UVM Theses and Dissertations
Format:
Print
Author:
Mital, Jeffrey Thomas
Title:
Dept./Program:
Microbiology and Molecular Genetics
Year:
2006
Degree:
PhD
Abstract:
Like all members of the Phylum Apicomplexa, including Plasmodium spp. (which cause malaria) and Cr-yptosporidium pawum (a significant cause of waterborne illness), Toxoplasma gondii is an obligate intracellular parasite. The disease caused by an acute T. gondii infection, toxoplasmosis, can be life threatening in the congenitally-infected fetus and immunocompromised hosts. Host cell invasion is a critical step in the pathogenesis of diseases caused by Apicomplexan parasites. However, relatively little is known about the proteins that orchestrate invasion, due to the difficulty of studying essential genes in Apicomplexans. Apical membrane antigen 1 (AMA1) is a conserved transmembrane protein that localizes to the micronemes (secretory organelles discharged during invasion) and is a leading malaria vaccine candidate. Antibody inhibition experiments and the inability to knock out AMA1 suggest an essential, yet undefined, role during invasion. We have used a regulatable promoter to create a conditional knockout of T. gondii AMA1 (TgAMA1) and show that parasites depleted of TgAMA1 are severely compromised in host cell invasion. A TgAMA1 deficiency has no effect on motility, conoid extension, microneme secretion, or the parasite's ability to bind to host cells, but significantly inhibits rhoptry secretion. These data, together with ultrastructural analyses, suggest a two-step model for parasite attachment; the second, more intimate stage of attachment requires AMA1 and may be necessary for rhoptry secretion. In the course of this research novel Laser Scanning Cytometer (LSC)- based attachment and invasion assays were developed. These LSC-based assays have provided the field with robust, reproducible, and quantitative means to compare the invasiveness and attachment of different parasite populations. This research provides the first direct evidence that AMA1 plays a critical role during invasion and furthers our understanding of, and ability to study, T. gondii invasive mechanisms.