Toxoplasma gondii is a member of the phylum Apicomplexa that includes several 
important human pathogens, such as Cryptosporidium and Plasmodium falciparum, 
the causative agent of human malaria. It is an obligate intracellular parasite 
that can cause severe disease in congenitally infected neonates and 
immunocompromised individuals. Despite the importance of attachment and invasion 
to the success of the parasite, little is known about the underlying mechanisms 
that drive these processes. Here we describe a screen to identify small 
molecules that block the process of host cell invasion by the T. gondii 
parasite. We identified a small molecule that specifically and irreversibly 
blocks parasite attachment and subsequent invasion of host cells. Using tandem 
orthogonal proteolysis-activity-based protein profiling, we determined that this 
compound covalently modifies a single cysteine residue in a poorly characterized 
protein homologous to the human protein DJ-1. Mutation of this key cysteine 
residue in the native gene sequence resulted in parasites that were resistant to 
inhibition of host cell attachment and invasion by the compound. Further 
analysis of the invasion phenotype confirmed that modification of Cys127 on 
TgDJ-1 resulted in a block of microneme secretion and motility, even in the 
presence of direct stimulators of calcium release. Together, our results suggest 
that TgDJ-1 plays an important role that is likely downstream of the calcium 
flux required for microneme secretion, parasite motility, and subsequent 
invasion of host cells.