Mapping a Toxoplasma gondii interactome by crosslinking mass spectrometry and machine learning

Toxoplasma gondii, a widespread human parasite, persists in hosts through complex molecular interactions. Protein-protein interactions (PPIs) underpin essential biological processes, including parasite-host interactions and cellular invasion. Herein, we utilized advanced crosslinking mass spectrometry (XL-MS) techniques to map a T. gondii tachyzoite cytosolic extract interactome. By integrating MS-cleavable and non-cleavable analysis, we identified a total of 405 unique PPIs at medium confidence (FDR < 5%) and 226 at high confidence (FDR < 1%), revealing both known and novel interactions within critical cellular complexes such as the ribosome, proteasome, and dense granule proteins. Structural validation confirmed spatial proximity of crosslinked residues, while comparative analyses against existing datasets (hyperLOPIT, ToxoNET, STRING) corroborated the biological relevance of identified interactions. Furthermore, we introduced a machine learning approach leveraging biological annotations and experimental data to significantly enhance the detection and validation of PPIs. Our findings not only provide a refined view of T. gondiis molecular architecture, but also highlight the utility of XL-MS coupled with computational tools in dissecting complex parasite proteomes. The XL-MS interactome map provides a new valuable resource for understanding parasite biology and developing targeted therapeutic strategies.