Efeito da exposição à radiação ionizante no ciclo celular, morfologia e crescimento do Trypanosoma cruzi
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Data
2024-01-29
Autores
Marchiano, Fernanda Sycko [UNIFESP]
Orientadores
Silveira Filho, José Franco da [UNIFESP]
Tipo
Tese de doutorado
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Resumo
A radiação ionizante induz quebras na dupla fita de DNA e rearranjos cromossômicos em T. cruzi. Para investigar os efeitos da radiação na organização dogenoma de T. cruzi, epimastigotas de CL Brener foram expostos a doses de 100 e 500Gy de radiação gama e analisados quanto ao crescimento em cultura e recuperação do cariótipo. Entre 18 clones isolados por diluição seriada, epimastigotas do clone F9 isolado de uma população exposta à radiação de 500 Gy, apresentou notáveis alterações no crescimento, motilidade e alterações morfológicas no flagelo em meio de cultura axenico. Por coloração de Giemsa e ensaios de imunofluorescência com anticorpos específicos contra proteínas da estrutura flagelar observamos alterações no padrão de fluorescência e morfologia do flagelo que está parcialmente descolado do corpo celular. Análise por microscopia eletrônica corroborou os dados acima e revelou que o cinetoplasto está disposto lateralmente ao núcleo. Para entender o déficit decrescimento do clone F9, estimamos a duração das fases do ciclo celular em epimastigotas em proliferação sincronizados com a hidroxiureia e verificamos que a duração da fase G1 foi significativamente reduzida no mutante. Por meio de ensaios de invasão de células de mamíferos com tripomastigotase multiplicação intracelular foi possível observar redução significativa na invasão pelo clone F9, apesar do parasita ser ainda capaz de perpetuar a infecção. Para compreender as alterações observadas ao nível genômico, utilizamos a técnica de aCGH (“array Comparative Genomic Hybridization”) onde observamos uma série de alterações de ganho e em menor número de perda de DNA. Diversos genes envolvidos em processos biológicos como estrutura flagelar e motilidade, “splicing”, transcrição, tradução, reparo de DNA e ciclo celular foram alterados, sendo a maioria das alterações relacionadas ao ganho de DNA. Estes resultados sugerem que o aumento no número de cópias dos genes por amplificação genica (aneuploidia segmentar ou cromossômica) foi a solução encontrada pelo parasita para compensar os efeitos da radiação. Nas famílias multigênicas envolvidas na invasão de células de mamíferos (MASP, TcMUC, Trans-Sialidase, DGF-1, GP63 e SAP), as perdas foram predominantes e podem ter afetado a capaciade invasiva de tripomastigotas. T. cruzi demonstra ter eficiente mecanismo de reparo, capaz de reparar a degradação cromossômica observada nos parasitas irradiados com dosagens de até 500 Gy. Além de reparo eficiente, o parasita se demonstra ser resiliente às alterações morfológicas em uma organela multifuncional em patógenos, como o flagelo.
The ionizing radiation induces DNA double-strand breaks and chromosomal rearrangements in T. cruzi. To investigate the effects of radiation on the organization of T. cruzi genome, CL Brener epimastigotes were exposed to doses of 100 and 500 Gy of gamma radiation and then analyzed for growth in and karyotype recovery. Among 18 clones isolated by serial dilution, epimastigotes of clone F9 isolated from a population exposed to 500 Gy radiation showed notable phenotypic traits in culture axenic medium, slow growth, altered motility and morphological changes in the flagellum. By using Giemsa staining and immunofluorescence assays with specific antibodies against proteins of the flagellar structure, we observed changes in the fluorescence pattern and morphology of the flagellum, which is partially detached from the cell body. Electron microscopy analysis corroborated the above data and revealed that the kinetoplast is arranged laterally to the nucleus. To understand the growth deficit of clone F9, we estimated the duration of cell-cycle phases of proliferating epimastigotes synchronized with hydroxyurea and found that the length of the G1 phase was significantly reduced in the mutant. Through invasion assays of mammalian cells with trypomastigotes and intracellular multiplication, it was possible to observe a significant reduction in invasion by the F9 clone, despite the parasite being still capable of perpetuating the infection. To understand the changes observed at the genomic level, we used the aCGH technique (“array Comparative Genomic Hybridization”) where we observed a series of changes in DNA gain and, to a lesser extent, loss. Several genes involved in biological processes such as flagellar structure and motility, splicing, transcription, translation, DNA repair and cell cycle were altered, with the majority of changes related to DNA gain. These results suggest that the increase in the number of gene copies through gene amplification (segmental or chromosomal aneuploidy) was the solution found by the parasite to compensate for the effects of radiation. Regarding the multigene families involved in the invasion of mammalian cells (MASP, TcMUC, Trans-Sialidase, DGF-1, GP63 and SAP), losses were predominant and may have affected the invasive capacity of trypomastigotes. T. cruzi demonstrates an efficient repair mechanism, capable of repairing the chromosomal degradation observed in parasites irradiated with doses of up to 500 Gy. In addition to efficient repair, the parasite has been shown to be resilient to morphological changes in a multifunctional organelle in pathogens, such as the flagellum.
The ionizing radiation induces DNA double-strand breaks and chromosomal rearrangements in T. cruzi. To investigate the effects of radiation on the organization of T. cruzi genome, CL Brener epimastigotes were exposed to doses of 100 and 500 Gy of gamma radiation and then analyzed for growth in and karyotype recovery. Among 18 clones isolated by serial dilution, epimastigotes of clone F9 isolated from a population exposed to 500 Gy radiation showed notable phenotypic traits in culture axenic medium, slow growth, altered motility and morphological changes in the flagellum. By using Giemsa staining and immunofluorescence assays with specific antibodies against proteins of the flagellar structure, we observed changes in the fluorescence pattern and morphology of the flagellum, which is partially detached from the cell body. Electron microscopy analysis corroborated the above data and revealed that the kinetoplast is arranged laterally to the nucleus. To understand the growth deficit of clone F9, we estimated the duration of cell-cycle phases of proliferating epimastigotes synchronized with hydroxyurea and found that the length of the G1 phase was significantly reduced in the mutant. Through invasion assays of mammalian cells with trypomastigotes and intracellular multiplication, it was possible to observe a significant reduction in invasion by the F9 clone, despite the parasite being still capable of perpetuating the infection. To understand the changes observed at the genomic level, we used the aCGH technique (“array Comparative Genomic Hybridization”) where we observed a series of changes in DNA gain and, to a lesser extent, loss. Several genes involved in biological processes such as flagellar structure and motility, splicing, transcription, translation, DNA repair and cell cycle were altered, with the majority of changes related to DNA gain. These results suggest that the increase in the number of gene copies through gene amplification (segmental or chromosomal aneuploidy) was the solution found by the parasite to compensate for the effects of radiation. Regarding the multigene families involved in the invasion of mammalian cells (MASP, TcMUC, Trans-Sialidase, DGF-1, GP63 and SAP), losses were predominant and may have affected the invasive capacity of trypomastigotes. T. cruzi demonstrates an efficient repair mechanism, capable of repairing the chromosomal degradation observed in parasites irradiated with doses of up to 500 Gy. In addition to efficient repair, the parasite has been shown to be resilient to morphological changes in a multifunctional organelle in pathogens, such as the flagellum.
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Citação
MARCHIANO, Fernanda Sycko. Efeito da exposição à radiação ionizante no ciclo celular, morfologia e crescimento do Trypanosoma cruzi. 2024. 146 f. Tese (Doutorado em Microbiologia e Imunologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2024.