Functional dissection of prenyltransferases reveals roles in endocytosis and secretory vacuolar sorting in type 2-ME49 strain of <i>Toxoplasma gondii</i>

Prenyltransferases act essential roles in the prenylation modification, which is significant for proteins, like small GTPases to execute various important activities in <i>Toxoplasma gondii</i> (<i>T.gondii</i>). The structures and partial functions of prenyltransferases (FTase, GGTase-I, and GGTase-II) in prenylation process have been dissected in <i>T. gondii</i>. However, the cellular effects of prenyltransferases on type 2-ME49 strain of <i>Toxoplasma</i> are largely unknown. To address this gap, CRISPR/Cas9-based gene-editing technology was employed to construct conditional knockdown strains of prenyltransferases in ME49 strain. Subsequent observation of ingestion ability of host cytosolic molecules (e.g, green fluorescent protein [GFP]) and status of secretory vacuolar sorting post-knockdown of prenyltransferases revealed significant findings. Our study demonstrated that degradation of FTase and GGTase-II notably affected the trafficking of endocytic GFP and vacuolar secretory trafficking to rhoptry bulb. Additionally, depletion of GGTase-II led to disordered endoplasmic reticulum and microtubules, as well as impaired gliding motility. The integrity of mitochondrion was damaged after degradation of GGTase-I. These findings underscore the critical functions of prenyltransferases in endocytosis and secretory vacuolar sorting in ME49 strain of <i>T. gondii</i>, thereby enhancing our understanding of prenyltransferases as potential drug targets.