The regulation of the sulfur amino acid biosynthetic pathway in Cryptococcus neoformans: the relationship of Cys3, Calcineurin, and Gpp2 phosphatases

Autor(es)

de Melo, Amanda Teixeira ^[1] ; Martho, Kevin Felipe ^[1] ; Roberto, Thiago Nunes ^[1] ; Nishiduka, Erika S. ^[2] ; Machado Jr, Joel ; Brustolini, Otavio J. B. ^[3] ; Tashima, Alexandre K. ^[2] ; TerezaVasconcelos, Ana ^[3] ; Vallim, Marcelo A. ^[4] ; Pascon, Renata C. ^[4]

Afiliação do(s) autor(es)

^[1] Univ Fed Sao Paulo, Campus Diadema, Sao Paulo, SP - Brazil

^[2] Univ Fed Sao Paulo, Escola Paulista Med, Dept Bioquim, Campus Sao Paulo, Sao Paulo, SP - Brazil

^[3] Labinfo Lab Bioinformat, LNCC, Petropolis, RJ - Brazil

^[4] Machado Jr, Jr., Joel, Univ Fed Sao Paulo, Campus Diadema, Sao Paulo, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: SCIENTIFIC REPORTS; v. 9, AUG 15 2019.
• Citações Web of Science: 2

Resumo

Cryptococcosis is a fungal disease caused by C. neoformans. To adapt and survive in diverse ecological niches, including the animal host, this opportunistic pathogen relies on its ability to uptake nutrients, such as carbon, nitrogen, iron, phosphate, sulfur, and amino acids. Genetic circuits play a role in the response to environmental changes, modulating gene expression and adjusting the microbial metabolism to the nutrients available for the best energy usage and survival. We studied the sulfur amino acid biosynthesis and its implications on C. neoformans biology and virulence. CNAG\_04798 encodes a BZip protein and was annotated as CYS3, which has been considered an essential gene. However, we demonstrated that CYS3 is not essential, in fact, its knockout led to sulfur amino acids auxotroph. Western blots and fluorescence microscopy indicated that GFP-Cys3, which is expressed from a constitutive promoter, localizes to the nucleus in rich medium (YEPD); the addition of methionine and cysteine as sole nitrogen source (SD-N + Met/Cys) led to reduced nuclear localization and protein degradation. By proteomics, we identified and confirmed physical interaction among Gpp2, Cna1, Cnb1 and GFP-Cys3. Deletion of the calcineurin and GPP2 genes in a GFP-Cys3 background demonstrated that calcineurin is required to maintain Cys3 high protein levels in YEPD and that deletion of GPP2 causes GFP-Cys3 to persist in the presence of sulfur amino acids. Global transcriptional profile of mutant and wild type by RNAseq revealed that Cys3 controls all branches of the sulfur amino acid biosynthesis, and sulfur starvation leads to induction of several amino acid biosynthetic routes. In addition, we found that Cys3 is required for virulence in Galleria mellonella animal model. (AU)

• Processo FAPESP: 16/14542-8 - Caracterização genética dos elementos regulatórios que controlam a biossíntese e a assimilação de aminoácidos e o seu papel na virulência de Cryptococcus neoformans.
• Beneficiário: Renata Castiglioni Pascon
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 18/20390-1 - Estudo dos elementos regulatórios que controlam a captação de aminoácidos em Cryptococcus neoformans
• Beneficiário: Kevin Felipe Cruz Martho
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 17/20106-9 - Peptidômica dos venenos de serpentes e aranhas brasileiras
• Beneficiário: Alexandre Keiji Tashima
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 15/04400-9 - O papel da autofagia no crescimento a alta temperatura (37°C) e virulência em Cryptococcus neorformans
• Beneficiário: Marcelo Afonso Vallim
• Modalidade de apoio: Auxílio à Pesquisa - Regular