A toxoplasmose afeta 1/3 da população humana e só existe uma vacina para uso veterinária. A radiação gama altera as proteínas tornando-as mais imunogênicas, por oxidação e melhor apresentação de antígenos na ausência de adjuvantes. Irradiamos extrato solúvel de taquizoítos da cepa RH de T. gondii (AgTg), e avaliamos seu uso como vacina em camundongos BALB/c. Doses abaixo de 500Gy não afetavam e doses acima de 2000Gy destruíam o extrato, sendo que animais imunizados com extrato irradiado a 1000, 1500 e 2000Gy tinham mais IgG especifica de maior avidez, comparado ao AgTg nativo (p<0.05). Animais imunizados pelo AgTg 1500Gy tiveram aumento da proliferação de esplenócitos, fenotipados como CD3+CD4+, CD3+CD8+ e de linfócitos B, comparados com animais imunizados pelo AgTg nativo. Animais imunizados pelo AgTg 1500Gy após desafio com cepa ME-49 cistogênica apresentaram menor numero de cistos cerebrais e maior sobrevida pós-desafio com cepa virulenta RH. A radiação ionizante em extratos de T.gondii aumenta a resposta imune e a memória imunológica na ausência de adjuvantes. / Toxoplasmosis affects 1/3 of the human population and only a vaccine for veterinary use. Gamma radiation alters the proteins making them more immunogenic by oxidation and better antigen presentation in the absence of adjuvants. Radiate soluble extract of RH strain tachyzoites of T. gondii (AgTg ) , and evaluate its use as a vaccine in BALB/c . Doses below 500Gy not affected and destroyed 2000Gy doses above extract, whereas animals immunized with irradiated extract at 1000, 1500 and 2000Gy had more of specific IgG avidity, compared to native AgTg (p < 0, 05). AgTg 1500GY the immunized animals had increased proliferation of splenocytes, phenotyped as CD3+CD4+,CD3+CD8+ and B-lymphocytes immunized animals compared to the native AgTg . Animals immunized by AgTg 1500GY after challenge with strain ME- 49 cystogenic showed lower number of brain cysts and greater survival after challenge with virulent RH. Ionizing radiation in extracts of T. gondii increases the immune response and immune memory in the absence of adjuvants.

Made available in DSpace on 2014-08-20T14:31:31Z (GMT). No. of bitstreams: 1 dissertacao_beatris_cademartori.pdf: 636391 bytes, checksum: 43565196211ab5284ee321f2b82e0430 (MD5) Previous issue date: 2007-06-28 / The toxoplasmosis acquired during pregnancy of benign course in imunocompetentes patients and asymptomatic in 85% of the cases may infect the fetus and result in congenital infection. Although most of the newborns do not present symptoms or clinical signs, there may be sequelae during the childhood and adult life, being the corioretinitis the most frequent one. Among the strategies to prevent congenital toxoplasmosis we can find the education for health and the preconception trial, which aim is to reduce the exposition to the risk factors that may lead to an infection by T. gondii, to detect and to treat acute infection in pregnant woman. The aims of this study were to evaluate the prevalence and risk of congenital transmission by Toxoplasma gondii, through serological analysis of the pregnant women and also through their knowledge about the zoonose transmission routes, to check the possible association between the soropositivity of the pregnant women and their exposition to the infection risk factors, and also to compare the Chemiluminescence and Indirect Immunofluorescence techniques in the diagnosis of the toxoplasmosis in the studied sample. A transversal study was conducted, with 425 pregnant women who were attending prenatal care in public health centres of Pelotas (RS-Brazil), during the period of May to November 2006. It was verified a T. gondii soroprevalence of (IgG) 54,8% and it was not detected the presence of IgM antibodies. The analysis of the risk factors showed a significant association (p<0.05) between the soropositivity of the pregnant women and their age, the handle of raw meat, the intake of raw vegetables, raw or undercooked meat and raw or nonpasteurized milk and direct contact with the soil. The Logistic Regression Model confirmed the above associations to the intake of raw or undercooked meat (OR=1.85) and to the direct contact with the soil (OR=2.67). The interaction with cats did not present a significant association with the T. gondii soropositivity patients. It was observed that the pregnant women who were interviewed did not know much about the disease and its main sources of infection. The Chemiluminescence technique proved to be an adequate one in the routine diagnosis of the T. gondii infection as, besides being a practical technique, it showed results comparable to the ones of the Indirect Immunofluoresence, sensibility, specificity and accuracy. The results indicate the necessity of implementing T. gondii primary prevention programmes for the pregnant women who as a way of reducing the risks of congenital toxoplasmosis. / A toxoplasmose adquirida durante a gestação, de curso benigno em pacientes imunocompetentes e assintomática em 85% dos casos, pode ser transmitida para o feto e resultar em infecção congênita. Embora maioria dos recém- nascidos não apresente sintomas ou sinais clínicos, pode evoluir para seqüelas na infância e vida adulta, sendo que a coriorretinite é a lesão mais freqüente. Entre as estratégias de prevenção da toxoplasmose congênita incluem-se a educação em saúde e a triagem pré-natal, com o objetivo de reduzir a exposição aos fatores de risco para a infecção por Toxoplasma gondii, detectar e tratar a infecção aguda na gestante. Os objetivos foram avaliar a prevalência e o risco de transmissão congênita por T. gondii, através da análise do perfil sorológico das mesmas e do conhecimento sobre as formas de transmissão da zoonose, verificar possíveis associações entre soropositividade das gestantes e a exposição aos fatores de risco da infecção, além de comparar as técnicas de Quimioluminescência e Imunofluorescência Indireta para o diagnóstico sorológico da toxoplasmose na amostra estudada. Foi realizado um estudo transversal, com 425 gestantes que realizaram o pré-natal nos Postos de Saúde de Pelotas-RS, no período de maio a novembro de 2006. Constatou-se uma soroprevalência (IgG) de 54,8% para T. gondii e não foi detectado a presença de anticorpos IgM. Na análise dos fatores de risco foi verificada associação significativa (p<0,05) entre a soropositividade das gestantes e a idade, manuseio de carne crua, consumo de vegetais crus, de carnes cruas ou mal cozidas, de leite cru não pasteurizado e contato direto com o solo. Através do modelo de regressão logística, essa associação foi confirmada para o consumo de carnes cruas ou mal cozidas (OR=1,85) e o contato direto com o solo (OR=2,67). O convívio com gatos, não apresentou associação significativa com a soropositividade para T. gondii nas pacientes estudadas. Constatou-se elevado desconhecimento da doença e das principais fontes da infecção pelas gestantes entrevistadas. A técnica de Quimioluminescência mostrou-se adequada ao diagnóstico de rotina da infecção por T. gondii, pois, além da praticidade, apresentou de sensibilidade, especificidade e acurácia comparáveis aos da Imunofluorescência Indireta. Os resultados obtidos indicam a necessidade da implantação de programas de prevenção primária da infecção por T. gondii para gestantes, a fim de reduzir o risco da toxoplasmose congênita.

Made available in DSpace on 2014-08-20T14:31:33Z (GMT). No. of bitstreams: 1 dissertacao_jacqueline_radin.pdf: 589146 bytes, checksum: 57a338810c6c34920d4047b74e163eaf (MD5) Previous issue date: 2011-03-11 / The Toxoplasma gondii is a protozoan which causes opportunistic infections in individuals with a compromised immune system. There exist a lot of studies correlating toxoplasmosis with immunosuppressed patients, however, in neoplasia carriers, who may suffer a process of immunosuppression during the chemotherapy, few studies were conducted. The aim of the present study was to evaluate, through serological profile, the risk of acute infection (or reactivation) of T.gondii in cancer patients who had chemotherapy treatment in the School Hospital of Pelotas Federal University and in the Radiotherapy and Cancer Center of Pelotas Santa Casa de Misericórdia , as well as to correlate such cancer with toxoplasmosis, through clinical and epidemiological data. A longitudinal cohort prospective study was carried out with a total of 95 patients, who were followed during the antineoplastic treatment by approximately two cycles, from March to November of 2010. The serological search was performed through Indirect Immunofluorescence Techniques (IFI) and Enzyme Linked Immunosorbent Assays (ELISA), and positive results to antibodies IgM anti-T. gondii were confirmed through Electrochemiluminescence Immunoassay (ECLIA). The epidemiological data were obtained through a questionnaire and the clinical, from the analysis of previously authorized patient dossiers. It was verified that the seropositive rate for IgG anti-T.gondii antibodies was of 84.21% at the pre-treatment chemotherapy sample, and of 83.33% at the post-treatment sample through the IFI technique, observing the low titration of antibodies, while through ELISA, 100% of the samples in both analysis were reagents. Class M antibodies were detected in six of the patients (6.3%) through IFI or ELISA and confirmed by ECLIA in one of them. In the risk factor analysis, it was noted a significant association (p<0.05) among seropositive patients for IgM in relation to the variables death (OR= 4.78) and race (OR= 10.88), respectfully. The results obtained point towards the necessity to perform the monitoring toxoplasmosis in cancer patients who are undergoing chemotherapy treatment, in order to avoid grave or even fatal consequences. / Toxoplasma gondii é um protozoário oportunista causador de infecções em indivíduos com o sistema imunológico comprometido. Existem vários estudos correlacionando a toxoplasmose com pacientes imunocomprometidos, porém, em portadores de neoplasias, que podem vir a sofrer um processo de imunodepressão durante o tratamento quimioterápico, poucos estudos foram realizados. O objetivo deste trabalho foi avaliar, através do perfil sorológico, o risco de infecção aguda ou reativação de T. gondii em pacientes oncológicos, que realizaram tratamento quimioterápico no Hospital Escola da Universidade Federal de Pelotas e no Centro de Radioterapia e Oncologia da Santa Casa de Misericórdia de Pelotas, além de correlacionar o câncer com a toxoplasmose, através de dados clínicos e epidemiológicos. Foi realizado um estudo de coorte longitudinal prospectivo, com um total de 95 pacientes, os quais foram acompanhados por pelo menos duas sessões de quimioterapia, no período de março a novembro de 2010. A pesquisa sorológica foi realizada através das técnicas de Imunofluorescência Indireta (IFI) e ensaio imunoenzimático (ELISA), e os resultados positivos para IgM anti-T.gondii foram confirmados pela eletroquimioluminescência (ECLIA). Os dados epidemiológicos foram obtidos pela aplicação de um questionário e os clínicos através da análise dos prontuários dos pacientes, previamente autorizados. Constatou-se que a taxa de soropositivos para IgG anti-T.gondii foi de 84,21% na amostra pré-tratamento quimioterápico e 83,33% na amostra pós-tratamento pela técnica de IFI, observando-se baixas titulações de anticorpos, enquanto que por ELISA, 100% das amostras, nas duas análises, foram reagentes. Anticorpos da classe M foram detectados em seis pacientes (6,3%) através da técnica de IFI ou ELISA e confirmado por ECLIA em um dos pacientes. Na análise dos fatores de risco, foi verificada associação significativa entre os pacientes soropositivos para IgM em relação às variáveis óbito (OR=4,78) e raça (OR=10,88), respectivamente. Os resultados obtidos indicam a necessidade de se realizar o monitoramento da toxoplasmose em pacientes oncológicos, que estão realizando tratamento quimioterápico, para evitar consequências graves e até mesmo fatais.

Made available in DSpace on 2014-08-20T14:37:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-03-13 / Serum samples from 1840 sheep from Rio Grande do Sul south (area region), Brazil, were tested to verify the prevalence of IgG antibodies against Toxoplasma gondii by Immunofluorescent Antibody Test (IFAT), using sera dilution 1:64 as cut-off value. Four age groups were studied: one year old, two years old, three years old and > four years old sheep. The prevalence rate for T. gondii infection was 20,2% (450/1840) (15,2% < IC < 26,2%) in sheep, with titers ranging from 1:64 to 1:4096. The prevalence rate of sheep farms, that had at least one positive sheep, was 87,4% (83/95) (78,9% < IC < 92,8%). There was association between antibodies seroprevalence to T. gondii and sheep age, once the one year old group s prevalence was 9,7% (95/687) (6,2%<IC95%<15,0%) and > four years old group s was 35,3% (255/681) (27,5%<IC95%<43,8%). Multivariate logistic regression did not reveal any significant seroprevalence association between sex and other risk factors. The univariate analysis showed presence of housed cats (p=0,17) and not carcasses remotion (p=0,20) as variables with some epidemiological importance. Toxoplasma gondii infection occurs in almost every sheep farms and thus, can be responsable for ovine reproductive losses and human health problems in this region. / As amostras de soro sanguíneo de 1840 ovinos do sul do Rio Grande do Sul, foram testadas para determinar a prevalência de anticorpos da classe IgG para Toxoplasma gondii através do uso da Reação de Imunofluorescência Indireta (RIFI), com ponto de corte de 1:64. Os animais experimentais foram divididos em quatro grupos de acordo com a idade: um ano, dois anos, três anos e quatro anos ou mais. A soroprevalência para T. gondii foi 20,2% (450/1840) (15,2% < IC < 26,2%) nos ovinos, com variação de títulos de 1:64 até 1:4096. A prevalência das propriedades foco (com pelo menos um animal positivo) foi 87,4% (83/95) (78,9% < IC < 92,8%). Foi observada associação entre soroprevalência para T. gondii e idade dos ovinos, já que a prevalência no grupo de um ano de idade foi 9,7% (95/687) (6,2%<IC95%<15,0%) e no grupo dos animais com quatro anos ou mais foi 35,3% (255/681) (27,5%<IC95%<43,8%). Não foi observada associação significativa entre soroprevalência e sexo, assim como com nenhum dos fatores de risco avaliados na análise multivariada (regressão logística). Entretanto, na análise univariada, duas variáveis se mostraram com maior intensidade de associação com a soroprevalência que as outras: presença de gatos domésticos (p=0,17) e o não recolhimento de carcaças nas propriedades (p=0,20). Os resultados indicam que a infecção por T. gondii está amplamente distribuída entre os rebanhos ovinos da região sul do Rio Grande do Sul, podendo causar perdas reprodutivas nesses animais e representar riscos à saúde humana.

Toxoplasma gondii, l'agent causal de la toxoplasmose appartient au phylum des Apicomplexes. Comme son nom l'indique, le parasite possède un complexe unique d'organites sécrétoires apicaux, les micronèmes, rhoptries et le conoïde, qui jouent un rôle essentiel dans l’invasion de la cellule hôte et la survie du parasite. T. gondii est devenu un modèle populaire de biologie cellulaire et aussi un outil de référence pour l'étude de l’organisation ultra-structurale et des différentes fonctions des autres parasites du phylum Apicomplexa tel que Plasmodium, l’agent causal de la malaria. Cette thèse porte sur deux facteurs essentiels à la survie du parasite : le complexe adapteur de la clathrine AP1 et la petite GTPase Rab11A qui jouent un rôle crucial dans la régulation de certaines voies du trafic intracellulaire de T. gondii. Ainsi, nos travaux ont permis de démontrer un rôle pour AP1 dans le triage différentiel et le transport vésiculaire des protéines MIC et ROP depuis le Trans-Golgi-Network (TGN) et les compartiments endosomaux, respectivement. D’autre part, nos résultats ont révélé un rôle original de AP1 dans la division parasitaire aux stages tardifs de la cytokinèse. Nous avons également identifié un partenaire de AP1, la protéine unique de T. gondii possédant un domaine ENTH : EpsL (pour Espin-Like Protein). Dans les autres Eucaryotes, les protéines epsines sont connues pour activer la formation des vésicules à clathrine en co-opération avec les complexes AP1 et AP2. Nos résultats ont effectivement démontré un rôle de EpsL, similaire à AP1, pour la biogénèse des rhoptries et micronèmes. Nous avons, dans un deuxième temps, examiné les différentes fonctions de la petite GTPase Rab11A. Notre étude par vidéo-microscopie, semble indiquer que Rab11A régule le transport de vésicules depuis le TGN vers la périphérie cellulaire et en particulier, les pôles basal et apical du parasite. Après sur-production de la forme mutée inactive de Rab11A, nous avons démontré un nouveau rôle de la protéine dans la sécrétion des protéines membranaires de surface et dans l'exocytose des granules denses, lors de l'invasion de la cellule hôte mais aussi durant la réplication parasitaire. Finalement, des expériences de pull-down ont permis d’identifier un partenaire intéressant liant Rab11A seulement sous sa forme activée, la protéine unique de T. gondii contenant un domaine HOOK (TgHOOK), que nous avons caractérisée au niveau fonctionnel. Nos résultats suggèrent que TgHOOK régule le transport des vésicules positives pour Rab11A d’une manière dépendante des microtubules. Par conséquent, cette dernière étude a permis de révéler de nouveaux aspects encore inexplorés, bien qu’essentiels, des mécanismes régulant la sécrétion de molécules à la surface parasitaire. / Toxoplasma gondii, the causative agent for the disease Toxoplasmosis belongs to the phylum Apicomplexa. As the name implies, the parasite possesses a unique complex of apical secretory organelles namely the micronemes, rhoptries and conoid, which favor host cell invasion and intracellular survival. T.gondii has become a popular cell biology model and also a reference tool for studying the structure and functions of other important parasites that belong to the same phylum, such as plasmodium, but also higher eukaryotes. The recent advances in dissecting protein trafficking pathways have led to a better understanding of the biogenesis of apical organelles and also to the identification of crucial protein molecules that could determine the fate of the parasite. This thesis focuses on two different molecules, the Clathrin Adaptor complex AP1 and the small GTPase Rab11A that play a crucial role in distinct trafficking pathways of the parasite contributing to a wide range of functions. First, we reveal a role of AP1 in the differential sorting of microneme and rhoptry proteins at the Tran-Golgi-Network and endosomal level, respectively. Accordingly, depletion of AP1 leads to a defect in apical organelle biogenesis. In addition, we reveal an original role of AP1 in parasite division by regulating late stages of cytokinesis. We also identified and studied a partner of AP1, the unique ENTH domain containing protein of the parasite, EpsL (for Espin-like protein). In other Eukaryotes, epsin proteins are well known regulators of clathrin-mediated vesicular budding in co-operation with AP1 and AP2. We demonstrated that EpsL shares similar functions to AP1 in regulating rhoptry and microneme formation. We next worked on the small GTPase Rab11A and defined the dynamics of the protein within the parasite by live imaging. In addition to its known role in cytokinesis, we unravelled a novel function for the molecule in the secretion of surface membrane proteins and the exocytosis of dense granules during both, parasite invasion and replication. Further, pull down experiments on active Rab11A helped us fish an interesting partner molecule, the unique HOOK-domain containing protein that we functionally characterized for the first time in T.gondii. Our data suggest a role of Rab11A in microtubule-dependent transport of vesicules in a HOOK-regulated manner. Therefore, our study provides novel molecular insights into a yet unexplored but essential aspect of constitutive secretion in the parasite.

Toxoplasma gondii est un parasite intracellulaire obligatoire potentiellement capable d’infecter tous les animaux homéothermes. L’Amérique du Sud, et plus particulièrement le Brésil occupe une place particulière dans l’épidémiologie de ce parasite cosmopolite en raison d’une part de formes cliniques sévères observées chez l’Homme et d’autre part d’une diversité génétique du parasite, sans équivalent jusqu’à présent sur d’autres continents. L’Etat de Rio de Janeiro présente des environnements très contrastés allant d’une ville capitale comportant différents degrés d’urbanisation à des zones isolées de haute altitude, habitat d’une faune très riche.Nous avons cherché à étudier l’influence de facteurs anthropiques et physiques sur l’épidémiologie du parasite dans différents environnements de l’Etat de Rio de Janeiro. Pour cela, nous avons d’une part étudié la séroprévalence chez les chats dans deux environnements avec différents degrés d’urbanisation de la ville de Rio, d’autre part mené des études d’épidémiologie moléculaire à l’aide de prélèvements sur des animaux de la ville de Rio, d’un parc national situé dans les zones de hautes altitudes et des zones rurales avoisinantes.L’étude de séroprévalence chez les chats dans la ville de Rio a permis de montrer l’intérêt de l’utilisation des prélèvements sur papier-filtre pour la réalisation de la technique de Modified-Agglutination-Test (MAT). Elle a révélé une différence significative de prévalence entre les chats errants du quartier résidentiel privé (4/107- 3,74%) et ceux du refuge municipal (32/265-12,08%). La densité des animaux et d’autres facteurs écologiques peuvent expliquer cette différence.La détection d’ADN toxoplasmique a été positive chez 8/16 chats de la ville de Rio, 14/18 animaux domestiques et 23/33 animaux sauvages de la zone d’amortissement du parc, 31/38 petits rongeurs ou marsupiaux piégés dans les zones de haute altitude. La détection a été positive sur les tissus de 3 félidés sauvages de la zone d’amortissement et sur les fèces d’un Puma yagouaroundi retrouvées dans le parc témoignant du rôle de ces félidés sauvages dans la contamination du sol aussi bien en zone préservée que dans les zones rurales proches du parc.Le génotypage par 15 marqueurs microsatellites a été possible pour 6 échantillons (dont une souche viable). Il a révélé une grande diversité génétique, mais aussi la présence aussi bien dans le parc qu’au centre-ville de Rio de souches appartenant aux lignées brésiliennes majeures BRII et BRIII. / Toxoplasma gondii is a protozoan parasite, potentially infecting all warm-blooded animals. In South America, and especially in Brazil, this cosmopolitan parasite presents some peculiarities due to an exceptional genetic diversity and the existence of severe clinical forms in Human, probably linked to the genetic background of some strains. In Brazil, the state of Rio de Janeiro presents highly contrasted environments from the city of Rio de Janeiro with different degrees of urbanizaton to isolated “sky islands”, hosting a rich and endemic fauna.We looked for physical and anthropic factors that may have an impact on the epidemiology of T. gondii in different environments of the state of Rio de Janeiro. We performed a seroprevalence study in cat populations living in 2 districts of Rio de Janeiro city with different degrees of urbanization, and molecular epidemiology studies on tissues of animals collected in Rio city, in a National Park characterized by “sky islands” and in intermediate and rural areas surrounding this park..The seroprevalence study on cats in Rio de Janeiro city allowed to show the value of blood sampling on filter-paper for performing the Modified-Agglutination-Test (MAT) for antibody detection in cats. There was a significant difference between seroprevalence observed in stray-cats from a residential district area with a relatively well-preserved natural environment (4/107- 3.74%) and in cats from a downtown public shelter (32/265-12.08%). Cat population density and other ecological factors may be involved in these differences.T. gondii DNA was detected in tissues of 8/16 cats from Rio city, 14/18 domestic and 23/33 wild animals in the area surrounding the park, and 31/38 small rodents or marsupials captured inside the National Park. DNA detection was positive in 3 tissues from wild felids found in the intermediate area surrounding the park and in feces from a Puma yagouaroundi found inside the park. This showed the role of wild felids in soil contamination in both environments.Genetic characterization using 15 microsatellite markers was successful for 6 samples, including one live strain (full genotyping for 5, and incomplete genotyping for one sample). This small sampling of strains was characterized by a large genetic diversity and revealed the presence of the main brazilian lineages, BRII and BRIII in isolated sky islands as well as in the city center of Rio de Janeiro.

Toxoplasma gondii est un parasite intracellulaire obligatoire responsable de la toxoplasmose. Il appartient au phylum des Apicomplexes, qui regroupe les espèces de Plasmodium responsables de la malaria. Les Apicomplexes ont un mécanisme d’invasion unique, impliquant la formation d’une interaction étroite entre la membrane plasmique du parasite et celle de la cellule hôte, appelée « jonction mobile » (MJ). Cette jonction est composée de la protéine AMA1 exposée à la surface du parasite qui interagit avec le complexe de protéines parasitaires RON2/RON4/RON5/RON8 injecté dans la cellule hôte. Les objectifs de ma thèse ont été d’étayer la composition moléculaire de la MJ et d’étudier la contribution de la cellule hôte dans l’invasion au travers de la recherche de partenaires de l’hôte pour les RONs. Quatre protéines de l’hôte, ALIX, TSG101, CD2AP et CIN85, ont été identifiées à la MJ. Les sites d’interactions ont été cartographiés sur RON2, RON4 et RON5. Des parasites mutants déficients pour le recrutement de ces partenaires ont été générés, et ont permis de montrer l’importance de l’association d’ALIX, TSG101, CD2AP et CIN85 à la MJ pour l’invasion de T. gondii et sa virulence dans un modèle murin. Le rôle de ces protéines dans le maintien des jonctions intercellulaires mais aussi leur participation à des processus de déformation membranaire, suggère que le parasite les recrute afin de favoriser son attachement et son internalisation. Nous avons également identifié un nouveau membre du complexe de RONs, appelé RON14. RON14 n’est pas essentielle pour la survie du parasite in vitro mais contribue à sa virulence in vivo. / Toxoplasma gondii is an obligate intracellular parasite responsible for toxoplasmosis. It belongs to the phylum Apicomplexa, which include Plasmodium species responsible for malaria. Apicomplexa has developed a unique invasion mechanism, involving the formation of a tight connection between the parasite and the host cell plasma membranes called moving junction (MJ). This junction is composed of AMA1 exposed at the surface of the parasite which interacts with a complex of parasite proteins RON2/RON4/RON5/RON8 injected into the host cell. The objectives of my thesis were to decipher the molecular composition of the MJ and to study the contribution of the host cell during invasion though the search of hosts partners for RONs. Four host proteins, ALIX, TSG101, CIN85 and CD2AP, have been identified at the MJ. Binding sites has been mapped on RON2, RON4 and RON5. By generating mutant parasites deficient for host partners recruitment, we show the importance of ALIX, TSG101, CIN85 and CD2AP association at the MJ for invasion and virulence in a mouse model. The role of these proteins in maintaining the intercellular junctions and inducing membrane deformation suggest that the parasite recruits them in order to favor its attachment and its internalization. We also identified a new member of the RON complex, called RON14. RON14 is not essential for the parasite survival in vitro but contributes to its virulence in vivo.

Toxoplasma gondii est un protiste apicomplexe responsable de la toxoplasmose. Ce parasite intracellulaire obligatoire possède des organites sécrétoires apicaux dont les rhoptries qui contiennent des facteurs de virulence essentiels à l'invasion et à la modulation de la cellule hôte qu'il infecte. Au cours de la division cellulaire de T. gondii, les protéines de rhoptries sont synthétisées selon la même cinétique. Dans le but d'identifier de nouvelles protéines dont la fonction est potentiellement liée aux rhoptries, nous avons recherché à partir des bases de données du génome de T. gondii, les protéines présentant ce profil particulier d'expression. La localisation subcellulaire de 12 candidats a été réalisée puis une caractérisation phénotypique de quatre d'entre eux a été entreprise. Nous avons identifié une nouvelle protéase de rhoptries, DegP, essentielle à la virulence du parasite in vivo. Nous montrons que DegP contrôle la phase aigüe de l'infection en modulant la réponse immune de l'hôte contribuant ainsi à la dissémination du parasite in vivo. Nous identifions également deux protéines homologues, Claw1 et Claw2, présentant une localisation atypique à l'extrémité apicale du parasite. Notre incapacité à déléter ces gènes pourrait indiquer un rôle essentiel de ces protéines au niveau du complexe apical de T. gondii. Enfin, bien que n'étant pas reliée aux rhoptries, ce crible a permis d'identifier la première protéine associée aux jonctions des vésicules constituant le complexe membranaire interne de Toxoplasma. La délétion de cette protéine, SIP, affecte la forme du parasite, entrainant un défaut de motilité, d'invasion et de virulence in vivo. / Toxoplasma gondii is an apicomplexan protist and the causative agent of toxoplasmosis. This obligate intracellular parasite harbors apical secretory organelles such as rhoptries that contain essential virulence factors responsible of the invasion and the modulation of the infected host cell. Along the cell cycle of T. gondii, rhoptry proteins share the same timing of expression. In order to identify new proteins involve in rhoptry content, biogenesis or secretion, we screened the genome database of T. gondii to isolate proteins that present this particular profile. We obtained the subcellular localization of 12 candidates and we investigated the biological functions for 4 of them. We showed that DegP, a rhoptry protease is essential for the in vivo virulence of T. gondii. DegP controls the acute phase during infection and modulate the host immune response leading to better parasite dissemination in vivo. Also, we identified Claw1 and its paralog Claw2 that present an atypical localization at the apical end of the parasite. To date, we were unable to disrupt the genes encoding these proteins suggesting that they may have an essential function related to the apical complex in T. gondii. Finally, we also examined a ‘hit' of this screening that was not related to rhoptries and we identified SIP, the first protein associated with the transversal junctions of the inner membrane complex in T. gondii. The disruption of SIP affects the shape of the parasite leading to an aberrant motility, defect in invasion and impaired parasite virulence in mice.

Toxoplasma gondii é um agente disseminado com um ciclo de vida complexo que envolve gatos e hospedeiros de sangue quente, incluindo o homem. Componentes vacinais são imunógenos pobres e vacinas atenuadas causam doenças crônicas. Taquizoítos irradiados à 255Gy induzem uma imunidade similar a infecção, com proteção parcial, sem estudos sobre memória. Estudamos a produção de IgG in vitro por células do baço e medula óssea de camundongos imunizados com taquizoítos irradiados e infectados. A medula óssea contém um número maior de células específicas do que o baço. As células de memória são de alta afinidade, mas IgG sérica apresentaram baixa avidez. Há também anticorpos específicos IgA nas fezes. Todas as vacinas indutoras de proteção parcial contra o desafio, que foi mais evidente em alta patogenicidade da cepa ME-49. A proteção foi proporcional tanto à quantidade de anticorpos IgG no soro ou produzidos por células da medula óssea. Nossos dados mostram claramente que os anticorpos de alta afinidade estão relacionados com a sobrevida específica de memória celular. / Toxoplasma gondii is a disseminated agent with a complex life cycle involving cats and warm blood hosts, including man. Component vaccines are poor immunogens and attenuated vaccines cause chronic disease. 255Gy irradiated tachyzoites induce the same immunity as natural infection, with partial protection, without studies on immunological memory. We studied in vitro production of specific IgG by spleen and bone marrow cells of mice immunized with irradiated tachyzoites, comparing with chronically infected animals. Bone marrow contains larger numbers of specific cells than spleen. Memory cells were of high affinity, but serum presented low avidity IgG. We also show presence of specific IgA antibodies in stools. All immunizations induce partial protection against challenge, which was more evident in highly pathogenic ME-49 strain. The protection was proportional both to the amount of IgG antibodies in serum or produced by bone marrow cells. Our data clearly shows that high affinity antibodies are related to specific memory cell survival.

Einleitung: Toxoplasma (T.) gondii zählt zu den häufigsten intrazellulären Parasiten weltweit. Alleinige Endwirte im fakultativ heteroxenen Lebenszyklus sind die Feliden. Als Zwischenwirte können jedoch zahlreiche Säugetier- und Vogelarten dienen, in denen sich parasitäre Gewebezysten entwickeln. Einer der Hauptübertragungswege auf den Menschen stellt der Verzehr von T. gondii-haltigem Fleisch infizierter Nutztiere dar. Inwieweit Putenfleisch ein potentielles Infektionsrisiko birgt und welche Bedeutung Puten in der Epidemiologie der humanen Toxoplasmose besitzen ist nicht ausreichend geklärt. Ziel der Untersuchungen: Ziel der vorliegenden Arbeit war es, ein reproduzierbares Infektionsmodell bei Puten für T. gondii zu entwickeln, um die Verteilung und Persistenz des Parasiten im Gewebe zu ermitteln. Es wurden verschiedene Parameter, wie Infektionsstadium, Infektionsdosis, Applikationsmodus und Untersuchungszeitpunkt hinsichtlich ihres Einflusses auf die Entwicklung parasitärer Gewebestadien verglichen. Material und Methoden: Insgesamt wurden 74 Puten nach einer Aufzuchtperiode von 4 bis 8 Wochen experimentell mit T. gondii-Tachyzoiten oder Oozysten infiziert. Je nach Versuchsgruppe wurden Tachyzoiten vom Stamm ME49 intravenös und/oder intramuskulär appliziert oder Oozysten vom Stamm ME49, DX oder Hannover 1 oral verabreicht. Die Verifikation der Infektion erfolgte über den Nachweis T. gondii-spezifischer Antikörper mit Hilfe eines kinetischen ELISA. Drei bis acht Puten jeder Versuchsgruppe wurden 6 bis 8 oder 10 bis 12 Wochen nach der Infektion getötet. Von jedem Tier wurden folgende Gewebeproben entnommen: Brust-, Oberschenkel- und Unterschenkelmuskulatur, Herz, Leber, Muskel- und Drüsenmagen, Gehirn, Lunge, Milz, Nieren, Darm, Pankreas und Hoden (sofern vorhanden). Die Organe wurden getrennt vollständig homogenisiert. Bei den Muskeln wurden Proben von verschiedenen Lokalisationen entnommen und ebenfalls einzeln homogenisiert. Der Nachweis von T. gondii-DNA in den Gewebeproben erfolgte mittels konventioneller PCR, basierend auf der Amplifizierung eines 469 bp Fragments des B1-Gens, und anschließender nested PCR (Länge Zielfragment: 375 bp). Zusätzlich wurden zu Beginn der Studie lichtmikroskopische Untersuchungen einzelner Organe in Form nativer Quetschpräparate (400fache Vergrößerung) auf T. gondii-Zysten durchgeführt. Ergebnisse: Ungeachtet der Infektionsdosis und des inokulierten Parasitenstadiums konnten bei keinem der Versuchstiere klinische Symptome einer Toxoplasmose beobachtet werden. Die unterschiedlich hohen Infektionsdosen hatten im Allgemeinen keinen signifikanten Einfluss auf die Anzahl positiv getesteter Puten oder Organproben. Lediglich die Anzahl positiver Gehirnproben nahm mit ansteigender Oozystendosis signifikant zu. Bei der Betrachtung aller Versuchsgruppen fiel auf, dass die Befallshäufigkeit der Organe sowohl zwischen den Tieren verschiedener Infektionsgruppen als auch innerhalb einer Infektionsgruppe stark schwankte. So variierte die Anzahl positiv getesteter Organe bei den Tachyzoiten-infizierten Puten zwischen 0 und 7, bei den Oozysten-infizierten Puten zwischen 0 und 9 Organen pro Tier. Die Untersuchungsergebnisse zeigen, dass sich T. gondii heterogen in der Pute verteilt und mindestens 12 Wochen persistieren kann. Bezogen auf alle Versuchstiere gab es kein Organ, dass durchgängig negativ blieb. Nach der Tachyzoiteninfektion waren am häufigsten Leber (43,3%), gefolgt von Brustmuskel (26,7%) und Herz (20,0%) infiziert, während bei den Oozysten-infizierten Tieren der Erreger am häufigsten im Gehirn (47,2%), gefolgt von Oberschenkelmuskulatur (25,0%) und Herz und Unterschenkelmuskulatur (je 22,2%) nachgewiesen werden konnte. Schlussfolgerungen: Tachyzoiten und Oozysten erwiesen sich als gleichermaßen geeignete Infektionsmedien und führten hinsichtlich der systemischen Verteilung des Parasiten in der Pute zu vergleichbaren Ergebnissen. Ein spezifischer Organtropismus des Erregers konnte nicht festgestellt werden. Aus Sicht der Lebensmittelhygiene und des Verbraucherschutzes bedeuten die Ergebnisse, dass im Fall einer T. gondii-Infektion ein potentielles Infektionsrisiko für den Menschen durch infiziertes Putenfleisch nicht ausgeschlossen werden kann.