Exploring Unique Aspects of Apicomplexan Cell Biology Using Molecular Genetic and Small Molecule Approaches

The Phylum Apicomplexa contains a number of devastating pathogens responsible for tremendous human suffering and mortality. Among these are Plasmodium, which is the causative agent of malaria, Cryptosporidium, which causes diarrheal illness in children and immuncompromised people, and Toxoplasma gondii, which causes congenital defects in the developing fetus and severe disease in immunocompromised people. T. gondii also serves as a model organism for other members of this phylum due to the relative ease of parasite culture and manipulation. Although effective treatments exist for some diseases caused by these apicomplexan parasites, drug resistance for others is widespread, perhaps most notably in Plasmodium species. Development of new therapeutic agents is needed to combat this resistance and alleviate disease burden. It is important that the drugs target parasitic cell components not conserved in humans in order to minimize side effects and drug toxicity. However, in order to target unique processes, a better understanding of apicomplexan biology must be gained. One approach toward understanding the unique biological processes of apicomplexan parasites is to study proteins conserved among the Phylum Apicomplexa, but not present in other organisms. One such protein, photosensitized INA-labeled protein 1 (TgPhIL1) was identified previously. The work presented in this dissertation describes targeted disruption of this gene in T. gondii, which results in parasites with an altered shape and a fitness defect in both tissue culture and a mouse model of infection. Another approach to understanding the unique processes of apicomplexan parasites is to perturb them using small molecules. By identifying the targets of the small molecules, a more detailed understanding of the process can be gained. To this end, a small molecule screen was performed in T. gondii in order to identify small molecules that inhibit the apicomplexan-specific and essential process of host-cell invasion. In addition to identifying 24 invasion inhibitors, 6 enhancers were also identified. One of these enhancers, compound 112762, was shown to enhance invasion of other apicomplexan parasites as well. Described herein are attempts to identify the target(s) of this compound. A derivative of this compound was linked to an affinity resin, and TgProfilin was identified as a putative target that may interact covalently with 112762. Additionally, affinity chromatography was used to demonstrate non-covalent binding of a T. gondii FK506-binding protein to 112762. Finally, based on a report in the literature of a compound nearly identical to 112762 that inhibits yeast and mammalian protein arginine methyltransferase 1 (PRMT1), it was hypothesized that 112762 might target TgPRMT1 in T. gondii. Supportive of this hypothesis, 112762 was shown to inhibit TgPRMT1 in vitro, to inhibit parasite protein methylation in vivo, and to bind the 112762 affinity resin. TgPRMT1 knockout parasites are being generated in order to determine whether they show resistance to compound 112762. As a result of this work, three potential targets of 112762 in T. gondii have been identified. This work opens the door for future studies aimed at understanding and controlling invasion by apicomplexan parasites and other processes specific to the Phylum Apicomplexa

Targeted Disruption of TgPhIL1 in Toxoplasma gondii Results in Altered Parasite Morphology and Fitness

The inner membrane complex (IMC), a series of flattened vesicles at the periphery of apicomplexan parasites, is thought to be important for parasite shape, motility and replication, but few of the IMC proteins that function in these processes have been identified. TgPhIL1, a Toxoplasma gondii protein that was previously identified through photosensitized labeling with 5-[125I] iodonapthaline-1-azide, associates with the IMC and/or underlying cytoskeleton and is concentrated at the apical end of the parasite. Orthologs of TgPhIL1 are found in other apicomplexans, but the function of this conserved protein family is unknown. As a first step towards determining the function of TgPhIL1 and its orthologs, we generated a T. gondii parasite line in which the single copy of TgPhIL1 was disrupted by homologous recombination. The TgPhIL1 knockout parasites have a distinctly different morphology than wild-type parasites, and normal shape is restored in the knockout background after complementation with the wild-type allele. The knockout parasites are outcompeted in culture by parasites expressing functional TgPhIL1, and they generate a reduced parasite load in the spleen and liver of infected mice. These findings demonstrate a role for TgPhIL1 in the morphology, growth and fitness of T. gondii tachyzoites

Chairperson

The Phylum Apicomplexa contains a number of devastating pathogens responsible for tremendous human suffering and mortality. Among these are Plasmodium, which is the causative agent of malaria, Cryptosporidium, which causes diarrheal illness in children and immuncompromised people, and Toxoplasma gondii, which causes congenital defects in the developing fetus and severe disease in immunocompromised people. T. gondii also serves as a model organism for other members of this phylum due to the relative ease of parasite culture and manipulation. Although effective treatments exist for some diseases caused by these apicomplexan parasites, drug resistance for others is widespread, perhaps most notably in Plasmodium species. Development of new therapeutic agents is needed to combat this resistance and alleviate disease burden. It is important that the drugs target parasitic cell components not conserved in humans in order to minimize side effects and drug toxicity. However, in order to target unique processes, a better understanding of apicomplexan biology must be gained. One approach toward understanding the unique biological processes of apicomplexan parasites is to study proteins conserved among the Phylum Apicomplexa