Dr. Gary E. Ward
Cell Biology of Host-Parasite Interaction
Nearly one third of the world’s population is infected with Toxoplasma gondii. Although most infections are asymptomatic, toxoplasmosis can be life-threatening during pregnancy and in people with immune systems weakened by immunosuppressants or AIDS. T. gondii can only multiply within cells of its host, and repeated cycles of host cell invasion, parasite multiplication and host cell lysis are a major cause of the tissue damage that occurs during acute infection. A better understanding of the mechanisms of host cell invasion by T. gondii is therefore important to the development of new approaches to treating toxoplasmosis. T. gondii is also a powerful model for studying the invasion of related apicomplexan parasites, including those that cause malaria and cryptosporidiosis.
The focus of our research is to identify T. gondii proteins that play a role in invasion and to determine exactly what these proteins do. We are studying the function of one protein, AMA1, in detail, and we have developed more general, small-molecule-based approaches to identifying new invasion-related proteins. The small molecule work has also led us to study the molecular mechanisms underlying parasite motility.
AMA1 Proteins secreted onto the parasite surface from apical organelles, the micronemes, are thought to play an important role in invasion. One highly conserved microneme protein that has received much attention as a malaria vaccine candidate is apical membrane antigen 1 (AMA1). AMA1 is a type I transmembrane protein with a large extracellular domain and a short cytosolic tail. AMA1 directly mediates the physical interaction between the parasite and host cell: remarkably, the extracellular domain of AMA1 on the parasite surface binds to a receptor on the host cell (RON2) that is inserted into the host cell membrane by the invading parasite. We are currently studying two aspects of T. gondii AMA1 critical to its role in mediating invasion: (a) the function of its cytosolic tail and (b) why and how its extracellular domain is cleaved and shed from the parasite surface during invasion.
Small-molecule-based approaches One approach we are taking to identify new invasion-related proteins is to screen large collections of small molecules for compounds that inhibit invasion and then determine how these compounds exert their effects. The screening part of this project has yielded a set of small molecules that affect invasion in a variety of interesting ways. The challenge now is to identify the targets of the compounds of interest. We have developed specific biochemical strategies for target identification tailored to particular small molecules, and we have gained new insights into invasion in the process (see below). More recently, we have developed a yeast-based approach to target identification that can be applied to a wide variety of structurally distinct small molecules. We are currently using this more general approach to identify targets of the compounds of most interest from our screens and from the screens of others. In addition to providing new insights into the mechanisms of host cell invasion, this work has clear drug development implications for T. gondii and other apicomplexan parasites. This project is being done in close collaboration with Dr. Nick Westwood at the University of St. Andrews.
Motility Parasite motility is important for parasite movement into and out of host cells, for crossing biological barriers and for dissemination through the body during infection. Parasite motility is therefore essential for virulence and pathogenesis. Proteins of the parasite’s motility apparatus, including the myosin motor protein, MyoA, are highly conserved across apicomplexan parasites but absent or divergent in higher eukaryotes, suggesting that they may be useful drug targets. Intriguingly, over 90% of the invasion inhibitors identified in our small molecule screens affect parasite motility. We have shown that one of the compounds we identified by high-throughput screening (tachypleginA) inhibits motility by covalently modifying myosin light chain, a protein that directly regulates MyoA activity. Using tachypleginA and other motility-inhibiting compounds as chemical probes, together with parasites containing mutations in proteins of the motility apparatus and a variety of assays we have developed to study motor function, we are attempting to develop a detailed mechanistic understanding of how T. gondii and related parasites move over, around and into cells of their hosts during infection.
A fibroblast with two parasitophorous vacuoles containing T. gondii (phase contrast microscopy).
Four intracellular parasites visualized by immunofluorescence microscopy.
Mital J, Meissner M, Soldati D and Ward GE. Conditional expression of Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion. Mol Biol Cell. 2005 Sep;16(9):4341-4349
Kato N, Sakata T, Breton G, Le Roch KG, Nagle A, Andersen C, Bursulaya B, Henson K, Johnson J, Kumar KA, Marr F, Mason D, McNamara C, Plouffe D, Ramachandran V, Spooner M, Tuntland T, Zhou Y, Peters EC, Chatterjee A, Schultz PG, Ward GE, Gray N, Harper J, Winzeler EA.Gene expression signatures and small-molecule compounds link a protein kinase to Plasmodium falciparum motility. Nat Chem Biol. 2008 Jun;4(6):347-56.
Sheiner L, Santos JM, Klages N, Parussini F, Jemmely N, Friedrich N, Ward GE, Soldati-Favre D. Toxoplasma gondii transmembrane microneme proteins and their modular design. Mol Microbiol. 2010 Aug77; (4):912-929.
Heaslip AT, Leung JM, Carey KL, Catti F, Warshaw DM, Westwood NJ, Ballif BA, Ward GE. A small-molecule inhibitor of T. gondii motility induces the posttranslational modification of myosin light chain-1 and inhibits myosin motor activity. PLoS Pathog. 2010 Jan 15;6(1):e1000720.
Barkhuff WD, Gilk SD, Whitmarsh R, Tilley LD, Hunter C, Ward GE. Targeted disruption of TgPhIL1 in Toxoplasma gondii results in altered parasite morphology and fitness. PLoS One. 2011;6(8):e23977
Hall CI, Reese ML, Weerapana E, Child MA, Bowyer PW, Albrow VE, Haraldsen JD, Phillips MR, Sandoval ED, Ward GE, Cravatt BF, Boothroyd JC, Bogyo M. Chemical genetic screen identifies Toxoplasma DJ-1 as a regulator of parasite secretion, attachment, and invasion. Proc Natl Acad Sci U S A. 2011 Jun 28;108(26):10568-10573
Parussini F, Tang Q, Moin SM, Mital J, Urban S, Ward GE. Intramembrane proteolysis of Toxoplasma apical membrane antigen 1 facilitates host-cell invasion but is dispensable for replication. Proc Natl Acad Sci U S A. 2012 May 8;109(19):7463-7468.