UVM Theses and Dissertations
Format:
Print
Author:
Carey, Kimberly Lynne
Dept./Program:
Microbiology and Molecular Genetics
Year:
2004
Degree:
Ph. D.
Abstract:
The promiscuous, obligate intracellular parasite, Toxoplasma gondii, invades host cells using a complex, multi-step process. Many surface, secretory and cytoskeletal proteins of T. gondii have been identified, but ascribing a function to most of these proteins has been problematic due to the lack of sequence homology with domains or proteins of known function and the limited genetic techniques available. Until the recent development of a regulatable promoter, disruption of genes essential to the process of T. gondii invasion resulted in a lethal phenotype. This dissertation involves the use of two alternative approaches to identify parasite proteins functioning in host cell invasion. To identify novel transmembrane proteins on the surface of the tachyzoite that could function in invasion, monoclonal antibodies were produced and screened for antigens with surface/apical localization by immunofluorescence microscopy and transmembrane domains by Western blotting extracts of Triton X-114 phase-partitioned tachyzoites. The two described herein, GRA8 and ROP4, are secreted from the dense granules and rhoptries, respectively, into the parasitophorous vacuol.
Instead of functioning in host cell invasion, these proteins are more likely involved in modifying the parasitophorous vacuole for intracellular survival, yet as with many of the previously identified proteins, the functions of GRA8 and ROP4 remain undetermined. A more direct approach to identify proteins important for host cell invasion is the use of small molecules in a screen analogous to a forward genetic screen. In the case of T. gondii host cell invasion, small molecules were added to the host cells and parasites, then screened for their ability to perturb invasion. Using this high-throughput assay, several small molecules were identified as inhibitors and enhancers of host cell invasion. Secondary assays have been used to determine which, if any, of the known steps of invasion, such as parasite gliding motility or microneme secretion, are affected by the invasion inhibitors and enhancers. The results from these assays demonstrate more complex relationships among the known mechanisms of invasion than previously recognized. Several approaches are underway to identify the target proteins for some of the small molecules with interesting biological activity profiles.
Instead of functioning in host cell invasion, these proteins are more likely involved in modifying the parasitophorous vacuole for intracellular survival, yet as with many of the previously identified proteins, the functions of GRA8 and ROP4 remain undetermined. A more direct approach to identify proteins important for host cell invasion is the use of small molecules in a screen analogous to a forward genetic screen. In the case of T. gondii host cell invasion, small molecules were added to the host cells and parasites, then screened for their ability to perturb invasion. Using this high-throughput assay, several small molecules were identified as inhibitors and enhancers of host cell invasion. Secondary assays have been used to determine which, if any, of the known steps of invasion, such as parasite gliding motility or microneme secretion, are affected by the invasion inhibitors and enhancers. The results from these assays demonstrate more complex relationships among the known mechanisms of invasion than previously recognized. Several approaches are underway to identify the target proteins for some of the small molecules with interesting biological activity profiles.