Made available in DSpace on 2014-06-11T19:27:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-08-25Bitstream added on 2014-06-13T20:35:35Z : No. of bitstreams: 1 oliveira_kr_me_botfmvz.pdf: 157744 bytes, checksum: f115a70ad010444fe6ca9960d82e76d8 (MD5) / A toxoplasmose é uma das mais freqüentes zoonoses parasitárias, de distribuição mundial, cujo agente etiológico é o Toxoplasma gondii. A infecção em suínos tem sido descrita em diversos países, demonstrando seu caráter cosmopolita e a susceptibilidade desta espécie. O T.gondii pode ser transmitido sob várias formas, e com relação à espécie suína, a ingestão de carne crua ou mal cozida e seus derivados são importantes vias de transmissão para o homem. Estudou-se a prevalência de anticorpos anti-Toxoplasma gondii em 550 amostras de soros de suínos de criações de fundo de quintal na microrregião de Registro - SP, utilizando como prova sorológica o método de aglutinação direta (MAD). Este mostrou-se uma ferramenta diagnóstica de fácil realização, baixo custo, não necessitando de equipamentos especiais para a leitura e interpretação dos resultados. Das 550 amostras de soro, 111(20,18%) reagiram positivamente com os títulos 64 (15,32%), 256 (27,93%), 1024 (39,64%), 4096 (10,81%), 16384 (4,50%) e 65536 (1,80%). Concluí-se que os resultados obtidos indicam alta prevalência desta enfermidade nas condições e região de origem dos animais. À medida que foram obtidos títulos altos de anticorpos contra o agente, pode-se concluir também que há risco potencial de transmissão da toxoplasmose para o homem, sugerindo que muitos desses animais poderiam apresentar bradizoítos em sua musculatura. / Toxoplasmosis is one of the most frequent parasitic zoonosis with worldwide distribution whose etiological agent is Toxoplasma gondii. The infection in swine has been described in many countries, showing the cosmopolitan character and susceptibility of this specie. T.gondii can be transmitted by many ways. The ingestion of swine raw or badly cocked meat and derivatives are important pathways of transmission to man. The anti-T.gondii antibodies prevalence was studied in 550 serum samples of swine under rustic condition (animals with no breed, popular named as backyard breeding) in Registro-SP, using the modified agglutination test (MAT). Such method is verified as an easy-appliance tool, with low costs and non necessity of special equipment in order to achieve interpretation of results. In 550 samples of serum, 111 (20.18%) were positive with titles of 64 (15.32%), 256 (27.93%), 1024 (39.64:%), 4096 (10.81%), 16384 (4.50%), 65536 (1.80%). These results achieved indicate a high prevalence of the disease on the region studied and under the specific conditions described. The high rate obtained as result also leads to the conclusion that there is potential risk of toxoplasmosis transmission to man due to the possibility of the animals to have bradyzoits in their musculature.

Made available in DSpace on 2014-06-11T19:29:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-05-28Bitstream added on 2014-06-13T20:38:48Z : No. of bitstreams: 1 costa_vm_me_botfmvz.pdf: 404473 bytes, checksum: 0e9d9447d55f3ca2e11ba7a40a45a785 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A toxoplasmose é similar em humanos e em ratos, sendo estes, o modelo experimental, mais utilizado para o estudo da doença. Há poucos relatos da transmissão desta enfermidade pelo leite materno, e desta forma o objetivo do presente estudo foi pesquisar a presença de Toxoplasma gondii no leite de ratas experimentalmente infectadas e a transmissão galactogênica. Utilizaram-se ratas Wistar (Rattus norvegicus), divididas em três grupos: G1, G2 e G3, contendo seis fêmeas cada um. Foram inoculadas via oral com 104 bradizoítos da cepa BTU4, genótipo I, isolada de cão com cinomose. As ratas do G1 e G2 foram infectadas 60 dias antes do acasalamento e as do G3 logo após o parto. Os animais do G2 foram submetidos à imunossupressão pós-parição. Para a detecção do parasito no leite utilizou-se a reação em cadeia pela polimerase (PCR) e para verificar a transmissão para os filhotes pesquisou-se nestes anticorpos anti-T. gondii pela imunofluorescência indireta (RIFI) e método de aglutinação direta (MAD), e a bioprova pela inoculação de camundongos com pool de tecidos (cerebral, pulmonar, hepático, cardíaco, muscular esquelético, língua e diafragma) de cada ninhada, bem como pela PCR nestes tecidos individualmente. A PCR foi positiva em três amostras de leite, duas provenientes da rata 1 do G1, e uma da rata 3 do G3. Os filhotes de todas as ratas do G1 soroconverteram, mas foram negativos na bioprova. Filhotes das ratas 1 e 3 do G3 soroconverteram, e a bioprova foi positiva. Amostras de fígado, musculatura esquelética e pulmão de filhotes do G1 foram positivas na PCR. Desta forma conclui-se que ocorreu a transmissão do T. gondii pelo leite, sugerindo-se novos estudos em lactentes, considerando-se a magnitude da doença em crianças e recém-nascidos. / Toxoplasmosis is similar in humans and rats, and the latter constitute the most used experimental model to study this disease. Few reports on toxoplasmosis transmission through maternal milk are available in literature; thus, the aim of the present study was to investigate whether Toxoplasma gondii is present in and transmitted through the milk of experimentally infected rats. Wistar (Rattus norvegicus) female rats were divided into three groups: G1, G2 and G3, with six animals each. They were orally inoculated with 104 bradyzoites of BTU4 strain, genotype I, isolated from a dog with distemper. G1 and G2 rats were infected 60 days before mating and those of G3, soon after the parturition. G2 animals were subjected immunosuppression just after parturition. Polymerase chain reaction (PCR) was used to detect the parasite in the milk. To verify the parasite transmission to the offspring, these anti-T. gondii antibodies were investigated through indirect immunofluorescence assay (IFA) and direct agglutination test (DAT). Bioassay consisted of inoculating mice with a pool of tissues (brain, lungs, liver, heart, skeletal muscle, tongue and diaphragm) from each litter, as well as PCR in these tissues individually. PCR was positive in three milk samples, two from rat 1 (G1) and one from rat 3 (G3). The pups of all G1 rats seroconverted but were negative in the bioassay. The pups of rats 1 and 3 (G3) seroconverted and their bioassay was positive. Liver, skeletal muscle and lung samples were PCR-positive in G1 pups. Thus, we can conclude that T. gondii was transmitted through milk, suggesting the need of new studies in breast-feeding mothers as this disease is highly severe in children and newborns.

A toxoplasmose é uma infecção comum nos animais causada pelo Toxoplasma gondii configurando-se como uma importante zoonose. O homem infecta-se, principalmente, através da ingestão de carne contaminada com cistos do protozoário. São encontradas altas taxas de prevalência de toxoplasmose nos rebanhos de ovinos do mundo todo, sendo a ingestão de alimentos e água contaminados com oocistos as mais importantes fontes de infecção da espécie. No ovino, a toxoplasmose pode ser assintomática ou causar distúrbios reprodutivos, notadamente abortos, levando a perdas econômicas consideráveis. Com o objetivo de contribuir com dados sobre a freqüência de anticorpos para o T. gondii em ovinos na região da Grande Porto Alegre-RS, foram estimadas as freqüências de anticorpos para T. gondii da classe IgG em soros de ovinos provenientes de propriedades da região citada, utilizando a técnica de Hemaglutinação Indireta (HAI) e Imunofluorescência Indireta (IFI). A freqüência estimada de anticorpos para T. gondii em uma amostragem de 250 ovinos foi de 13,6% pela técnica de HAI e 15,2% pela técnica de IFI. A amostragem foi estratificada em dois grupos experimentais de acordo com o sexo e a idade dos animais. No grupo I, composto de 127 machos, obteve-se 4,8% de positivos para HAI e 7,6% de positivos para IFI. Enquanto nas 123 fêmeas, detectou-se um percentual de 8,8% de positivas pela técnica de HAI e 7,6% reagiram para IFI. Em relação a faixa etária se encontrou: 11 positivos (4,4%) nos animais com menos de um ano e 23 (9,2%) para aqueles com mais de um ano na reação de HAI, num total de 34 (13,6%) animais; enquanto que para técnica de IFI obteve-se o resultado de 19 (7,6%) ovinos jovens e 19 (7,6%) ovinos adultos em 38 (15,2%) animais analisados. A percentagem de co-positividade entre as técnicas utilizados foi de 55,26% e a co-negatividade foi de 93,865, e a concordância total ficou em 88%, enquanto o índice de Kappa calculado foi de 0,513. / Toxoplasmosis is a serious zoonosis. It is a common infection in animals caused by a parasite called Toxoplasma gondii. Human infection occurs mainly for ingestion of meat contaminated with T. gondii cysts. A high prevalence rate of toxoplasmosis is found in sheep cattle all over the world, being the consumption of food and water contaminated with oocystes the major source of infection of the species. In ovine, toxoplasmosis can be assymptomatique or cause reproduction disorders, especially abortions, which leads to significant economic losses.With the objective of providing data about the frequency of T. gondii antibodies in sheep from the Greater Porto Alegre area, we estimated the frequency of IgG class antibodies to T. gondii in sheep sera. The techniques employed were the Indirect Hemagglutination Technique (HAI) and the Indirect Imunofluorescence Test (IFI). The estimated frequency of antibodies to T. gondii in a sample of 250 sheep sera was 13,6% (HAI), and 15,2% (IFI). The sample was stratified into two experimental groups, according to gender and age. In group one, composed by 127 males, 4,8% were HAI positive, and 7,6% were IFI positive, whereas in the group composed by 123 females, 8,8% were HAI positive, and IFI 7,6%. Concerning age group, there were 11 HAI positives (4,4%) among the animals with less than one year of age, and 23 HAI positive (9,2%) among the animals over one year, totalizing 34 animals. For the IFI technique, the results obtained showed 19 (7,6%) young sheep and 19 (7,6%) adults in 38 animals analyzed. The co-positivity between the techniques was 55,26% and the co-negativity was 93,865. Total agreement was 88% while the Kappa index calculated was 0,513.

Orientador: Claudio Lucio Rossi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-07-25T17:20:41Z (GMT). No. of bitstreams: 1 Suzuki_LisandraAkemi_M.pdf: 14558191 bytes, checksum: c85c5e0b4751c7cbf7f83293db450f8a (MD5) Previous issue date: 1999 / Resumo: O diagnóstico da toxoplasmose aguda adquirida é, freqüentemente, baseado na detecção de anticorpos específicos IgM anú-T.gondii e/ou elevação significativa dos títulos de anticorpos IgG. Entretanto, a elevada ocorrência de altos títulos de IgG anti-T. gondii em pessoas sadias e a persistência, em alguns casos, dos anticorpos IgM por muito tempo após infecção com o parasita pode dificultar a interpretação dos resultados dos testes sorológicos quando se suspeita de toxoplasmose aguda. Em nosso primeiro estudo, são apresentados os resultados da detecção de anticorpos específicos da classe IgA e da determinação da avidez dos anticorpos IgG em amostras seqüenciais de soros de um paciente apresentando níveis significativos de anticorpos IgM anú-T.gondii durante sete anos após o início dos sintomas clínicos da toxoplasmose. Os anticorpos IgA foram quantificados por uma técnica de ELISA de captura (ETI-TOXOK-A). O paciente ainda apresentou um resultado positivo (>10 UA/ml) em uma amostra de soro coletada dois anos após o inicio das manifestações clínicas. A avidez dos anticorpos IgG foi determinada com o sistema Falcon assay screening test (F.A.S.T.®) - ELISA, utilizando somente uma diluição da amostra de soro. índices de avidez compatíveis com uma infecção aguda foram encontrados nas amostras de soros obtidas durante os cinco primeiros meses após o início dos sintomas clínicos. O objetivo do segundo estudo foi avaliar vários marcadores sorológicos para o diagnóstico da toxoplasmose aguda adquirida, tais como a detecção de anticorpos IgA e IgE anti-T. gondii, o teste de aglutinação diferencial (AC/HS) e os testes baseados na determinação da avidez dos anticorpos IgG. Foram testadas, num total de 64 amostras de soros, 31 amostras de soros de pacientes com infecção adquirida recentemente e 33 amostras de soros de pacientes com infecção crônica. Os anticorpos IgA foram quantificados por meio de dois "kits" de ELISA de captura (Platelia® Toxo-IgA e ETI-TOXOK A) e um "kit" de ELISA automatizado (IMx Toxo-IgA). Níveis de anticorpos IgA compatíveis com infecção recente foram detectados em todas as amostras de soros dos pacientes com toxoplasmose aguda, com os três "kits". Por outro lado, a análise das amostras de soros dos 33 pacientes com toxoplasmose crônica mostrou que níveis significativos de anticorpos IgA podem ser detectados com alta freqüência durante a fase crônica da infecção, com os três "kits". Anticorpos IgE anti-T. gondii foram detectados, pela técnica de imunocaptura-aglutinação (ISAGA), em 26 (84%) dos 31 pacientes com toxoplasmose aguda e em duas amostras de soros de pacientes com toxoplasmose crônica coletadas mais de um ano após o início das manifestações clínicas. Vinte e nove (94%) dos 31 pacientes com infecção aguda e 15 (45%) dos 33 pacientes com infecção crônica apresentaram, no teste AC/HS, um padrão compatível com toxoplasmose aguda. A avidez dos anticorpos IgG anti-T. gondii foi determinada por dois métodos diferentes. Um dos métodos, utilizando o "kit" comercial Platelia Toxo-IgG, foi baseado na titulação de cada amostra de soro e cálculo dos títulos, com e sem o tratamento com uréia, em relação a um valor de "cut-off" definido. No outro método, utilizando o sistema F.A.S.T.- ELISA, foi feita apenas uma única diluição da amostra de soro, e foram comparadas as absorbâncias das reações na presença e ausência de uréia. Todas as 31 amostras de soros de pacientes com infecção aguda apresentaram índices compatíveis com toxoplasmose aguda pelo método de titulação, ao passo que, com o método de diluição única, 4 destas amostras apresentaram resultados duvidosos, mostrando que o método de titulação das amostras foi mais sensível para o diagnóstico de infecção recente. Resultados semelhantes foram obtidos no grupo de 33 pacientes com toxoplasmose crônica pelos dois métodos; apenas uma amostra de soro apresentou um índice de avidez não compatível com a fase crônica, pelo método de titulação. Os resultados obtidos no presente estudo mostraram que os marcadores sorológicos, atualmente utilizados para o diagnóstico da toxoplasmose aguda adquirida, possuem limitações significativas. Nossos dados sugerem que a determinação da avidez dos anticorpos IgG anti-Z gondii, principalmente pelo método de titulação das amostras de soros, pode ser bastante útil para um diagnóstico mais confiável do estágio em que se encontra a infecção, nos pacientes que apresentam níveis significativos de anticorpos IgM. / Abstract: The diagnosis of acute acquired toxoplasmosis has been frequently based on the detection of specific IgM antibodies and on the demonstration of a significant increase in specific IgG antibodies titers. However, the prevalence of high Toxoplasma IgG antibody titers among normal subjects and the sustained persistence, in some persons, of specific IgM antibodies have complicated the interpretation of serological tests when acute toxoplasmosis is suspected. In our first study, we report the detection of specific IgA antibodies and the determination of the avidity of Toxoplasma-specific IgG in sequential serum samples from a patient exhibiting significant levels of specific IgM antibodies for seven years after the onset of the clinical symptoms of toxoplasmosis. Anti-T.gondii IgA was quantified by an antibody capture ELISA (ETI-TOXOK-A). The patient still had a positive IgA result (> 10 AU/ml) in a serum sample taken two years after the beginning of the clinical manifestations. The IgG avidity was determined by a single serum dilution method, using the Falcon assay screening test (F.A.S.T.®) - ELISA system. Avidity indices compatible with an acute infection were found only in serum samples taken during the first five months after the onset of the clinical symptoms. The purpose of the second study was to assess the usefulness of newer serological markers, such as the detection of specific IgA and IgE antibodies, the differential agglutination (AC/HS) test using formalin and acetone-fixed tachyzoites of T. gondii and the determination of the avidity of Toxoplasma-specific IgG, in the diagnosis of acute acquired toxoplasmosis. Sixty-four serum samples, 31 from patients with a recently acquired Toxoplasma infection and 33 from patients with chronic toxoplasmosis, were tested. Anti-Z gondii IgA was measured by two antibody capture ELISA tests (ETI-TOXOK-A and Platelia® Toxo IgA) and an automated direct ELISA (IMx® Toxo IgA). The three kits detected antibody levels compatible with a recent infection in serum samples of all patients with acute toxoplasmosis. On the other hand, the analysis of serum samples from 33 patients with chronic toxoplasmosis revealed that significant levels of IgA may be detected with a high frequency by the three kits during the chronic phase of infection. IgE antibodies detected by ISAGA were present in 26 (84%) of 31 patients with acute toxoplasmosis and in serum samples from two subjects with chronic infection taken more than one year after the beginning of the clinical symptoms of infection. Twenty-nine (94%) of 31 patients with a recent Toxoplasma infection and 15 (45%) of 33 subjects with chronic toxoplasmosis had an AC/HS pattern compatible with acute toxoplasmosis. The avidity of Toxoplasma IgG was evaluated by two methods. One method was based on titration of each serum sample and calculation of the titers, in the absence and presence of urea, in relation to a defined cut-off value. In the other method, a single serum dilution was used and the absorbances of the reactions in the presence and absence of urea were compared. The titration method was more sensitive for diagnosing a recent primary Toxoplasma infection: All 31 serum samples from patients with acute toxoplasmosis had avidity indices compatible with acute toxoplasmosis by the titration method whereas with the single dilution method serum samples from four patients had equivocal results. In the 33 patients with chronic toxoplasmosis, similar results were obtained with the two avidity methods; only one serum sample had a non compatible avidity value with the titration method. The results obtained in the present study show that the current serological markers used for diagnosing acute acquired toxoplasmosis have significant limitations. Our data suggest that the determination of the avidity of Toxoplasma-specific IgG, mainly by the titration method, in patients with detectable IgM antibodies could permit a more efficient diagnosis of the stage of infection by T gondii. / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Orientador: Katia Denise Saraiva Bresciani / Resumo: O objetivo foi verificar a soroprevalência de anticorpos IgG contra T. gondii e identificar pela primeira vez os fatores associados ao risco de infecção em gestantes com acompanhamento pré-natal em Araçatuba. Em parceria com a Secretaria Municipal de Saúde de Araçatuba/SP, foram analisados 428 questionários no período de maio de 2018 a março de 2019, em quatro Unidades Básicas de Saúde (UBS) deste município. O questionário epidemiológico foi aplicado por enfermeiras de cada unidade e continha perguntas relacionadas aos fatores associados ao risco de infecção. Em 236/428 (54,91%) foram detectados anticorpos da classe IgG e a análise multivariada verificou que a soroprevalência foi associada a gestantes maior de 36 anos, casadas, ensino médio incompleto e gestação anterior. Aquelas que relataram gestação anterior têm 1,99 vezes mais chance de ser sororeagente para toxoplasmose neste município. Pela primeira vez, foi caracterizado o perfil epidemiológico de gestantes atendidas com toxoplasmose em Unidades Básicas de Saúde deste município do Estado de São Paulo. / Abstract: The objective was to verify the seroprevalence of IgG antibodies against T. gondii and to identify for the first time the factors associated with the risk of infection in pregnant women with prenatal care in Araçatuba. In partnership with the Municipal Health Secretariat of Araçatuba / SP, 428 questionnaires were analyzed from May 2018 to March 2019, in four Basic Health Units (BHU) of this municipality. The epidemiological questionnaire was administered by nurses from each unit and contained questions related to factors associated with risk of infection. In 236/428 (54.91%) IgG class antibodies were detected and the multivariate analysis found that seroprevalence was associated with pregnant women older than 36 years, married, incomplete high school and previous pregnancy. Those who reported previous pregnancy are 1.99 times more likely to be seropositive for toxoplasmosis in this municipality. For the first time, the epidemiological profile of pregnant women treated with toxoplasmosis in Basic Health Units of this municipality of the State of São Paulo was characterized. / Mestre

Indiana University-Purdue University Indianapolis (IUPUI) / Toxoplasma gondii is an obligate intracellular eukaryotic pathogen that causes severe neurologic disease in immunocompromised adults and congenitally infected neonates. Events critical to the propagation of T. gondii, such as invasion and egress, are regulated by calcium-dependent signaling. In order to identify unique components of the parasite’s calcium signaling networks, members of the Arrizabalaga laboratory have used a forward genetics approach to isolate mutants with altered sensitivity to the calcium ionophore A23187. Exposing extracellular parasites to A23187 induces protein secretion, motility and cytoskeletal rearrangements and prolonged treatment causes exhaustion of factors required for invasion, which results in what is referred to as ionophore induced death (iiDeath). Mutants capable of surviving this treatment were isolated from a chemically mutagenized population. Whole genome sequencing of one such mutant, MBD2.1, identified a nonsense mutation in a protein of unknown function (TGGT1_069070, ToxoDBv7.2) Complementation of MBD 2.1 with a wild-type copy of TGGT1_069070 restored sensitivity to iiDeath treatment. Endogenous tagging of this locus revealed that the encoded protein is secreted from a unique parasite secretory organelle known as the dense granule into the parasitophorous vacuole, leading to its designation as TgGRA41. Complete knockout of TgGRA41 recapitulates the resistance to iiDeath observed in MBD2.1 but also exhibits a dramatic decrease in propagation in tissue culture not seen in the original mutant. The knockout shows defects in multiple steps of the lytic including compromised invasion efficiency and premature egress of parasites from host cells. Cytosolic calcium measurements of extracellular parasites show enhanced uptake of calcium in the knockout strain as compared to parental and complemented, suggesting that the loss of TgGra41 results in calcium dysregulation. Together, these results provide a novel insight into the role that the parasitophorous vacuole of T. gondii plays in calcium homeostasis and calcium-dependent signaling processes.

Indiana University-Purdue University Indianapolis (IUPUI) / The pathogenesis associated with toxoplasmosis is attributed to repeated rounds of the parasite lytic cycle, which has been shown to be regulated by calcium fluxes. However, little is known about the calcium homeostatic mechanisms utilized by T. gondii. Recently, our lab has identified a novel protein-TgEFP1 (TGGT1_255660), which is predicted to bind Ca2+ through its two EF-hand domains. Interestingly, TgEFP1 showed a unique dual localization at the PLV/ELC and the PV of the parasite. Previous work showed that the PLV/ELC harbors other ion binding and conducting proteins that are important for parasite survival and propagation. However, the function of this compartment in the parasite is unknown. Therefore, I hypothesize that the PLV/ELC, through the function of TgEFP1, plays a key role in calcium homeostasis of T. gondii. To test this hypothesis, we sought to characterize the function of TgEFP1 during the parasite lytic cycle and determine TgEFP1 interacting proteins that also localize to the PLV/ELC. Partial permeabilization and ultrastructure expansion microscopy techniques confirmed the dual localization of TgEFP1 at the PLV/ELC and the PV. TgEFP1 knockout parasites exhibited several phenotypic defects including a faster lytic rate, shorter intracellular cycle, and were more sensitive to calcium ionophore treatment. Signal peptide deletion led to a mislocalization of TgEFP1 as cytosolic puncta, while mutations at key calcium coordinating residues lead to exclusive localization of TgEFP1 at the PV. Lastly, immunoprecipitation assays followed by LC-MS/MS identified a novel lectin-like protein- TgLectin (TGGT1_258950) as a direct interactor of TgEFP1-HA. Collectively, these findings support that through the function of TgEFP1, the PLV/ELC, plays a key role in calcium-dependent processes during the lytic cycle of the parasite.

Toxoplasma gondii is a protozoan parasite that causes life-threatening disease in congenitally infected infants and immunocompromised patients, such as those inflicted with AIDS. Toxoplasmic encephalitis (TE) is a common presenting condition in an AIDS infection. People become infected with T. gondii by ingesting tissue cysts in undercooked meats or by ingesting oocysts excreted by cats. Methylmercury (MeHg) is a well-documented neurotoxicant that accumulates in the brain and causes severe mental and visual dysfunction, including chronic encephalopathy. Consumption of contaminated fish, grains, and seeds are common sources of human exposure to methylmercury. Studies from our laboratory suggest that oral exposure to a single high dose of 20 mg/kg MeHg does not increase the susceptibility to acute toxoplasmosis in CBA/J mice. Therefore, we further investigated endpoints associated with immunotoxicity and neurotoxicity in 6-week old, female CBA/J mice exposed to both MeHg and T. gondii during a chronic T. gondii infection. We examined both single and multiple doses of MeHg exposure in a chronic parasitic infection model. In the single high dose study, four groups of six-week-old, female CBA/J mice were either fed 25 T. gondii tissue cysts of the ME-49 strain or given vehicle. Six weeks later, two out of the four groups (T. gondii and vehicle control) were orally gavaged with a single dose of 20 mg/kg body weight of MeHg and sacrificed seven days post exposure. Experiments from the multiple MeHg dose study were performed under similar conditions with the same number of groups and dosed by oral gavage with 8 mg/kg body weight of MeHg on days 0, 2,4,7,10,13. These mice were sacrificed on day 17 or 18 after initiating MeHg exposure. Flow cytometry following exposure to a single dose of MeHg in mice with a chronic T. gondii infection revealed significant changes (P < 0.05) within the T cell subpopulation percentages caused by exposure to MeHg. For example, the thymic CD4+CD8+ T cell subpopulations were increased (P <0.05). However, MeHg had no significant effect on the CD4+CD8-, CD4-CD8+, or non-T cell subpopulations in the spleen. Furthermore, MeHg increased splenic cellularity and spleen-to-body-weight ratios with or without a concurrent T. gondii infection. MeHg also caused a significant decrease in mouse body weight. There was a significant (P <0.05) increase in brain tissue cyst counts within the group exposed to both MeHg and T. gondii (16 ± 4, mean ± SE, n=7) versus T. gondii alone (4 ± 1, n=8). Histopathological examination demonstrated that the brain was affected, as lesions, gliosis, and meningitis were notable in mice given T. gondii. Exposure of mice to multiple doses of MeHg also resulted in effects on the immune system of CBA/J mice with and without chronic toxoplasmosis. Total cellularity and numbers of CD4+CD8+, CD4+CD8-, CD4-CD8+, and CD4-CD8- T-cell subpopulations show a marked decrease in number in the thymus, while total cellularity was also decreased in the spleen following concurrent exposure to T. gondii and MeHg. Flow cytometric examination of lymphocyte populations (CD4+ and CD8+ lymphocytes) in the spleen and thymus demonstrated differences from control in the groups exposed to T. gondii and MeHg. Histopathological examination did not reveal any significant lesions. The data from experiments in which single or multiple doses of MeHg were given to mice with a chronic T. gondii infection indicate that concurrent exposure, to both MeHg and T. gondii, dependent on dose and time of exposure had notable effects, especially on the immune system (Supported by NIH Grant F36GM20301). / Ph. D.

Caractérisation moléculaire et fonctionnelle de la jonction mobile contrôlant l'invasion de la cellule hôte par Toxoplasma gondii. Les apicomplexes sont des parasites eucaryotes responsables d'infections humaines et animales, dont le paludisme et la toxoplasmose. La plupart sont des parasites intracellulaires obligatoires ; l'entrée dans la cellule hôte est donc un évènement crucial dans leur cycle de développement. Ce processus, conservé au sein du phylum, implique la sécrétion séquentielle du contenu de deux organites : les micronèmes et les rhoptries. Lors de l'invasion, le parasite établit un contact étroit entre son extrêmité apicale et la membrane plasmique de la cellule hôte, appelé la jonction mobile (JM). La JM est un point d'ancrage à la cellule hôte qui est initié chez Toxoplasma par la sécrétion de protéines du col des rhoptries appelées TgRON2/RON4/RON5/RON8 (complexe de RONs). Ces protéines sont sécrétées dans la cellule hôte et TgRON2 est insérée dans la membrane de la cellule hôte. TgRON2 peut servir de récepteur à la protéine TgAMA1 (Apical Membrane Antigen 1) qui est une protéine de micronèmes sécrétée à la surface du parasite durant l'invasion. L'interaction AMA1-RON2 est également conservée chez Plasmodium, mais il n'existe pas de réactivité croisée entre espèces d'apicomplexes. La résolution de la structure de la protéine recombinante TgAMA1 en complexe avec un peptide TgRON2 nous a permis de déterminer des résidus critiques à l'interaction entre ces deux protéines in vitro et à l'invasion du parasite in vivo, et de définir les bases structurales de la spécificité intra-espèce de l'interaction AMA1-RON2. Par l'obtention d'une souche dépourvue de TgAMA1, nous montrons qu'AMA1 n'est pas essentielle à la survie du toxoplasme, comme il avait été supposé depuis longtemps. Nous confirmons le rôle clé de cette protéine dans l'invasion et la formation de la JM. Les mutants dépourvus d'AMA1 sont capables d'insérer le complexe de RONs dans la cellule hôte mais se détachent plus fréquemment, entrainant des invasions abortives. L'invasion résiduelle observée en absence d'AMA1 pourrait impliquer des protéines homologues à TgAMA1, TgRON2 et TgRON4, dont nous avons entamé la caractérisation moléculaire et fonctionnelle.Mot-clés : Apicomplexes, Toxoplasma gondii, invasion, jonction mobile, micronèmes, rhoptries / Molecular and functional characterisation of the moving junction controlling host cell invasion by Toxoplasma gondiiAbstract:Apicomplexa are eukaryotic parasites responsible for a variety of human and animal diseases, including malaria or toxoplasmosis. Most of them have an obligatory intracellular stage; thus, the invasive process is a crucial step in their developmental cycle. It implies the sequential secretion of two organelles: micronemes and rhoptries. During invasion, the parasite establishes a structure called the moving junction (MJ), which is a close apposition between the apical end and the plasma membrane of host cell. The MJ is an anchoring point for invasion that is initiated in Toxoplasma by the secretion of rhoptry neck proteins named TgRON2/RON4/RON5/RON8 (the RONs complex). These proteins are exported to the host cell cytoplasm and TgRON2 spans the host cell membrane. There, TgRON2 will function as a receptor to Apical Membrane antigen 1 (TgAMA1), which is a micronemal protein displayed on the surface of the parasite during the invasion process. The AMA1-RON2 interaction is conserved in Plasmodium but there is no interspecies cross-binding.We have determined the structure of a TgAMA1 recombinant protein in complex with a TgRON2 peptide, which allowed us to determine which residues are critical for the interaction between both proteins in vitro and for parasite invasion in vivo. Moreover, the co-structure explains at the structural level the evolutionary constraint of the AMA1-RON2 interaction. By generating an AMA1 null strain in T. gondii, we demonstrate that TgAMA1 is not an essential gene, as claimed before. We confirm the importance of AMA1 in invasion and its key role in MJ formation. AMA1 null parasites insert the RON complex into the host cell but are more frequently detached from it, causing abortive invasions. The residual invasion might involve proteins homologous to TgAMA1, TgRON2 and TgRON4, for which the molecular and functional characterization is undertaken.Keywords: Apicomplexes, Toxoplasma gondii, invasion, moving junction, micronemes, rhoptries

Toxoplasma gondii est un parasite protozoaire intracellulaire obligatoire appartenant à la famille des Apicomplexa. Chez les protozoaires, les protéases possèdent des rôles clés au niveau du cycle parasitaire, et sont ainsi considérées comme des facteurs de virulence. Chez T. gondii, les métallopeptidases pourraient être impliquées dans la traversée des différentes barrières biologiques. A l'heure actuelle, seules cinq métallopeptidases de T. gondii ont été décrites : une aminopeptidase N, deux toxolysines, une leucine aminopeptidase et une FtsH1 peptidase. Lors de l'étude de l'influence de l'invasion de monocytes humains par T. gondii sur le profil d'expression des métalloprotéases matricielles monocytaires, nous avons mis en évidence une protéase parasitaire présentant à la fois des propriétés gélatino- et élastinolytique.Le but de ce travail est de caractériser, purifier et identifier cette gélatinase de T. gondii.Dans un premier temps, nous avons caractérisé la gélatinase d'environ 100 kDa sécrétée par T. gondii comme étant une MMP-9 like (métallo-endopeptidase à zinc) mais ne possédant pas le même processus d'activation que les MMPs humaines.Dans un deuxième temps, après purification partielle par une série de chromatographie chélatrice de zinc, nous avons identifié par spectrométrie de masse la gélatinase comme étant la TGME49_227948 annotée dans ToxoDB. Cette protéase présente les domaines protéiques d'une métalloprotéase à zinc de la sous-famille M16C, et est proche au niveau de sa structure 3D de la chaine A de la 2FGE présente chez Arabidopsis Thaliana. Afin de confirmer l'annotation de ToxoDB, nous avons séquencé l'extrémité 5' de l'ARNm de cette protéase. Cependant, nos résultats expérimentaux ne sont pas en concordance avec l'annotation prédite dans la base de données ToxoDB.La séquence en acides aminés de cette protéase, nous a permis de synthétiser deux anticorps polyclonaux spécifiques afin de mettre en évidence deux formes de 140 kDa et 100 kDa et donc d'émettre l'hypothèse que cette protéase pourrait être clivée afin d'être activée. De plus, cette métalloprotéase a été détectée par western blot dans le cytosol des parasites mais elle est aussi secrétée dans le milieu conditionné. Par immunolocalisation, la protéase est présente au sein du parasite, au niveau du cytosol sans localisation préférentielle dans un organite particulier.Dans ce travail, nous avons montré que la gélatinase parasitaire sécrétée par T. gondii pourrait dégrader des composés de la matrice extracellulaire, d'où son rôle potentiel dans le mécanisme de traversée des barrières biologiques. / Toxoplasma gondii is an intracellular protozoan parasite which belongs to the Apicomplexa phylum. In protozoans, proteases have key roles in the parasitic cycle. They thereby are considered as virulence factors. In T. gondii, metallopeptidases may be involved in the crossing of biological barriers. Currently, only five metallopeptidases from T. gondii are described: an aminopeptidase N, two toxolysins, one leucine aminopeptidase and one FtsH1 peptidase. During the study of the influence of human monocytes invasion by T. gondii on the monocytic matrix metalloproteases expression profile, we brought out a parasitic protease showing gelatino- and elastinolytic properties.The aim of this study is to characterize, purify and identify this gelatinase from T. gondii.First we characterized the 100 kDa-gelatinase secreted by T. gondii as an MMP-9-like (zinc metalloendopeptidase) but its activation process is different from human MMPs.Then, after the partial purification by a series of zinc-chelate chromatographies, we identified by mass spectrometry the gelatinase as the TGME49_227948 annotated in ToxoDB. This protease has M16C subfamily zinc-metalloprotease protein domains and is close to the 3D structure of the A chain of the 2FGE in Arabidopsis thaliana. In order to confirm the ToxoDB annotation, we sequenced the 5' end of the mRNA of this protease. Nevertheless our experimental results are not in the line with the predicted annotation in ToxoDB database.The amino acids sequence of this protease allowed us to synthesize two specific polyclonal antibodies in order to highlight two forms, 140 kDa and 100 kDa, and to emit the hypothesis that this protease could be cleaved to be activated. Moreover this metalloprotease was detected by Western Blot in the cytosol of the parasites but it can also be secreted in the conditioned medium. By immulocalization, the protease is present in the cytosol of the parasite without any preferential localization in a particular organelle.In this study, we showed that the parasitic gelatinase secreted by T. gondii could degrade extracellular matrix compounds, which could explain its potential role in the mechanism involved in the crossing of biological barriers.