Regulação transcricional, metabólica e perfil de microRNAs dos biofilmes de Histoplasma capsulatum: genes, metabólitos e rnas não codificantes como alvos terapêuticos e/ou biomarcadores
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2017-03-24
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Universidade Estadual Paulista (Unesp)
Resumo
Histoplasma capsulatum variedade capsulatum é um patógeno fúngico dimórfico causador de uma importante micose sistêmica, denominada histoplasmose. A patogenia da histoplasmose ocorre como resultado da inalação dos microconídios da fase miceliar, que afetam primariamente os pulmões onde ocorre a diferenciação em leveduras, que posteriormente, induzem uma infecção pulmonar e disseminação para outros órgãos, particularmente em indivíduos imunocomprometidos. Recentemente, foi estabelecida uma correlação entre o modo de infecção de H. capsulatum e a formação de biofilmes, estruturas caracterizadas como redes tridimensionais complexas que induzem, entre outros, a resistência antifúngica. Por esta razão, é emergente a identificação de um novo alvo e/ou biomarcador que possa ser selecionado, a fim de estabelecer um novo regime terapêutico e/ou ferramenta diagnóstica para a histoplasmose. Com base nisto, este trabalho tem como objetivo analisar a regulação transcricional codificante e metabólica diferencial entre as duas formas de crescimento de H. capsulatum, em biofilmes e em crescimento planctônico, bem como determinar o perfil de microRNAs (miRNAs) aberrantes em células hospedeiras em resposta à infecção com leveduras livres e biofilmes. O sequenciamento completo das regiões transcritas foi realizado utilizando a plataforma HiSeq da Illumina e a investigação do padrão de miRNAs em macrófagos (Mø) hospedeiros infectados foi conduzida empregando uma técnica de RT-qPCR (reverse transcription-quantitative PCR). Subsequentemente, a produção de polissacarídeos totais foi determinada em amostras de culturas planctônicas, biofilmes e sobrenadante dos biofilmes e, adicionalmente, a abundância dos metabólitos de natureza não proteica e pequenos peptídeos nestas formas de crescimento foi estabelecida por LC-MS/MS. Em resumo, os dados obtidos revelaram que a estruturação das leveduras em biofilmes induz uma regulação negativa nos processos transcricionais direta (genoma codificante – RNAm) e indiretamente (genoma não-codificante – miRNAs) e, nos processos metabólicos das leveduras, resultante da: a) expressão reprimida da maioria dos genes (≈80% down-regulated) quando as leveduras livres passam a organizarem-se em biofilmes; b) superexpressão de todos os miRNAs alterados na comparação de células infectadas com biofilmes vs. leveduras planctônicas; c) intensidade relativa reduzida (DOWN) da maioria dos metabólitos (57%) produzidos pelas células em biofilmes em relação à intensidade nas homólogas livres, considerando os metabólitos com a mesma massa molecular entre biofilmes e culturas planctônicas e; d) produção significativamente menor de metabólitos exclusivos pelas culturas em biofilmes (8%) vs. células planctônicas (18%). Por conseguinte, no presente estudo, através das abordagens desenvolvidas foram indicadas as moléculas (genes, miRNAs e metabólitos) que constituem uma fonte potencial para delinear: a) um marcador de biofilmes na infecção às células hospedeiras; b) novos protótipos para a terapêutica da histoplasmose e; c) novos biomarcadores como ferramentas diagnósticas para a histoplasmose.
Histoplasma capsulatum variety capsulatum is a dimorphic fungal pathogen that causes an important systemic mycosis, called histoplasmosis. The pathogenesis of histoplasmosis occurs as a result of the inhalation of mycelial microconidia, which primarily affect the lungs where differentiation occurs in yeast, which subsequently induce pulmonary infection and dissemination to other organs, particularly in immunocompromised individuals. Recently, a correlation was established between the mode of infection of H. capsulatum and the formation of biofilms, structures characterized as complex three-dimensional networks that induce, among others, antifungal resistance. For this reason, it is emerging the identification of a new target that can be selected in order to establish a new therapeutic strategy for histoplasmosis. Based on this, this work aims to analyze the differential transcriptional and coding transcriptional regulation between the two forms of growth of H. capsulatum in biofilms and planktonic growth, as well as to determine the profile of aberrant microRNAs (miRNAs) in host cells post-infection with free yeasts and biofilms. Complete sequencing of the transcribed regions was performed using the Illumina HiSeq platform and the investigation of the miRNA pattern in infected host macrophages (Mø) was conducted using a reverse transcription-quantitative PCR (RT-qPCR) technique. Subsequently, the production of total polysaccharides was determined in samples of planktonic cultures, biofilms and supernatant of the biofilms and, additionally, the abundance of non-protein metabolites and small peptides in these growth forms was established by LC-MS/MS. In summary, the data obtained showed that the structure of yeasts in biofilms induces a negative regulation in the direct (coding genome - mRNA) and indirectly (non-coding genome - miRNAs) transcriptional processes and in the yeasts metabolic processes, resulting from: a) suppressed expression of most genes (≈80% down-regulated) when the free yeasts are organized in biofilms; b) overexpression of all altered miRNAs in the comparison of cells infected with biofilms vs. planktonic yeasts; c) reduced relative intensity (DOWN) of most of the metabolites (57%) produced by cells in biofilms compared to the intensity in the free homologues, considering the metabolites with the same molecular mass between biofilms and planktonic cultures; d) significantly lower production of exclusive metabolites by cultures in biofilms (8%) vs. planktonic cells (18%). Accordingly, in the present study, the approaches performed indicated molecules (genes, miRNAs and metabolites) that are a potential source for delineating: a) a marker of biofilms in infection to host cells; b) new prototypes for histoplasmosis therapy and; c) new biomarkers as diagnostic tools for histoplasmosis.
Histoplasma capsulatum variety capsulatum is a dimorphic fungal pathogen that causes an important systemic mycosis, called histoplasmosis. The pathogenesis of histoplasmosis occurs as a result of the inhalation of mycelial microconidia, which primarily affect the lungs where differentiation occurs in yeast, which subsequently induce pulmonary infection and dissemination to other organs, particularly in immunocompromised individuals. Recently, a correlation was established between the mode of infection of H. capsulatum and the formation of biofilms, structures characterized as complex three-dimensional networks that induce, among others, antifungal resistance. For this reason, it is emerging the identification of a new target that can be selected in order to establish a new therapeutic strategy for histoplasmosis. Based on this, this work aims to analyze the differential transcriptional and coding transcriptional regulation between the two forms of growth of H. capsulatum in biofilms and planktonic growth, as well as to determine the profile of aberrant microRNAs (miRNAs) in host cells post-infection with free yeasts and biofilms. Complete sequencing of the transcribed regions was performed using the Illumina HiSeq platform and the investigation of the miRNA pattern in infected host macrophages (Mø) was conducted using a reverse transcription-quantitative PCR (RT-qPCR) technique. Subsequently, the production of total polysaccharides was determined in samples of planktonic cultures, biofilms and supernatant of the biofilms and, additionally, the abundance of non-protein metabolites and small peptides in these growth forms was established by LC-MS/MS. In summary, the data obtained showed that the structure of yeasts in biofilms induces a negative regulation in the direct (coding genome - mRNA) and indirectly (non-coding genome - miRNAs) transcriptional processes and in the yeasts metabolic processes, resulting from: a) suppressed expression of most genes (≈80% down-regulated) when the free yeasts are organized in biofilms; b) overexpression of all altered miRNAs in the comparison of cells infected with biofilms vs. planktonic yeasts; c) reduced relative intensity (DOWN) of most of the metabolites (57%) produced by cells in biofilms compared to the intensity in the free homologues, considering the metabolites with the same molecular mass between biofilms and planktonic cultures; d) significantly lower production of exclusive metabolites by cultures in biofilms (8%) vs. planktonic cells (18%). Accordingly, in the present study, the approaches performed indicated molecules (genes, miRNAs and metabolites) that are a potential source for delineating: a) a marker of biofilms in infection to host cells; b) new prototypes for histoplasmosis therapy and; c) new biomarkers as diagnostic tools for histoplasmosis.
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Transcriptômica, Metabolômica, microRNAs, Histoplasma capsulatum, Biofilmes