Trypanosoma cruzi antioxidant system : protein expression in the different forms, along the growth curve and their involvement in mitochondrial bioenergetics

Orientador: Fernanda Ramos GadelhaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O Trypanosoma cruzi é o agente etiológico da doença de Chagas para a qual não há vacina e os tratamentos disponíveis têm eficácia limitada, sendo agravada pela heterogeneidade das cepas do parasita. Na busca por novos alvos terapêuticos, destaca-se o sistema antioxidante do parasita. As proteínas desse sistema como as triparedoxinas peroxidases citosólica e mitocondrial e as superóxido dismutases A e B (TcCPx, TcMPx, SODA e SODB, respectivamente) são fundamentais para a sobrevivência do parasita. Neste sentido, analisou-se o envolvimento da TcCPx e TcMPx na bioenergética mitocondrial de T. cruzi que superexpressavam estas enzimas (CPx e MPx, respectivamente) e também em células controle (pTEX), bem como a expressão dessas enzimas e das SODA e SODB, a liberação de H2O2 e a produção de O2 .- ao longo da curva de proliferação nas cepas Tulahuen 2 e Y. Paralelamente, foi verificado o nível de expressão da TcCPx e TcMPx na forma tripomastigota de cultura sob incubação com H2O2, tanto intracelularmente quanto no meio de incubação e a expressão dessas enzimas comparada com a da forma epimastigota. Em relação à CPx e MPx, foi possível observar que na CPx há um aumento na expressão da TcMPx, e a mesma correlação para a MPx foi observada. Diferenças na liberação de H2O2 entre CPx e MPx foram detectadas apenas quando a cadeia respiratória mitocondrial foi inibida. A MPx teve maiores taxas de consumo de oxigênio do que a pTEX e CPx, entretanto em todas as células, o potencial de membrana mitocondrial e os níveis de ATP foram similares. Em relação às cepas Tulahuen 2 e Y, diferenças foram observadas em relação à liberação de H2O2 e produção de O2.- não somente entre as cepas, mas também ao longo da curva de proliferação. Todas as enzimas estudadas nessas cepas variaram sua expressão em função do tempo em cultura. Contudo, na cepa Y, o nível de expressão de todas as enzimas foi menor do que da Tulahuen 2, exceto para a SODA. Em relação à forma tripomastigota, tanto a TcCPx quanto a TcMPx tiveram padrões de expressão similares, com um aumento em baixas concentrações (10?M) seguido por uma redução até concentrações maiores de H2O2. No sobrenadante do meio de incubação a TcMPx não foi detectada, contudo a TcCPx foi e em níveis que aumentaram concomitantemente com a redução da sua expressão no compartimento intracellular. Adicionalmente, o nível de expressão de ambas peroxiredoxinas foi maior na forma tripomastigota em relação à epimastigota. Devido a um leve desacoplamento observado nas MPx, a presença ou indução de um transportador na membrana mitocondrial é sugerido quando a TcMPx é expressa em maiores níveis. Além disso, houve uma modulação das enzimas antioxidantes ao longo da curva de proliferação e entre as diferentes cepas, formas do parasita e tratamento com H2O2. Dada a importância das espécies reativas de oxigênio na sinalização redox do parasita, a expressão diferencial das enzimas antioxidantes nos diferentes parâmetros analisados sugere que as mesmas desempenham diversos papéis na sobrevivência, proliferação e diferenciação do parasitaAbstract: Trypanosoma cruzi is the etiologic agent of Chagas disease, which has no vaccine and the available treatments have limited efficacy that is aggravated by the heterogeneity of parasite strains. In the search for new therapeutic targets, the antioxidant system of the parasite has attracted attention. The proteins of this system such as the cytosolic and mitochondrial tryparedoxin peroxidases and superoxide dismutases A and B (TcCPx, TcMPx, SODA and SODB, respectively) are essential for parasite survival. In this sense, analysis of the involvement of TcCPx and TcMPx in the mitochondrial bioenergetics in T. cruzi overexpressing these enzymes (CPx and MPx, respectively) and also in control cells (pTEX) as well as the expression of these enzymes and SODA and SODB, H2O2 release and O2 .- production along the growth curve in two strains Tulahuen 2 and Y were performed. In parallel, the expression level of TcCPx and TcMPx was determined in tissue culture-derived trypomastigotes under incubation with H2O2, both intracellularly and in the incubation medium and the expression of these enzymes compared to the epimastigote form. Regarding CPx and MPx, it was observed that in CPx the expression of TcMPx was increased, and the same correlation was observed for MPx. Differences in H2O2 release between CPx and MPx were detected only when the mitochondrial respiratory chain was inhibited. MPx had higher oxygen consumption rates than pTEX and CPx, however in all cells, the mitochondrial membrane potential and ATP levels were similar. Regarding Tulahuen 2 and Y strains, differences were observed in relation to H2O2 release and O2.- production not only between strains but also along the growth curve. All the enzymes studied in these strains varied their expression in function of time in culture. However, in the Y strain, the expression level of all enzymes was lower than Tulahuen 2, except for SODA. Regarding trypomastigotes, not only TcCPx but also TcMPx had similar expression patterns, with an increasing in lower concentrations (10?M) followed by a reduction till higher H2O2 concentrations. In the supernatant of the incubation medium TcMPx was not detected, but TcCPx was detected and its levels increased concomitantly with the reduction in its expression in the intracellular compartment. Additionally, the level of expression of both peroxiredoxinas was higher in trypomastigote compared to epimastigote. Because of the mild uncoupling observed in MPx, the presence or induction of a carrier in the mitochondrial membrane is suggested when the TcMPx is expressed at higher levels. In addition, there was a modulation of the antioxidant enzymes along the growth curve and between different strains, forms of the parasite and treatment with H2O2. Given the importance of reactive oxygen species in parasite redox signaling, the differential expression of the antioxidant enzymes in the different parameters analyzed suggests that they play different roles in the survival, proliferation and differentiation of the parasiteDoutoradoBioquimicaDoutor em Biologia Funcional e Molecula

HSP70 of Leishmania amazonensis alters resistance to different stresses and mitochondrial bioenergetics

The 70 kDa heat shock protein (HSP70) is a molecular chaperone that assists the parasite Leishmania in returning to homeostasis after being subjected to different types of stress during its life cycle. In the present study, we evaluated the effects of HSP70 transfection of L. amazonensis promastigotes (pTEX-HSP70) in terms of morphology, resistance, infectivity and mitochondrial bioenergetics. The pTEX-HSP70 promastigotes showed no ultrastructural morphological changes compared to control parasites. Interestingly, the pTEX-HSP70 promastigotes are resistant to heat shock, H2O2-induced oxidative stress and hyperbaric environments. Regarding the bioenergetics parameters, the pTEX-HSP70 parasites had higher respiratory rates and released less H2O2 than the control parasites. Nevertheless, the infectivity capacity of the parasites did not change, as verified by the infection of murine peritoneal macrophages and human macrophages, as well as the infection of BALB/c mice. Together, these results indicate that the overexpression of HSP70 protects L. amazonensis from stress, but does not interfere with its infective capacity

Using a Chagas disease hospital database: a clinical and epidemiological patient profile

Abstract INTRODUCTION: Chagas disease (CD) prevention and control rely on studies of its distribution, characteristics of individuals affected and mode of transmission. CD data in Brazil are scarce; a retrospective analysis of the clinical characteristics of 80 patients treated at the Clinical Hospital of UNICAMP, Campinas, Brazil, was performed. METHODS: Patient data records were analyzed. RESULTS: Thirty percent of the patients probably got infected through vector-borne transmission, 65% came from endemic areas, a predominance of cardiac and cardiodigestive forms was found among males, and the cardiac form prevailed (51%). CONCLUSIONS: The results update the view on the epidemiology of CD in Campinas, Brazil

Comparison among three polymerase chain reaction assays on detection of DNA from Leishmania in biological samples from patients with american cutaneous leishmaniasis

INTRODUCTION: The study analyzed positivity of polymerase chain reaction (PCR) on detection of DNA from Leishmania in patients' samples. METHODS: Extracted DNA was submitted to L150/L152, 13Y/13Z, and seminested PCR (snPCR). RESULTS: Results were evidenced by bands of approximately 120, 720, and 670 bp for L150/L152, 13Y/13Z, and snPCR, respectively. L150/L152, 13Y/13Z, and snPCR positivity indexes were 76.9, 56.4, and 9.2 (p>0.05), respectively, for suspected and 93.7, 68.7, and 84.4 (p<0.05), respectively, for confirmed. CONCLUSIONS: Preliminary results showed that these assays, mainly L150/L152 and snPCR, can detect Leishmania DNA and carry potential on laboratory diagnosis of leishmaniasis