii invasion mechanisms and host immune responses.
