Secreted protein kinases regulate cyst burden during chronic toxoplasmosis

Area of Science:

• Parasitology
• Molecular Biology
• Infectious Diseases

Background:

• Toxoplasma gondii, an apicomplexan parasite, secretes numerous rhoptry kinases (ROPKs) and pseudokinases.
• While some ROPKs are known virulence factors, many remain uncharacterized, necessitating further investigation into their functions.

Purpose of the Study:

• To characterize unannotated rhoptry kinases (ROPs) in Toxoplasma gondii.
• To investigate the in vivo role of specific ROPKs in parasite virulence and cyst formation.

Main Methods:

• Predicted ROPKs were assessed for bradyzoite expression and prioritized for reverse genetic analysis.
• CRISPR/Cas9 technology was employed to engineer epitope-tagged ROP21 and ROP27 and to generate knockout parasite lines (ROP21, ROP27, ROP28, ROP30).
• Localization studies and in vitro/in vivo assessments of parasite growth, bradyzoite differentiation, and tissue cyst burden in mice were performed.

Main Results:

• ROP21 and ROP27 were localized to the parasitophorous vacuole and cyst matrix, secreted via a constitutive pathway.
• Individual gene knockouts did not affect in vitro growth or bradyzoite differentiation.
• A combined knockout of ROP21 and ROP17 (∆rop21/∆rop17) resulted in a 50% reduction in tissue cyst burden in vivo.

Conclusions:

• The study challenges existing ROPK annotations based solely on bioinformatics.
• Secreted kinases play a crucial role in determining the severity of chronic toxoplasmosis.
• ROP21 and ROP17 are important for maintaining parasite burden in vivo.