Submitted by Tatiana Oliveira (tsilva@icict.fiocruz.br) on 2012-06-05T18:40:11Z No. of bitstreams: 1 lucio_andre_v_dias_ioc_bp_0006_2010.pdf: 4820130 bytes, checksum: fcb218258dc3edcfe6059416e4b6d952 (MD5) / Made available in DSpace on 2012-06-05T18:40:11Z (GMT). No. of bitstreams: 1 lucio_andre_v_dias_ioc_bp_0006_2010.pdf: 4820130 bytes, checksum: fcb218258dc3edcfe6059416e4b6d952 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil / Na presente tese, foram estudados aspectos da relação parasito-hospedeiro, constituída pelo protozoário parasito Hepatozoon caimani (Apicomplexa: Adeleorina) e seu hospedeiro vertebrado, o jacaré Caiman yacare (Crocodylia: Alligatoridae), na região do Pantanal Sul, Brasil. Com o objetivo de esclarecer os componentes epizootiológicos desta relação procuramos no primeiro momento examinar os níveis de prevalência e distribuição da parasitemia do H. caimani em uma população do C. yacare, procurando por fatores que poderiam estar associados ao maior risco de infecção, tais como gênero, tamanho/idade, e condição corporal, no período de julho de 2006 a fevereiro de 2008. Encontramos prevalência de 76% para toda a população amostrada (n=229) e o risco de infecção foi associado somente com o tamanho/idade dos jacarés, onde os animais filhotes não apresentaram infecção, os juvenis apresentaram 63%, adulto jovens e adultos foram 100% infectados. O H. caimani apresentou ainda uma distribuição agregada na população do C. yacare. De maneira a esclarecer os fatores responsáveis pela alta quantidade de jacarés infectados na região do Pantanal Sul, iniciamos estudos para investigar os possíveis fatores responsáveis por este fenômeno. Em um segundo momento, realizamos o levantamento dos culicídeos que realizam repasto sanguíneo nos C. yacare e verificamos infecções naturais pelo H. caimani somente em exemplares do gênero Culex, sobretudo no subgênero Melanoconion. Obtivemos infecções experimentais de C. yacare limpos após a ingestão de mosquitos Culex spp. com oocistos esporulados. A espécie incriminada como vetora primária do parasito foi Cx. (Mel.) theobaldi, devido a sua elevada abundância, preferência alimentar pelos jacarés e elevados níveis de infecção natural pelo H. caimani. Verificamos que alguns gêneros de anuros silvestres, e os próprios C. yacare, mediante o canibalismo, podem ser considerados hospedeiros intermediários do H. caimani, provavelmente contribuindo para a transmissão do mesmo no ambiente silvestre. Por fim, verificamos que gametócitos do H. caimani em jacarés C. yacare e C. latirostris, experimentalmente infectados, podem ser encontrados por períodos que variaram de três a seis meses nos crocodilianos, sugerindo que os jacarés podem servir como fonte de infecção para os vetores no campo por períodos relativamente longos / We studied aspects of the host-parasite relation beteween the protozoan parasite Hepatozoon caimani (Apicomplexa: Adeleorina) and its vertebrate host, the caiman Caiman yacare, in the Pantanal region, Brazil. We investigated the eTpizootiological components of this relation by surveying the prevalence and parasitemia of TH. caimani in a population of C. yacare and searched for the factors that could influence the infection rates, such as sex, size/age and Tbody condition from July 2006 to February 2008. We sampled 229 caimans and found a prevalence of 76% and the infection rate was related to caimans size/age. The young ones were not infected, the prevalence was 63% in the juveniles and 100% among the adult ones. THepatozoon caimani showed an aggregated pattern of distribuition. We also investigated the factors likely to have caused the high prevalence of H. caimani among caimans. By sampling the culicids that bite C. yacare, we found that the natural infection occurs only by Culex species, especially those belonging to the subgenus Melanoconion. Experimental infections of clean caimans C. yacare was retrieved after they ingested Culex spp. with sporulated oocysts. Culex (Mel.) theobaldi was pointed as the main vector because of its great abundance, preferance for biting caimans and high natural infection levels by H. caimani. Some wild anuran genera may also be pointed as paratenic hosts, as well as caimans C. yacare themselves through canibalism. Finally, H. caimani can be found in experimentally infected caimans C. yacare and C. latirostris for three up to six months, suggesting caimans as a perennial source of infection for the vectors in the environment

L'autophagie est un processus d'auto-dégradation conservé chez la plupart des eucaryotes. Généralement induit par un stress nutritif, il requiert la formation d'un compartiment à double membrane appelé l’autophagosome qui séquestre et transporte des composants intracellulaires dégradés et recyclés dans le lysosome. La protéine ATG8, qui occupe une position centrale dans ce processus, est recrutée aux membranes de l’autophagosome par un système de conjugaison très régulé. Toxoplasma gondii est un protozoaire parasite appartenant au phylum des Apicomplexes, qui contient une machinerie d'autophagie réduite. Suite à un stress nutritif, ce parasite intracellulaire obligatoire est néanmoins capable de générer des autophagosomes décorés par TgATG8. De façon surprenante, en condition normale de croissance intracellulaire, cette protéine se localise principalement à l’apicoplaste, un plaste non photosynthétique acquis par endosymbiose secondaire qui contient des voies métaboliques essentielles à la survie du parasite. Le but de ma thèse a été d’élucider les fonctions canoniques et non canoniques d‘ATG8 chez Toxoplasma. La première partie de cette étude porte sur la caractérisation fonctionnelle et spatio-temporelle de l'association de TgATG8 avec l’apicoplaste. Nous avons montré que TgATG8 est recrutée aux extrémités de l’apicoplaste en élongation, ce qui permet le maintien de l’organelle à travers les générations en le connectant aux centrosomes pour une répartition dans les deux cellules filles. La deuxième partie de ce travail vise à isoler et identifier par spectrométrie de masse des partenaires putatifs de TgATG8 qui seraient impliqués dans l’autophagie ou dans le rôle non-canonique à l’apicoplaste. Nous avons analysé la localisation subcellulaire de neuf candidats et des caractérisations fonctionnelles ont été entreprises pour trois protéines. Bien que nous n’ayons pas pu confirmer leurs interactions avec TgATG8, cela a permis l'identification de nouvelles protéines parasitaires: une phospholipase à l’apicoplaste essentielle à la survie du parasite, un régulateur potentiel du cycle cellulaire et un composant du cytosquelette du parasite. / Autophagy is a self-degradative process evolutionary conserved among eukaryotes. Typically induced by starvation, it involves the formation of a double membrane compartment called the autophagosome to sequester and deliver intracellular components for lysosomal degradation and recycling. The protein ATG8 occupies a central position in this process and is recruited to autophagosomal membranes by a highly regulated conjugation system. Toxoplasma gondii is a parasitic protist belonging to the Apicomplexa phylum, which possesses a reduced autophagy machinery. This obligate intracellular parasite is nevertheless able to generate TgATG8-decorated autophagosomes upon nutrient stress. Surprisingly, during normal intracellular parasite growth, TgATG8 mainly localizes to the apicoplast, a non-photosynthetic plastid acquired by secondary endosymbiosis which hosts essential metabolic pathways. My thesis aimed to elucidate the canonical and non-canonical roles of ATG8 in Toxoplasma. The first part of this study is the functional and spatio-temporal characterization of TgATG8 association with the apicoplast. We showed TgATG8 is recruited to both ends of the elongating plastid during parasite division, and allows the maintenance of the organelle across generations by permitting its centrosome-driven distribution into the two daughter cells. The second part of this work is the isolation and mass spectrometry-based identification of putative TgATG8-interacting proteins that would be involved in autophagy-related or non-canonical functions. We analyzed the subcellular localization of nine candidates and functional studies were conducted for three proteins. Although we were unable to confirm their interactions with TgATG8, this approach allowed the identification of novel and important parasite proteins: an essential apicoplast phospholipase, a potential regulator of the cell cycle, and a component of the parasite cytoskeleton.

Phylum Apicomplexa comprises several thousand parasitic protozoans that cause significant disease in humans and animals worldwide. Of particular relevance to human health are Plasmodium spp., the causative agents of malaria; and Toxoplasma gondii, which infects approximately 30% of all humans on earth, and causes serious disease in immunocompromised individuals and neonatally infected fetuses. Central to the pathogenesis of apicomplexans is a unique form of substrate-dependent locomotion termed “gliding motility”, which is essential for traversing the environment and actively invading host cells. Driving motility is the class-XIV unconventional myosin motor (MyoA), which is notably divergent from canonical myosins in that it lacks a “tail” and conventional sequence motifs in both the neck and motor regions. Thus, the mechanisms that enable MyoA to function with a step size and velocity similar to canonical human myosins are not well understood. Over the past 2 decades, the apicomplexan research community has identified many of the components involved in gliding motility, resulting in a functional model of MyoA and accessory proteins forming the “glideosome” macromolecular complex. However, there was still relatively little known about the unique physical processes that drive force production and transduction in the apicomplexan motor complex. Thus, I set out to use structural and biophysical methods to interrogate this divergent molecular motor, and provide the first high-resolution model of apicomplexan motility. Towards this goal, I first used structural and biophysical methods to establish the most complete model to date of class-XIV motor complex assembly, answering key questions about the interface between MyoA and its accessory proteins. To understand the unique molecular basis of force production in apicomplexan motors, I then solved the first ever crystal structure of a class-XIV myosin, MyoA from T. gondii. Supplementing this structure with further biophysical data, I was able to determine the functional consequences of class-defining sequence polymorphisms, and elucidate the basis of phosphorylation-dependent motor regulation. The systematic dissection of apicomplexan motor complexes described herein provides crucial insight into a fundamental biological process, and may help overcome existing barriers for targeted therapeutic development. / Graduate

No description available.

Coccidia are obligate intracellular parasites belonging to the phylum Apicomplexa. Many coccidia are of medical and veterinary importance such as Cystoisospora species and Toxoplasma gondii. The need to discover new anticoccidial therapies has increased due to development of resistance by the parasite or toxicity issues in the patient. The goals of this work were to develop a model for canine coccidiosis while proving that Cystoisospora canis is a true primary pathogen in dogs and to determine the efficacy of a new anticoccidial agent. A canine coccidiosis model would be useful in evaluating new anticoccidial treatments. Oral infections with 5 X 104 (n=2) and 1 X 105 (n=20) sporulated C. canis oocysts were attempted in 22 purpose bred beagle puppies. Clinical signs associated with disease were observed in all dogs. Bacterial and viral pathogens were ruled out by transmission electron microscopy (TEM) and bacterial growth assays. Development of C. canis in cell culture was also evaluated. The efficacy of ponazuril, a new anticoccidial drug, was examined in T. gondii. In vitro studies were conducted to determine the activity of ponazuril on tachyzoites and how this agent affects development of apicomplexcan parasites. The tachyzoite production assay was conducted. Ponazuril at a dose of 1.0 µg/ml had a significant affect on tachyzoite reproduction. Comparisons were made on how ponazuril affects T. gondii and Neospora caninum. Inhibition of T. gondii tachyzoites occurred after the second round of replication and with N. caninum tachyzoites after 4 rounds of replication. Results of TEM revealed ponazuril affects replication of T. gondii and N. caninum differently. The efficacy of ponazuril to prevent and treat acute and chronic toxoplasmosis was investigated. Mice treated prophylactically with ponazuril were completely protected from developing an acute T. gondii infection. Fatal toxoplasmosis was prevented in mice starting treatment 3 and 6 days post infection at a dose of 20 mg/kg. Immunohistochemistry was used to evaluate ponazuril's effect on chronic toxoplasmosis. Sections of brain were scored according to the number of tissue cysts present. Ponazuril also proved to be highly active against toxoplasmic encephalitis in an interferon-gamma knockout mouse model. / Ph. D.

Neospora caninum (filo Apicomplexa) é um parasita obrigatório intracelular como todos os membros deste filo, alguns reconhecidos por causarem doenças com impacto relevante na saúde humana (Plasmodium e Toxoplasma) e veterinária (Babesia, Eimeria e Cryptosporidium). Causador da neosporose, N. caninum vem emergindo como um dos maiores causadores de abortos infecciosos em bovinos, levando a consideráveis perdas econômicas na bovinocultura mundial. Devido à sua recente descoberta, o conhecimento sobre diversos processos bioquímicos de N.caninum ainda é limitado, demandando novas pesquisas para a compreensão de seus mecanismos de sobrevivência e consequente identificação de alvos para intervenção terapêutica. O processo de invasão celular é bastante investigado em pesquisas envolvendo apicomplexas, uma vez que a sobrevivência desses parasitas depende do sucesso de sua entrada na célula hospedeira. Proteínas secretadas de organelas filo-específicas (micronemas, roptrias e grânulos densos) estão intimamente envolvidas com a invasão celular. Elas são responsáveis pela interação inicial com a célula hospedeira, participam da junção de movimento formada no momento da invasão, e contribuem para a estabilização do vacúolo parasitóforo. Neste trabalho as proteínas secretadas por taquizoítas de N. caninum foram investigadas de duas formas: (1) por caracterização molecular de proteínas com domínio Apple; e (2) por estudo do secretoma do parasita. Os domínios proteicos do tipo Apple são caracterizados pela capacidade de interação proteína-proteína e proteína-carboidrato, e estão presentes em algumas proteínas micronêmicas com propriedades adesivas. Neste trabalho três proteínas de N. caninum contendo domínios Apple foram caracterizadas: MIC17A, MIC17B e MIC17C. A análise das sequências proteicas e das estruturas dos domínios Apple, obtidas por modelagem molecular, mostraram alta identidade sequencial e estrutural entre MIC17A e MIC17C. Apesar de ser paráloga às outras duas, MIC17B apresenta diferenças importantes em sua sequência e estrutura. Para MIC17B e MIC17C foram realizados experimentos de detecção das proteínas nativas nos extratos total e secretado do taquizoíta que sugerem diferentes formas de processamento entre essas proteínas no parasita. Para MIC17B foi confirmada a localização em micronemas, num padrão diferente do observado para MIC17C. Os ensaios de invasão combinados aos de localização indicam que estas proteínas estejam relacionadas ao processo de invasão celular, porém, suas funções permanecem desconhecidas. O secretoma é o conjunto de proteínas secretadas pelo parasita e, para explorar a composição deste extrato (ESA) no taquizoíta de N. caninum, duas abordagens complementares foram utilizadas. Na primeira abordagem foram identificadas as proteínas presentes no ESA por espectrometria de massas. Na segunda abordagem realizou-se uma ii quantificação relativa das proteínas, marcadas por dois isótopos, nos extratos totais de taquizoítas submetidos ou não ao estímulo secretório. O resultado esperado seria com as proteínas secretadas diminuídas no parasita estimulado. Em ambas as abordagens foram utilizadas técnicas de espectrometria de massas de alta resolução (nanoLC-MS/MS), o que resultou num alto número de identificações; 615 proteínas no ESA e 2011 proteínas quantificadas. A comparação das duas abordagens permitiu o reconhecimento de proteínas com maior probabilidade de secreção. Uma rede de interação entre as proteínas diferencialmente expressas foi predita, gerando resultados que, associados às informações sobre as proteínas aumentadas, permitiram uma investigação sobre proteínas potencialmente envolvidas com a regulação do metabolismo relacionado à secreção. Os resultados obtidos por ambos os estudos aqui demonstrados somam conhecimento acerca do parasita N. caninum e demonstram ser úteis para guiar a busca e seleção de alvos a serem investigados para o desenvolvimento de terapêutica contra a neosporose. / Neospora caninum (Apicomplexa phylum) is an obligatory intracellular parasite like all members from this phylum, some causing diseases with relevant impact on human (Plasmodium and Toxoplasma) and veterinary (Babesia, Eimeria and Cryptosporidium) health. Causative agent of neosporosis, N. caninum has emerged as one of the leading causes of infectious abortion in cattle, generating huge economical losses in worldwide livestock. Due to its recent discovery, knowledge of N. caninum biochemical processes remains scarce, demanding new research for comprehending its survival mechanisms and, consequently, identifying new targets for therapeutic intervention. The invasion process has often been investigated in apicomplexans since their survival depends on the success of their entry into the host cell. Proteins secreted from phylum-specific organelles (micronemes, rhoptries and dense granules) are deeply involved with invasion. They are responsible for the initial interaction with the host cell; participate of the moving junction formed in the moment of invasion; and contribute for the stabilization of the parasitophorus vacuole. In this study, the proteins secreted by N. caninum tachyzoites were investigated in two ways: (1) the molecular characterization of Apple domaincontaining proteins; and (2) exploring the parasite secretome. The Apple protein domains are characterized by the ability to interact as protein-protein and proteincarbohydrate, and are present in some microneme proteins with adhesive properties. Here three N. caninum proteins containing Apple domains were characterized: MIC17A, MIC17B and MIC17C. Analyses of the Apple domains sequences and structures, obtained by molecular modeling, revealed high sequential and structural identities between MIC17A and MIC17C. Although being a paralog of the other two proteins, MIC17B presents significant differences in its sequence and structure. Experiments were performed for native MIC17B and MIC17C detection in the total and secreted tachyzoite extracts, suggesting different processing forms for these proteins in the parasite. For MIC17B, the microneme localization was confirmed, differently from the pattern observed for MIC17C. Invasion and localization assays indicated that these proteins are related to the cell invasion process; nevertheless, their functions remain unknown. The secretome is the set of proteins secreted by the parasite and, to explore this extract (ESA) composition in N. caninum, two complementary approaches were used. Firstly proteins present in ESA were identified by mass spectrometry. In the second approach, a relative quantification was performed on the proteomes of ethanol stimulated/non stimulated tachyzoites, expecting that the secreted proteins would be down regulated at the stimulated parasite. Both approaches were performed with high resolution mass spectrometry techniques (nanoLC-MS/MS), reaching a high number of identifications: 615 proteins iv in ESA and 2011 quantified proteins. The comparison between both approaches allowed the recognition of the most likely secreted proteins. An interaction network was predicted, involving the differentially expressed proteins. These results, associated with the information of up regulated proteins, allowed the investigation of proteins potentially involved with the secretion metabolism regulation. The findings from our two studies add up knowledge about N. caninum and demonstrate to be useful in guiding the search and selection for new targets for therapeutic development against neosporosis.

Wir nutzten in dieser Studie den apikomplexen Parasiten Eimeria falciformis als Modell. Unsere Ergebnisse zeigen, dass das in infizierten Wildtypmäusen dominierende Zytokin IFN-γ für Immunschutz und für die Entwicklung der Darmpathologie entbehrlich war. E. falciformis-infizierte IFN-γR-/- and IFN-γ-/- Mäuse zeigten extremen Körpergewichtsverlust und starke Pathologie im Darm. Die Entwicklung des Parasiten in diesen Mäusen war überraschenderweise reduziert. Diese Beobachtungen gingen mit einer drastisch erhöhten Produktion von parasiten-spezifischem IL-17A und IL-22 durch CD4+ T Zellen einher. Gleichzeitige Neutralisierung von IL-17A und IL-22 in E. falciformis-infizierten IFN-γR-/- Mäusen verringerte den Körpergewichtsverlust und die Darmpathologie, und führte zu einer erhöhten Ausscheidung von Parasiten. Die Behandlung einer E. falciformis-infizierten intestinalen Epithelzelllinie mit IL-17A oder IL-22 führte zu einer signifikant reduzierten Entwicklung von E. falciformis in vitro. Diese Daten demonstrieren erstmalig einen anti-parasitären Effekt von IL-22 im Darm und deuten auf redundante Rollen von IL-17A und IL-22 im Hinblick auf die Förderung von Darmpathologie in Abwesenheit von IFN-γ hin. Um E. falciformis als Modellsystem weiter zu entwickeln, haben wir die Transfektion von E. falciformis Sporozoiten mit verschiedenen Plasmiden die den Reporter YFP und den Resistenzmarker DHTS enthalten etabliert. Rektal in Mäuse injizierte Sporozoiten entwickelten sich erfolgreich zu Oocysten, wenn auch mit geringerer Effizienz im Vergleich zur oralen Infektion mit Oozysten. Wiederholte in vivo Selektion YFP-exprimierender Oozysten führte zu Populationen mit maximal 34 % YFP-exprimierenden Parasiten. Wir demonstrieren in dieser Arbeit zum ersten Mal die Transfektion von E. falciformis und zeigen Perspektiven im Hinblick auf die Etablierung einer stabil transgenen Parasitenlinie auf. / The roles of Th1 and Th17 responses as mediators of host protection and pathology in the intestine are the subjects of intense research. Here we investigated a model of intestinal inflammation driven by the intracellular apicomplexan parasite Eimeria falciformis. Although IFN-γ was the predominant cytokine during E. falciformis infection in wild type mice, it was found to be dispensable for host defence and the development of infection-driven intestinal inflammation. E. falciformis-infected IFN-γR-/- and IFN-γ-/- mice developed dramatically exacerbated body weight loss and intestinal pathology, but surprisingly harboured fewer parasites. This was associated with a striking increase in parasite-specific IL-17A and IL-22 production in the mesenteric lymph nodes and at the site of infection. Concurrent neutralisation of IL-17A and IL-22 in E. falciformis infected IFN-γR-/- mice resulted in a reduction in infection induced body weight loss and inflammation and significantly increased parasite shedding. Taken together these data demonstrate for the first time an anti-parasitic effect of IL-22 during an intestinal infection and suggest that IL-17A and IL-22 have redundant roles in driving intestinal pathology in the absence of IFN-γ signalling. To further develop E. falciformis as a model system, we established transfection of E. falciformis sporozoites using various plasmids that contain the fluorescent reporter YFP and the resistance marker DHTS. Sporozoites applied rectally to mice were shown to complete their life cycle, albeit with a lower efficiency in comparison to oral infection with oocysts. Repeated in vivo selection using pyrimethamine and/or FACS and manual sorting led to a maximum percentage of 34 % YFP-expressing oocysts. Taken together, we demonstrate for the first time transfection of E. falciformis and provide perspectives for further work on the establishment of a stable transgenic parasite line.

Neospora caninum (filo Apicomplexa) é um parasita obrigatório intracelular como todos os membros deste filo, alguns reconhecidos por causarem doenças com impacto relevante na saúde humana (Plasmodium e Toxoplasma) e veterinária (Babesia, Eimeria e Cryptosporidium). Causador da neosporose, N. caninum vem emergindo como um dos maiores causadores de abortos infecciosos em bovinos, levando a consideráveis perdas econômicas na bovinocultura mundial. Devido à sua recente descoberta, o conhecimento sobre diversos processos bioquímicos de N.caninum ainda é limitado, demandando novas pesquisas para a compreensão de seus mecanismos de sobrevivência e consequente identificação de alvos para intervenção terapêutica. O processo de invasão celular é bastante investigado em pesquisas envolvendo apicomplexas, uma vez que a sobrevivência desses parasitas depende do sucesso de sua entrada na célula hospedeira. Proteínas secretadas de organelas filo-específicas (micronemas, roptrias e grânulos densos) estão intimamente envolvidas com a invasão celular. Elas são responsáveis pela interação inicial com a célula hospedeira, participam da junção de movimento formada no momento da invasão, e contribuem para a estabilização do vacúolo parasitóforo. Neste trabalho as proteínas secretadas por taquizoítas de N. caninum foram investigadas de duas formas: (1) por caracterização molecular de proteínas com domínio Apple; e (2) por estudo do secretoma do parasita. Os domínios proteicos do tipo Apple são caracterizados pela capacidade de interação proteína-proteína e proteína-carboidrato, e estão presentes em algumas proteínas micronêmicas com propriedades adesivas. Neste trabalho três proteínas de N. caninum contendo domínios Apple foram caracterizadas: MIC17A, MIC17B e MIC17C. A análise das sequências proteicas e das estruturas dos domínios Apple, obtidas por modelagem molecular, mostraram alta identidade sequencial e estrutural entre MIC17A e MIC17C. Apesar de ser paráloga às outras duas, MIC17B apresenta diferenças importantes em sua sequência e estrutura. Para MIC17B e MIC17C foram realizados experimentos de detecção das proteínas nativas nos extratos total e secretado do taquizoíta que sugerem diferentes formas de processamento entre essas proteínas no parasita. Para MIC17B foi confirmada a localização em micronemas, num padrão diferente do observado para MIC17C. Os ensaios de invasão combinados aos de localização indicam que estas proteínas estejam relacionadas ao processo de invasão celular, porém, suas funções permanecem desconhecidas. O secretoma é o conjunto de proteínas secretadas pelo parasita e, para explorar a composição deste extrato (ESA) no taquizoíta de N. caninum, duas abordagens complementares foram utilizadas. Na primeira abordagem foram identificadas as proteínas presentes no ESA por espectrometria de massas. Na segunda abordagem realizou-se uma ii quantificação relativa das proteínas, marcadas por dois isótopos, nos extratos totais de taquizoítas submetidos ou não ao estímulo secretório. O resultado esperado seria com as proteínas secretadas diminuídas no parasita estimulado. Em ambas as abordagens foram utilizadas técnicas de espectrometria de massas de alta resolução (nanoLC-MS/MS), o que resultou num alto número de identificações; 615 proteínas no ESA e 2011 proteínas quantificadas. A comparação das duas abordagens permitiu o reconhecimento de proteínas com maior probabilidade de secreção. Uma rede de interação entre as proteínas diferencialmente expressas foi predita, gerando resultados que, associados às informações sobre as proteínas aumentadas, permitiram uma investigação sobre proteínas potencialmente envolvidas com a regulação do metabolismo relacionado à secreção. Os resultados obtidos por ambos os estudos aqui demonstrados somam conhecimento acerca do parasita N. caninum e demonstram ser úteis para guiar a busca e seleção de alvos a serem investigados para o desenvolvimento de terapêutica contra a neosporose. / Neospora caninum (Apicomplexa phylum) is an obligatory intracellular parasite like all members from this phylum, some causing diseases with relevant impact on human (Plasmodium and Toxoplasma) and veterinary (Babesia, Eimeria and Cryptosporidium) health. Causative agent of neosporosis, N. caninum has emerged as one of the leading causes of infectious abortion in cattle, generating huge economical losses in worldwide livestock. Due to its recent discovery, knowledge of N. caninum biochemical processes remains scarce, demanding new research for comprehending its survival mechanisms and, consequently, identifying new targets for therapeutic intervention. The invasion process has often been investigated in apicomplexans since their survival depends on the success of their entry into the host cell. Proteins secreted from phylum-specific organelles (micronemes, rhoptries and dense granules) are deeply involved with invasion. They are responsible for the initial interaction with the host cell; participate of the moving junction formed in the moment of invasion; and contribute for the stabilization of the parasitophorus vacuole. In this study, the proteins secreted by N. caninum tachyzoites were investigated in two ways: (1) the molecular characterization of Apple domaincontaining proteins; and (2) exploring the parasite secretome. The Apple protein domains are characterized by the ability to interact as protein-protein and proteincarbohydrate, and are present in some microneme proteins with adhesive properties. Here three N. caninum proteins containing Apple domains were characterized: MIC17A, MIC17B and MIC17C. Analyses of the Apple domains sequences and structures, obtained by molecular modeling, revealed high sequential and structural identities between MIC17A and MIC17C. Although being a paralog of the other two proteins, MIC17B presents significant differences in its sequence and structure. Experiments were performed for native MIC17B and MIC17C detection in the total and secreted tachyzoite extracts, suggesting different processing forms for these proteins in the parasite. For MIC17B, the microneme localization was confirmed, differently from the pattern observed for MIC17C. Invasion and localization assays indicated that these proteins are related to the cell invasion process; nevertheless, their functions remain unknown. The secretome is the set of proteins secreted by the parasite and, to explore this extract (ESA) composition in N. caninum, two complementary approaches were used. Firstly proteins present in ESA were identified by mass spectrometry. In the second approach, a relative quantification was performed on the proteomes of ethanol stimulated/non stimulated tachyzoites, expecting that the secreted proteins would be down regulated at the stimulated parasite. Both approaches were performed with high resolution mass spectrometry techniques (nanoLC-MS/MS), reaching a high number of identifications: 615 proteins iv in ESA and 2011 quantified proteins. The comparison between both approaches allowed the recognition of the most likely secreted proteins. An interaction network was predicted, involving the differentially expressed proteins. These results, associated with the information of up regulated proteins, allowed the investigation of proteins potentially involved with the secretion metabolism regulation. The findings from our two studies add up knowledge about N. caninum and demonstrate to be useful in guiding the search and selection for new targets for therapeutic development against neosporosis.

Indiana University-Purdue University Indianapolis (IUPUI) / Histone acetylation has been linked to developmental changes in gene expression and is a validated drug target of apicomplexan parasites, but little is known about the roles of individual histone modifying enzymes and how they are recruited to target genes. The protozoan parasite Toxoplasma gondii (phylum Apicomplexa) is unusual among invertebrates in possessing two GCN5-family lysine acetyltransferases (KATs). While GCN5a is required for gene expression in response to alkaline stress, this KAT is dispensable for parasite proliferation in normal culture conditions. In contrast, GCN5b cannot be disrupted, suggesting it is essential for Toxoplasma viability. To further explore the function of GCN5b, we generated clonal parasites expressing an inducible HA-tagged form of GCN5b containing a point mutation that ablates enzymatic activity (E703G). Stabilization of this dominant-negative form of GCN5b was mediated through ligand-binding to a destabilization domain (dd) fused to the protein. Induced accumulation of the ddHAGCN5b(E703G) protein led to a rapid arrest in parasite replication. Growth arrest was accompanied by a decrease in histone H3 acetylation at specific lysine residues as well as reduced expression of GCN5b target genes in GCN5b(E703G) parasites, which were identified using chromatin immunoprecipitation coupled with microarray hybridization (ChIP-chip). We also demonstrate that GCN5b interacts with AP2-domain proteins, which are plant-like transcription factors in Apicomplexa. The interactions between GCN5b, AP2IX-7, and AP2X-8 were confirmed by reciprocal co-immunoprecipitation and revealed a “core complex” that includes the co-activator ADA2-A, TFIID subunits, LEO1 polymerase-associated factor (Paf1) subunit, and RRM proteins. The dominant-negative phenotype of ddHAGCN5b(E703G) parasites, considered with the proteomics and ChIP-chip data, indicate that GCN5b plays a central role in transcriptional and chromatin remodeling complexes. We conclude that GCN5b has a non-redundant and indispensable role in regulating gene expression required during the Toxoplasma lytic cycle.

Small molecule screening is commonly used to discover lead compounds for drug development, but it can also be a powerful way to identify chemical probes for studying biological mechanisms. Our lab uses small molecules to study the mechanisms by which the protozoan parasite Toxoplasma gondii infects and replicates within its hosts. In this work, we employed a fluorescence-based assay to screen the Medicines for Malaria Venture (MMV) Open Access Malaria box for compounds that affect T. gondii growth. The box contains 400 previously identified small-molecule inhibitors of the related parasite, Plasmodium falciparum. We identified 79 hits, including a 2,4-diaminoquinazoline (MMV006169; IC50=1.15µM) that strongly inhibits T. gondii intracellular replication and invasion with no evidence of toxicity to mammalian cells. Extensive structure-activity relationship analyses with T. gondii identified a number of analogs with changed potency and altered effects on replication and invasion. These structure-activity analyses provided the information necessary to synthesize a bivalent chemical inducer of dimerization (CID) containing MMV006169 for use in yeast three-hybrid experiments. Yeast growth competition assays showed that this CID is capable of entering the yeast nucleus, as required for yeast three-hybrid screening. Yeast three-hybrid was used in a targeted format to test the hypothesis that MMV006169 works by inhibiting parasite CDC48, an ATPase involved in trafficking and the degradation of misfolded proteins. Large-scale cDNA library screening by yeast three-hybrid suggests that the compound may instead be working through inhibition of a host cell target. This work has provided insight into how MMV006169 affects the parasite's lytic cycle and generated a testable hypothesis for the biologically relevant target of the compound.