Comunicação celular in vitro mediada por vesículas extracelulares isoladas de fungos patogênicos cultivados em condições de estresse
Data
2024-11-18
Autores
Silva, Camila Pereira da 
Orientadores
Puccia, Rosana
Tipo
Dissertação de mestrado
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ISSN da Revista
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Resumo
Vesículas extracelulares (EVs) são estruturas arredondadas delimitadas por uma membrana lipídica de camada dupla. Elas transportam moléculas de natureza diversa e de forma protegida para o meio extracelular, com potencial de estabelecer comunicação à distância. Dados prévios do laboratório mostraram a capacidade de EVs do patógeno fúngico Paracoccicioides brasiliensis modular o fenótipo de células do mesmo isolado e apontaram para uma ampla alteração no proteoma e transcriptoma de EVs de P. brasiliensis (isolado vPb18, virulento) e Candida albicans (cepa 90028) cultivados em condições de estresse oxidativo e nitrosativo subletal. O presente trabalho teve como objetivo avaliar o efeito de EVs produzidas nessas condições no fenótipo de outras células fúngicas. Os experimentos de dose-resposta mostraram que concentrações de 5 mM de NaNO2 e 0,5 mM de H2O2 são subletais, porém causam estresse em células de vPb18 cultivadas na presença de estressor por 24 h, a 36oC, em meio sólido de Ham’s F-12 acrescido de 1,5% de glicose (Ham/glc). Para C. albicans, a concentração de 5 mM de H2O2 é subletal e causa estresse nas mesmas condições de cultura, como sugerido pela incorporação do reagente di-hidroetílico. As vEVs produzidas por vPb18 cultivado em condições subletais de estresse nitrosativo (vEVNO) e oxidativo (vEVOxi) não apresentaram diferença estatisticamente significante, em relação aos respectivos controles vEVcNO e vEVcOxi, no diâmetro médio estimado por rastreamento de nanopartículas (NTA), na variação do potencial Zeta, ou no conteúdo de proteína. O diâmetro hidrodinâmico avaliado por dispersão dinâmica de luz (DLS) não variou para vEVNO, mas foi estatisticamente superior para vEVOxi, assim como o índice de polidispersão, porém o conteúdo de esterol foi estatisticamente inferior ao do controle. As EVCaOxi, produzidas por C. albicans cultivada sob estresse oxidativo subletal, apresentaram valores estatisticamente semelhantes aos do controle EVcCa no diâmetro aferido por NTA, na variação do potencial Zeta e no índice de polidispersão. O valor médio de DLS e o conteúdo de proteína foram estatisticamente superiores para EVCaOxi em relação ao controle EVcCa. O efeito de EVs foi testado por co-incubação por 4 h, a 36oC, de vEVNO, vEVOxi e seus controles com células de P. brasiliensis aPb18 (variante atenuada) e Pb3, com subsequente cultivo em spots de diluição seriada em meio Ham/glc, acrescido de agentes estressores. Em nossas condições experimentais, observamos uma visível redução da resistência de aPb18 ao Congo red e ao NaCl após pré-incubação com vEVOxi. Outros efeitos foram discretos e independentes da origem das vEVs, a saber: a) aumento da resistência de aPb18 ao sorbitol após pré-incubação com vEVNO,vEVOxi e seus controles; b) sutil aumento da resistência do isolado Pb3 ao NaCl, mediado por vEVNO, seu controle e ao Congo red, mediado por vEVOxi e seu controle. A co-incubação de EVCaOxi e seu controle com células de C. albicans resultou em um discreto aumento de resistência ao Calcofluor white, porém não ao Congo red ou ao H2O2, nas condições testadas. Entretanto, EVCaOxi causou uma sutil diminuição de crescimento das leveduras mesmo na ausência de estressor. Em conjunto, os resultados deste trabalho sugerem que as EVs produzidas sob estresse oxidativo subletal sofrem uma variação na constituição da superfície tanto para P. brasiliensis como para C. albicans, resultando em aumento no diâmetro hidrodinâmico. Todavia, a carga superficial de potencial Zeta, que foi negativa sugerindo estabilidade das partículas, aparentemente não foi afetada. Por outro lado, a redução de resistência de aPb18 ao Congo red e ao NaCl, mediada por vEVOxi, assim como a redução da viabilidade de C. albicans, mediada por EVCaOxi, deve estar relacionada a alterações nas vEVs causadas pelo estresse oxidativo. Os resultados desta dissertação são originais e serão explorados mais amplamente no laboratório no futuro próximo.
Extracellular vesicles (EVs) are rounded structures limited by a bilayered lipid membrane. They carry molecules of diverse nature in a protected manner to the extracellular environment and potentially establish longdistance communication. Previous data from the laboratory showed the ability of EVs from the fungal pathogen Paracoccicioides brasiliensis to modulate the phenotype of cells from the same isolate and pointed to a broad change in the proteome and transcriptome of EVs from P. brasiliensis (isolate vPb18, virulent) and Candida albicans (strain 90028) cultivated under conditions of sublethal oxidative and nitrosative stress. The present work aimed to evaluate the effect of EVs produced under these conditions on the phenotype of other fungal cells. The doseresponse experiments showed that concentrations of 5 mM NaNO2 and 0.5 mM H2O2 are sublethal, but cause stress in vPb18 cells cultured in the presence of the stressor agents for 24 h, at 36oC, in solid Ham’s F12 medium plus 1.5% glucose (Ham/glc). For C. albicans, a concentration of 5 mM H2O2 is sublethal and causes stress under the same culture conditions. The vEVs produced by vPb18 cultivated under sublethal conditions of nitrosative (vEVNO) and oxidative (vEVOxi) stress did not show statistically significant differences, relative to the respective vEVcNO and vEVcOxi controls, in the mean diameter estimated by nanoparticle tracking (NTA), in the variation of Zeta potential, or in protein content. The hydrodynamic diameter assessed by dynamic light scattering (DLS) did not vary for vEVNO, but was statistically higher for vEVOxi, as well as the polydispersity index. The sterol content was statistically lower than that for vEVcOxi control. The EVCaOxi, produced by C. albicans cultivated under sublethal conditions of oxidative stress, presented values statistically similar to those of the EVcCa control in the diameter measured by NTA and in the variation of the Zeta potential. The average DLS value was statistically higher for EVCaOxi, as well as the polydispersity index. The estimated protein content for EVCaOxi was statistically higher than that for EVcCa. Taken together, these results suggest that EVs produced under sublethal oxidative stress showed variation in surface constitution for both P. brasiliensis and C. albicans, resulting in an increase in hydrodynamic diameter, but the surface charge of Zeta potential, which was negative and thus suggestive of particle stability, was apparently not affected. The effect of EVs was tested by coincubation for 4 h, at 36oC, of vEVNO, vEVOxi and their controls with P. brasiliensis aPb18 (attenuated variant) and Pb3 cells, with subsequent cultivation in serial dilution spots in Ham medium/glc, plus stress agents. Under our experimental conditions, we observed a visible reduction in the resistance of aPb18 to Congo red and NaCl after preincubation with vEVOxi. Other effects were discrete and independent of the origin of the vEVs, namely: a) increased resistance of aPb18 to sorbitol after preincubation with vEVNO, vEVOxi and their controls; b) subtle increase in the resistance of the Pb3 isolate to NaCl, mediated by vEVNO and its control, and to Congo red, mediated by vEVOxi and its control. Coincubation of EVCaOxi and its control with C. albicans cells resulted in subtle resistance to Calcofluor white, but not to Congo red or H2O2, under the conditions tested. However, EVCaOxi caused a slight decrease in yeast growth even in the absence of a stressor. Taken together, the results of this work suggest that EVs produced under sublethal oxidative stress showed variation in surface constitution for both P. brasiliensis and C. albicans, resulting in an increase in hydrodynamic diameter. However, the surface charge of Zeta potential, which was negative suggesting stability of the particles, apparently was not affected. On the other hand, the reduction in resistance of aPb18 to Congo red and NaCl, mediated by vEVOxi, as well as the reduction in viability of C. albicans, mediated by EVCaOxi, must be related to changes in vEVs caused by oxidative stress. The results of this dissertation are original and will be explored more extensively in the laboratory in the near future.
Extracellular vesicles (EVs) are rounded structures limited by a bilayered lipid membrane. They carry molecules of diverse nature in a protected manner to the extracellular environment and potentially establish longdistance communication. Previous data from the laboratory showed the ability of EVs from the fungal pathogen Paracoccicioides brasiliensis to modulate the phenotype of cells from the same isolate and pointed to a broad change in the proteome and transcriptome of EVs from P. brasiliensis (isolate vPb18, virulent) and Candida albicans (strain 90028) cultivated under conditions of sublethal oxidative and nitrosative stress. The present work aimed to evaluate the effect of EVs produced under these conditions on the phenotype of other fungal cells. The doseresponse experiments showed that concentrations of 5 mM NaNO2 and 0.5 mM H2O2 are sublethal, but cause stress in vPb18 cells cultured in the presence of the stressor agents for 24 h, at 36oC, in solid Ham’s F12 medium plus 1.5% glucose (Ham/glc). For C. albicans, a concentration of 5 mM H2O2 is sublethal and causes stress under the same culture conditions. The vEVs produced by vPb18 cultivated under sublethal conditions of nitrosative (vEVNO) and oxidative (vEVOxi) stress did not show statistically significant differences, relative to the respective vEVcNO and vEVcOxi controls, in the mean diameter estimated by nanoparticle tracking (NTA), in the variation of Zeta potential, or in protein content. The hydrodynamic diameter assessed by dynamic light scattering (DLS) did not vary for vEVNO, but was statistically higher for vEVOxi, as well as the polydispersity index. The sterol content was statistically lower than that for vEVcOxi control. The EVCaOxi, produced by C. albicans cultivated under sublethal conditions of oxidative stress, presented values statistically similar to those of the EVcCa control in the diameter measured by NTA and in the variation of the Zeta potential. The average DLS value was statistically higher for EVCaOxi, as well as the polydispersity index. The estimated protein content for EVCaOxi was statistically higher than that for EVcCa. Taken together, these results suggest that EVs produced under sublethal oxidative stress showed variation in surface constitution for both P. brasiliensis and C. albicans, resulting in an increase in hydrodynamic diameter, but the surface charge of Zeta potential, which was negative and thus suggestive of particle stability, was apparently not affected. The effect of EVs was tested by coincubation for 4 h, at 36oC, of vEVNO, vEVOxi and their controls with P. brasiliensis aPb18 (attenuated variant) and Pb3 cells, with subsequent cultivation in serial dilution spots in Ham medium/glc, plus stress agents. Under our experimental conditions, we observed a visible reduction in the resistance of aPb18 to Congo red and NaCl after preincubation with vEVOxi. Other effects were discrete and independent of the origin of the vEVs, namely: a) increased resistance of aPb18 to sorbitol after preincubation with vEVNO, vEVOxi and their controls; b) subtle increase in the resistance of the Pb3 isolate to NaCl, mediated by vEVNO and its control, and to Congo red, mediated by vEVOxi and its control. Coincubation of EVCaOxi and its control with C. albicans cells resulted in subtle resistance to Calcofluor white, but not to Congo red or H2O2, under the conditions tested. However, EVCaOxi caused a slight decrease in yeast growth even in the absence of a stressor. Taken together, the results of this work suggest that EVs produced under sublethal oxidative stress showed variation in surface constitution for both P. brasiliensis and C. albicans, resulting in an increase in hydrodynamic diameter. However, the surface charge of Zeta potential, which was negative suggesting stability of the particles, apparently was not affected. On the other hand, the reduction in resistance of aPb18 to Congo red and NaCl, mediated by vEVOxi, as well as the reduction in viability of C. albicans, mediated by EVCaOxi, must be related to changes in vEVs caused by oxidative stress. The results of this dissertation are original and will be explored more extensively in the laboratory in the near future.
Descrição
Citação
SILVA, Camila Pereira da. Comunicação celular in vitro mediada por vesículas extracelulares isoladas de fungos patogênicos cultivados em condições de estresse. 2024. 84 f. Dissertação (Mestrado em Microbiologia e Imunologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2024.