Characterization of transcription factors containing the basic leucine zipper (bZIP) ATF1 domain in Aspergillus fumigatus

Author(s)

Lilian Pereira Silva

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2021-02-10

Examining board members

Gustavo Henrique Goldman; Larissa Fernandes Matos; Marcio Lourenço Rodrigues; Charley Christian Staats

• Advisor: Gustavo Henrique Goldman

Abstract

The fungus Aspergillus fumigatus is an important opportunistic pathogen and allergen in mammals, causing diseases such as invasive aspergillosis, which mainly affects immunocompromised patients or those undergoing chemotherapeutic treatment. A. fumigatus has a great adaptation ability when faced with different stressful conditions. The fungus is able to modulate its biochemical machinery in a fast and robust way, thus guaranteeing its survival. The high osmolarity glycerol (HOG) pathway is involved in these adaptation processes. The MpkC and SakA kinases of A. fumigatus, orthologous of Saccharomyces cerevisiae Hog1p, constitute the primary regulator of the response to hyperosmotic stress that is activated by the HOG pathway, one of the pathways responsible for biochemical and transcriptional modulation under stress. After performing RNAseq under osmotic stress, the transcriptional responses of wild type A. fumigatus and the strains ΔmpkC, ΔsakA and ΔmpkC ΔsakA were compared. The results strongly indicate that MpkC and SakA have independent and collaborative roles during the transcriptional response to transient osmotic stress. In addition, null mutants for four transcription factors containing the basic leucine zipper (bZIP) domain were identified and characterized. The involvement of the bZIP transcription factors (TF) AtfA, -B, -C and -D during adaptation to different stressful conditions was assessed. These TFs are involved in the ability of A. fumigatus to adapt to different stressful agents, such as congo red (CR) and calcofluor white (CFW), which damage the cell wall, and they are also involved in the response to oxidative stress caused by hydrogen peroxide. First, we investigate the interactions, genetic interactions between ΔatfA, ΔatfB, ΔatfC and ΔatfD. Our results indicate that there are several levels of genetic interactions between these mutants. However, the ΔatfA and ΔatfB mutations show epistasis in most of the conditions investigated here. All double mutants have reduced growth in the presence of acetate, glycerol and ethanol as the sole sources of carbon, and have affected the accumulation of glycogen and trehalose in the mycelium. In addition, it was demonstrated through fluorescence miscroscopy that the AtfA: GFP protein is constitutively located in the nucleus. AtfB, -C and D: GFP were constitutively located only 20% in the nucleus, increasing their translocation to the nucleus between 30 and 70% in the presence of stress conditions. It was also observed, through co-immunoprecipitation (Co-IP) experiments, the interaction of AtfA-D with SakA. The formation of heterodimers between AtfA-D also occurs in the presence and absence of osmotic and cell wall stresses. These data point to the importance of AtfA-D and also suggest that these TFs are involved not only in the response to stress, but also in metabolic processes of A. fumigatus. (AU)

• FAPESP's process: 16/21392-2 - "Characterization of transcription factors that have the basic domain" zipper "leucine (bZIP) ATF1 in Aspergillus fumigatus"
• Grantee: Lilian Pereira Silva
• Support Opportunities: Scholarships in Brazil - Doctorate