Apicomplexan parasites include the etiologic agents of many widespread infections of humans and livestock, such as malaria and toxoplasmosis. These diseases are caused by destruction of the host tissues in which the parasites replicate. Because apicomplexans only replicate inside host cells, the process of invasion is critical to their survival and pathogenesis. Invasion is mediated by the release of proteins from specialized secretory organelles at the apical end of the parasite, the micronemes and rhoptries. Microneme proteins include adhesins that support parasite motility and are therefore secreted as soon as parasites emerge from replication and start migrating towards new host cells to infect. Rhoptries, by contrast, secrete their contents only upon host cell recognition once parasites have committed to invasion. Evidence suggests that the release of rhoptry contents depends on the prior secretion of microneme proteins; however, the molecular events that link these two processes are unknown. Based on a genome-wide screen in the model apicomplexan Toxoplasma gondii, our lab recently identified a conserved microneme protein necessary for invasion of human cells, which we named CLAMP. Our preliminary studies show that CLAMP is necessary for rhoptry secretion and stably associates with two microneme proteins: SPATR, which was previously implicated in invasion, and an uncharacterized protein we call CLIP. We hypothesize that these interactions represent a novel invasion complex necessary for rhoptry protein secretion.
Our first aim i s to compare the functions of CLAMP, SPATR, and CLIP; investigate how they oligomerize; and elucidate the relationship between complex formation and rhoptry secretion. In our second aim, we will identify and characterize host and parasite proteins that interact with the CLAMP invasion complex. Finally, our third aim will take an unbiased look at the host cell factors that stimulate rhoptry protein secretion, which may intersect with the CLAMP complex to regulate this key step in apicomplexan invasion. Based on the conservation of the parasite proteins involved, we expect that the principles uncovered will be generalizable to the phylum and broadly inform our understanding of these infectious agents.
Toxoplasma gondii is an important opportunistic pathogen, NIAID category B Biodefense agent, and a model organism for other apicomplexan parasites including Plasmodium spp., the etiologic agents of malaria. The proposed study will characterize a newly discovered protein complex that plays a key role in the ability of parasites to enter the host cells in which they replicate. Characterizing the interactions between host and parasite factors during this initial step of infection will uncover new therapeutic and prophylactic approaches to treat these infectious agents.
