Biologia de sistemas no estudo do dimorfismo térmico: uma isolado de Paracoccidioides brasiliensis atípico na morfogênese

• Ano de defesa: 2016
• Autor(a) principal: Luciana Marcia de Oliveira
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; UFMG
• Palavras-chave em Português: Dimorfismo térmico; RNASeq; Paracoccidioides brasiliensis; Bioinformática
• Link de acesso: http://hdl.handle.net/1843/BUOS-APSRNY

Paracoccidioidomycosis, systemic mycosis endemic in Latin America, is caused by the thermodymorphic fungi of the Paracoccidioides complex. The disease is contracted by inhaling infectious conidia which, at body temperature (37°C), trigger the morphogenetic transition by developing yeast forms (L). From the isolate of P. brasiliensis Pb339 (ATCC32069), under a selective pressure of 400 g/mL of the sulfamethoxazole, an isolate defective in thermal dimorphism (YRT, Yeast at Room Temperature) was selected. At room temperature (RT), even in the absence of the drug, the YRT isolate grows as a beige cerebriform mass. L cell aggregates, 1 to 3 buddings, ovoid, irregular and catenulate,suggestive of pseudohyphae, were recorded under optical and electronic microscopy (scanning and transmission). Unlike most isolates of P. brasiliensis that are readily removable from the surface of the culture medium at 37°C, it adheres strongly. The YRT isolate survived the infection challenge in mice, being recovered from the pulmonary lavagemaintaining an atypical L phenotype at RT; induced significant increase and accumulation (P <0.05) of neutrophils, but less accumulation of macrophages than the control. In YPD agar (0.1% or absence of D-glucose) and in minimal medium, a reversal was observed for themycelial form (M) at RT and no growth at 37oC. The levels of chitin in the atypical isolate were lower (p <0.05) than in the L controls, but higher than in the M forms. At 37oC and under drug pressure, it presented higher levels (p <0.05) of reactive oxygen derivatives (ROS) than controls. RNA sequencing technology, RNASeq (Next-generation sequencing - Illumina), was applied for the first time to investigate the transcripts of the L and M stages of the Pb339 isolate and the atypical YRT isolate. We obtained 5x108 readings (PE 2x100, Q Phred 25), of which 3.2x108 were mapped to the reference genome Pb18. The estimatedFPKM values of the biological replicates were positively correlated. The L and M phases of the PbB339 isolate have distinct transcripts, respectively, presenting 248 and 802 differentially expressed genes. The oxidative stress pathways were the most enriched. At 37oC the YRT and Pb339 isolates presented a similar transcriptional profile, involving 462positively regulated genes. The transcriptase of the defective isolate YRT at RT was dramatically altered when compared to the M-phase of the reference isolate. We identified 387 differentially expressed genes in the YRT when compared to the M phase (Pb339). Oxidative and protein synthesis routes were the most representative; we observed overrepresentation of the genes with the cofactor binding function, ABC transporters and kinase signaling pathways. 125 genetic variants (SNP / Indel) were identified in the YRT isolate, 2 of which were associated with non-synonymous substitutions (MAPK3 and TAO3 / PAG1 genes involved in the regulation of mycelial growth of pathogenic fungi) and 4 with loss offunction, highlighting the gene encoding the USP protein (universal stress protein) and a hypothetical gene containing repetitive regions of tetratricopeptides and glycosyltransferase domains, both involved in the response to environmental stress and in the phenomenon ofdimorphism. In this work, we present the first transcriptoms of P. brasiliensis specific phases performed by RNASeq technology, as well as unpublished transcriptoms of a defective isolate for thermal dimorphism, performed at RT and at 37ºC. In this context, theverticalization of the analyzes has the potential to provide new information on the thermal dimorphism and morphogenesis of the fungus Paracoccidioides so far little explored.