Chemical genetic screen identifies Toxoplasma DJ-1 as a regulator of parasite secretion, attachment, and invasion

Abstract

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.

Serum Albumin Stimulates Protein Kinase G-dependent Microneme Secretion in Toxoplasma gondii

“…A variety of alcohols in addition to acetaldehyde show this activity in T. gondii, although we have chosen to use ethanol here as it is the most potent secretagogue (7). The downstream effectors that regulate Ca 2ϩ -dependent microneme secretion include the kinases TgCDPK1 (8) and TgCDPK3 (9 -11) as well as other downstream Ca 2ϩ effectors including TgPRP1 (12), TgDJ-1 (13), and TgDOC2.1 (14). Genetic or chemical ablation of TgCDPK1 activity blocks microneme release and inhibits motility, invasion, and egress (8).…”

A Focused Small-Molecule Screen Identifies 14 Compounds with Distinct Effects on Toxoplasma gondii

“…To date, other studies have been carried out using small-molecule libraries (7,13), but in both of these, the authors were looking for inhibitors of parasite invasion.…”

“…Successful parasite invasion requires the ability of the parasite to glide on, attach to, and penetrate host cells (7,13,23). Any defect in any one of these aspects leads to defective invasion and subsequent parasite death.…”

“…Cell-based small-molecule screens have the potential to identify new inhibitors of key parasite processes, including invasion, replication, and egress (7,13,20). The advantage of cell-based assays is that the compounds are screened against their potential targets in their native environment, and the success of this approach has been well documented in identifying previously uncharacterized gene products essential for specific parasite processes.…”

“…The advantage of cell-based assays is that the compounds are screened against their potential targets in their native environment, and the success of this approach has been well documented in identifying previously uncharacterized gene products essential for specific parasite processes. For example, the T. gondii gene DJ-1 was found to be crucial for Toxoplasma invasion on the basis of a small-molecule screen (7,13). Hits from cell-based assays also offer a scaffold for studying parasite mechanisms, which in turn aid in drug development processes using the small molecule as a lead (22).…”