Exploring Parasites' Secrets
The phylum Apicomplexa includes several of the most prevalent and important human eukaryotic pathogens, such as Plasmodium spp., which causes malaria, and Toxoplasma gondii, which causes toxoplasmosis. To enter a host cell, the parasites deploy a remarkable machine at their anterior end known as the apical complex (AC), for which the phylum is named, and consists of cytoskeleton elements and specialized secretory organelles.
The exact means by which the various components of the AC coordinate invasion have been a mystery due, in part, to a lack of tools capable of resolving the structure of this extraordinary apparatus in its natural context. To address this, we couple cryogenic electron tomography with molecular and biochemical tools to study the invasion machinery of Toxoplasma gondii.
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The Segev-Zarko lab is studying the identity, structure, and function of parasite and host cell proteins that play a role in invasion. We are particularly focused on studying a novel eukaryotic secretion mechanism that is essential for the close association between parasite and host cell membranes, which is necessary for successful invasion.
Current Studies
How rhoptry proteins are injected into host cells?
During the invasion process, the parasite injects effectors from its secretory organelles, known as rhoptries, into the host cell. Rhoptry proteins facilitate a tight interaction with the host cell, which is essential for invasion. Following this, various effector proteins are introduced to modulate host cell pathways and functions, creating a customized intracellular environment conducive to the parasite's survival and replication.
In eukaryotes, the transport of macromolecules across two membranes involves the well-studied SNARE-mediated fusion. In addition to this conventional secretion system, apicomplexan parasites have had to evolve a way to move proteins across three membranes (rhoptry membrane, parasite membrane, and host cell membrane), a major biophysical challenge known to exist in only a very few instances in nature. Gram-negative bacteria utilize several protein secretion systems to transport proteins across three membranes; however, no homology to these systems has yet to be identified in Toxoplasma. Recently, an AV that is sandwiched between the parasite’s plasma membrane and the rhoptries has been implicated to facilitate rhoptry injection.
We are working to identify the components and the intricate mechanism of protein injection from the rhoptries into the host cell.
Resolving the structural and molecular organization of the conoid fibrils
The conoid, a dynamic structure within the AC of Toxoplasma gondii and related coccidia, comprises a barrel of uniquely arranged tubulin filaments that protrudes during invasion. Numerous proteins have been shown to localize to the conoid, but many of their functions and precise locations are yet to be determined. Using cryogenic electron tomography, a powerful tool to study cellular architecture in situ, the parasite’s AC was visualized in a near-native state. By averaging subvolumes of interest, a density map was generated that allowed accurately assigning tubulins in the conoid fibrils and revealing the organization of tubulin-associated proteins. Using single-molecule super-resolution fluorescence microscopy, we now work to determine with high resolution the molecular organization of Myosin H (Myo-H), a motor protein that associates with the conoid and is indispensable for parasite invasion.