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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.

No description available.

Toxoplasma gondii est un parasite protozoaire intracellulaire obligatoire, et est l’agent responsable de la toxoplasmose, une parasitose habituellement bénigne chez le sujet immunocompétent ou en dehors d'une grossesse. Lorsqu’elle est congénitale, la toxoplasmose peut se manifester par de malformations neurologiques sévères. Cette maladie se développe sous deux formes. La première comprend à la phase aigüe provoquée par l’expansion de la population de tachyzoites qui peuvent provoquer des malformations chez le fœtus. La seconde est dite chronique et asymptomatique, le bradyzoite est y présent sous forme de kystes. Une réactivation des bradyzoites en tachyzoites peut être fatale pour les patients immunodéprimés. L’interconversion tachyzoïte-bradyzoïte est donc au centre de la pathogénèse de cette zoonose. L’interconversion est régulée au niveau transcriptionnel, avec un contrôle épigénétique strict. In vitro, il a été montré que l’inhibition de l’histone déacétylase TgHDAC3 par FR235222 induit la conversion. Dans cette thèse, nous avons étudiés un nouveau composé I2, ayant des propriétés agissant également sur les HDACs. Nous démontrons que ce composé inhibe la croissance chez toutes les souches de T. gondii, sans pour autant induire la différenciation tachyzoïte-bradyzoïte. Par contre, le composé I2 induit une déformation de la vacuole, qui prend l’apparence d’une bulle, uniquement chez certaines souches de T. gondii. D’après les expériences effectuées la distorsion de la vacuole n’interfère ne s’apparente pas à une paroi kystique. Un criblage génétique a permis de définir une région génomique responsable du phénotype « bulle » de la vacuole. En l’état actuel le gène responsable reste à être identifié ainsi que les mécanismes qui participent à la distorsion de la vacuole. Parallèlement, un autre projet a été initié sur la base d’une étude de gènes impliqués dans la croissance et/ou dans la résistance à l’IFNγ, cytokine principale de défense contre le parasite. Un gène a été étudié. La délétion de ce celui-ci chez le parasite provoque un défaut de croissance et de manière surprenante une résistance à un traitement à l’IFNγ. Ce gène et son mode de fonctionnement restent à être étudié. Ensemble, ces travaux nous montrent une adaptation de T. gondii à son environnement et le développement de mécanismes de survie qui restent à être élucidés. / Toxoplasma gondii is an obligate intracellular protozoan parasite, and is the causative agent of toxoplasmosis, a benign parasitosis in immunocompetent or non-pregnant subjects. When congenital, toxoplasmosis can manifest as severe neurological malformations. This disease develops in two forms. The first includes the phase caused by the rise of the population of tachyzoites that can cause malformations in the fetus. The second is chronic and asymptomatic, bradyzoite is in the form of cysts. Reactivation of bradyzoites into tachyzoites may be fatal for immunocompromised patients. Tachyzoite-bradyzoite interconversion is therefore at the center of the pathogenesis of this zoonosis. Interconversion is regulated at the transcriptional level, with strict epigenetic control. In vitro, inhibition of histone deacetylase TgHDAC3 by FR235222 has been shown to induce conversion. In this thesis, we have studied a new compound I2, with properties that also act on HDACs. We demonstrate that this compound inhibits growth in all T. gondii strains, but does not induce tachyzoite-bradyzoite differentiation. On the other hand, the compound I2 induces a deformation of the vacuole, which takes the appearance of a bubble, only in certain strains of T. gondii. From the experiments carried out the distortion of the vacuole does not interfere with a cystic wall. Genetic screening has defined a genomic region responsible for the bubble phenotype of the vacuole. In the current state the responsible gene remains to be identified as well as the mechanisms that participate in the distortion of the vacuole. In parallel, another project was initiated on the basis of a study of genes involved in the growth and / or resistance to IFNγ, the main cytokine of defense against the parasite. A gene has been studied. The deletion of this one in the parasite causes a growth defect and, surprisingly, resistance to treatment with IFNγ. This gene and its mode of operation remain to be studied. Together, these works show us an adaptation of T. gondii to its environment and the development of mechanisms of survival that remain to be elucidated.

The humoral response of humans, calves and horses to Cryptosporidium sporozoite antigens was evaluated using a western blot technique. Sera from calves, humans and horses were obtained at various times following the detection of infection. Sera were reacted with detergent-solubilized, sporozoite antigens form sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The number of antigens recognized by immune sera from humans and animals increased with time post infection (P.I.). A 20 kDa antigen appeared to be a major sporozoite surface determinant since it was labelled via membrane protein biotinylation and recognized by mouse monoclonal antibodies using indirect immunofluorescence and western blotting. Detectable recognition of the 20 kDa band occurred in 3 week post infection (P.I.) sera from all species tested. Sera reactivity to the 20 kDa band diminished significantly within 5 months P.I. when infected humans had no further recurrence of cryptosporidial diarrhea. In contrast, 12 month P.I. sera from an individual constantly exposed to oocysts under working conditions was as strongly reactive as the 3 week convalescent sera. Therefore, reactivity to the 20 kDa antigen appeared to be a good indicator of exposure to Cryptosporidium. Anti-sporozoite indirect immunofluorescent titers decrease in reactivity from convalescent to post convalescent sera which correlated with western blot results. Chromosomal DNA of five Cryptosporidium parvum isolates and one Cryptosporidium baileyi isolate were compared by field inversion gel electrophoresis (FIGE). FIGE analyses of parasite DNA prepared from purified sporozoites versus intact oocysts showed no observable differences. Chromosomal DNA migration patterns of the five Cryptosporidium parvum isolates were indistinguishable. Distinct differences in chromosomal DNA were evident between the Cryptosporidium baileyi and Cryptosporidium parvum isolates, yet the overall pattern was similar. Five C. parvum isolates were also compared using two dimensional electrophoretic analyses. Silver stained patterns of sporozoite proteins showed a shift in a 106 kDa protein in three of the isolates. One isolate (Mexico) showed a complete absence of this protein (106 kDa) and the presence of an additional 40 kDa protein not found in any other isolate.

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