UVM Theses and Dissertations
Format:
Online
Author:
Garudathri, Jayanthi
Dept./Program:
Microbiology and Molecular Genetics
Year:
2008
Degree:
MS
Abstract:
Toxoplasma gondii is an obligate intracellular pathogen, and belongs to the phylum Apicomplexa. The pathogenesis of the parasite is thought to be due to, in part, repeated cycles of invasion of host cells, replication and lysis of host cells. Tgondii is a haploid organism and forward genetic methods to study invasion are hard to employ, because if genes essential to invasion are disrupted, they will be lethal to the parasite. Although conditional mutants have been generated, it is hard to generate a large library of conditional mutants. As an alternate approach, a library of small molecules can be 'screened in an assay for a particular phenotype. Then the target of the small molecule responsible for the phenotype can be identified. Two different small molecules screens can be performed: a non-presumptive approach using small molecules with no known targets or a hypothesis based approach using small molecules with known or hypothesized targets. Approximately 12,000 structurally diverse small molecules with no known target were tested in a high throughput screen in our lab to identify inhibitors of Tgonclii invasion.
In this thesis I have described two different projects using small molecule inhibitors identified from this screen. My first project was hypothesis driven. One of the inhibitors identified, 118793, is structurally similar to a known inhibitor of mammalian cGMP Phosphodiesterase. The goal of the project was to test the hypothesis that cGMP Phosphodiesterase is the target of the compound 118793 during invasion. This hypothesis is also supported by the fact, that cGMP dependent protein kinase is important for invasion and motility of Zgondii. An in-vitro phosphodiesterase assay using tritiated cGMP as substrate and anion exchange chromatography was adopted to test the hypothesis. The effect of compound 118793 on the cGMP hydrolytic activity of phosphodiesterase in T.gondii extracts and purified bovine phosphodiesterase was tested. From our assay, compound 118793 does not appear to inhibit cGMP phosphodiesterase in T.gondii or purified cGMP Phosphodiesterase. In addition, I also generated a FRET based cGMP indicator called CYGNET 2.1 to test the hypothesis in vivo.
The second project was to test the effect of the invasion inhibitors identified in our screen on Tgondii egress. The dogma in the field is that the process of invasion and egress are mechanistically similar. I thought it would of interest to determine if any of the small molecules inhibited invasion but not egress. I adopted an ionophore induced egress assay to address this question. Ionophore-induced egress is thought to involve active motility. I tested invasion inhibitors that inhibit motility and some that do not inhibit motility. From my assay, it appears that one of the motility inhibitors does not inhibit egress. In addition, two of the non-motility inhibitors inhibit egress. These results prompt us to revisit current theories about invasion and egress. Given the limitations we have to study Tgondii, small molecule approach has proven to be very useful and advantageous in identifying proteins and pathways involved in invasion.
In this thesis I have described two different projects using small molecule inhibitors identified from this screen. My first project was hypothesis driven. One of the inhibitors identified, 118793, is structurally similar to a known inhibitor of mammalian cGMP Phosphodiesterase. The goal of the project was to test the hypothesis that cGMP Phosphodiesterase is the target of the compound 118793 during invasion. This hypothesis is also supported by the fact, that cGMP dependent protein kinase is important for invasion and motility of Zgondii. An in-vitro phosphodiesterase assay using tritiated cGMP as substrate and anion exchange chromatography was adopted to test the hypothesis. The effect of compound 118793 on the cGMP hydrolytic activity of phosphodiesterase in T.gondii extracts and purified bovine phosphodiesterase was tested. From our assay, compound 118793 does not appear to inhibit cGMP phosphodiesterase in T.gondii or purified cGMP Phosphodiesterase. In addition, I also generated a FRET based cGMP indicator called CYGNET 2.1 to test the hypothesis in vivo.
The second project was to test the effect of the invasion inhibitors identified in our screen on Tgondii egress. The dogma in the field is that the process of invasion and egress are mechanistically similar. I thought it would of interest to determine if any of the small molecules inhibited invasion but not egress. I adopted an ionophore induced egress assay to address this question. Ionophore-induced egress is thought to involve active motility. I tested invasion inhibitors that inhibit motility and some that do not inhibit motility. From my assay, it appears that one of the motility inhibitors does not inhibit egress. In addition, two of the non-motility inhibitors inhibit egress. These results prompt us to revisit current theories about invasion and egress. Given the limitations we have to study Tgondii, small molecule approach has proven to be very useful and advantageous in identifying proteins and pathways involved in invasion.