The apicomplexan parasite Toxoplasma gondii is the causative agent of life-threatening encephalitis in immunocompromised patients and in addition can cause a variety of birth defects if the infection is contracted congenitally. The pathology associated with disease originates in fast rounds of lytic intracellular replication cycles. Using genetic approach we recently identified a role for a DOC2 protein (TgDOC2.1) in Ca2+- mediated microneme secretion. Micronemes contain adhesion molecules required for successful host cell invasion and egress, which is essential to complete the lytic cycle. The goal of this proposal is to unravel the role and function of TgDOC2.1 in microneme secretion. This will provide exciting new insights into a poorly understood mechanism critical to the pathogenesis of not only Toxoplasma, but to all other apicomplexan parasites since Ca2+-dependent microneme secretion is a conserved across the phylum. At the same time, this will provide insights into the potential of this pathway as a new specific drug target (secretion is not targeted by currently approved drugs). In other systems DOC2-domain containing proteins recruit the membrane fusion machinery (e.g. SNARE and MUNC proteins) to facilitate fusion of the secretory vesicle with the plasma membrane. However, no conserved domains interacting with the secretory machinery are conserved in TgDOC2.1. Comparative genomics of TgDOC2.1 identified four conserved sequence block across the Apicomplexa, highlighting a potentially crucial role for these domains (block 2 contains the DOC2 domain). To dissect TgDOC2.1's function we will first generate reagents, either a specific antiserum or fusion-reporter, to establish its spatio-temporal sub-cellular localization pattern throughout Ca2+-dependent excocytosis. This will indicate at which membrane TgDOC2.1 exerts its function. In addition we will establish a conditional TgDOC2.1 knock-down parasite line that can be used for functional complementation studies with TgDOC2.1 domain deletion mutants. This will identify which domains play a critical role in membrane translocation and/or microneme secretion. In the same model we will test point mutations in the Ca2+-binding domains. The Ca2+- binding Asp residues will first be mapped using a well-established heterologous mammalian tissue culture model. Simultaneously, to learn with which proteins TgDOC2.1 cooperates in the secretion process we will identify proteins interacting with TgDOC2.1 using two parallel approaches. The first is a genetic yeast two- hybrid system wherein we will use TgDOC2.1 as bait to screen a tachyzoite cDNA library. In the second we will explore a new method, named BioID. In this method we will fuse an E. coli BirA mutant protein to TgDOC2.1 and express it in the parasite. The BirA mutant results in promiscuous biotinilyation of proteins in the same complex, which subsequently can be easily identified by streptavidin enrichment and mass spectrometry. Putative TgDOC2.1 interaction partners identified by either method will be validated by co-localization studies in the parasite. Altogether, we will define the mechanism behind the conserved microneme secretion process.
Limited treatment options are available to treat opportunistic infection by Toxoplasma gondii. We propose to dissect the role of protein TgDOC2.1 in microneme organelle secretion, which is essential for host cell invasion and subsequent egress upon completion of intracellular Toxoplasma gondii replication. Microneme secretion is potentially druggable, is conserved across the Apicomplexa including the malaria causing Plasmodium parasites, and understanding its mechanism is important to understand the pathogenesis of these parasites.
|Farrell, Andrew; Coleman, Bradley I; Benenati, Brian et al. (2014) Whole genome profiling of spontaneous and chemically induced mutations in Toxoplasma gondii. BMC Genomics 15:354|