Aspergillus fumigatus G-Protein Coupled Receptors GprM and GprJ Are Important for the Regulation of the Cell Wall Integrity Pathway, Secondary Metabolite Production, and Virulence

Author(s):Show less -

da Costa Filho, Ailton Pereira ^[1] ; Pereira Brancini, Guilherme Thomaz ^[1] ; de Castro, Patricia Alves ^[1] ; Valero, Clara ^[1] ; Ferreira Filho, Jaire Alves ^[1] ; Silva, Lilian Pereira ^[1] ; Rocha, Marina Campos ^[2] ; Malavazi, Iran ^[2] ; de Moraes Pontes, Joao Guilherme ^[3] ; Fill, Taicia ^[3] ; Silva, Roberto Nascimento ^[4] ; Almeida, Fausto ^[4] ; Steenwyk, Jacob L. ^[5] ; Rokas, Antonis ^[5] ; dos Reis, Thaila F. ^[1] ; Ries, Laure N. A. ^[4] ; Goldman, Gustavo H. ^[1]

Affiliation

^[1] Univ Sao Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Ribeirao Preto - Brazil

^[2] Univ Fed Sao Carlos, Ctr Ciencias Biol & Saude, Dept Genet & Evolucao, Sao Carlos - Brazil

^[3] Univ Estadual Campinas, Inst Quim, Campinas, SP - Brazil

^[4] Univ Sao Paulo, Fac Med Ribeirao Preto, Ribeirao Preto - Brazil

^[5] Vanderbilt Univ, Dept Biol Sci, 221 Kirkland Hall, Nashville, TN 37235 - USA

• Document type: Journal article
• Source: MBIO; v. 11, n. 5 SEP-OCT 2020.
• Web of Science Citations: 1

Abstract

G-protein coupled receptors (GPCRs) are extracellular signaling receptors that sense environmental cues. Fungi sense their environment primarily through GPCR-mediated signaling pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. Aspergillus fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that present a wide range of clinical manifestations. Here, we investigate in detail the role of the GPCRs GprM and GprJ in growth and gene expression. GprM and GprJ are important for melanin production and the regulation of the cell wall integrity (CWI) pathway. Overexpression of gprM and gprJ causes a 20 and 50% reduction in growth rate compared to the wild-type (WT) strain and increases sensitivity to cell wall-damaging agents. Phosphorylation of the CWI protein kinase MpkA is increased in the Delta gprM and Delta gprJ strains and decreased in the overexpression mutants compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. Transcriptome sequencing suggests that GprM and GprJ negatively regulate genes encoding secondary metabolites (SMs). Mass spectrometry analysis confirmed that the production of fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, and fumitremorgin is reduced in the Delta gprM and Delta gprJ strains, at least partially through the activation of MpkA. Overexpression of grpM also resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signaling. Finally, we show that the Delta gprM and.gprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis. IMPORTANCE A. fumigatus is the main etiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immunocompromised humans. Withstanding the host environment is essential for A. fumigatus virulence, and sensing of extracellular cues occurs primarily through G-protein coupled receptors (GPCRs) that activate signal transduction pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. The A. fumigatus genome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GP-CRs GprM and GprJ regulate the phosphorylation of the mitogen-activated protein kinase MpkA and thus control the regulation of the cell wall integrity pathway. GprM and GprJ are also involved in the regulation of the production of the secondary metabolites fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, melanin, and fumitremorgin, and this regulation partially occurs through the activation of MpkA. Furthermore, GprM and GprJ are important for virulence in the insect model Galleria mellonella. This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial for A. fumigatus virulence. (AU)

• FAPESP's process: 17/14159-2 - The role of lactose and acetate metabolism in Aspergillus fumigatus virulence
• Grantee: Laure Nicolas Annick Ries
• Support type: Research Grants - Young Investigators Grants
• FAPESP's process: 19/06359-7 - Understanding the chemical factors involved in the endophytism x pathogenicity of different Penicillium brasilianum isolates
• Grantee: Taicia Pacheco Fill
• Support type: Regular Research Grants
• FAPESP's process: 16/12948-7 - The role of mitogen-activated protein kinase (MPKA) MpkA in regulating gliotoxin production in Aspergillus fumigatus.
• Grantee: Patrícia Alves de Castro Silva
• Support type: Scholarships in Brazil - Post-Doctorate
• FAPESP's process: 18/25217-6 - Organization and integration of large scale data to study the dynamics of expression of map kinases of the fungus Aspergillus fumigatus in diverse genetic and environmental conditions
• Grantee: Jaire Alves Ferreira Filho
• Support type: Scholarships in Brazil - Technical Training Program - Technical Training
• FAPESP's process: 18/10962-8 - The influence of G-protein-coupled receptors (GPCRs) on growth, morphogenesis and virulence of human pathogenic fungi
• Grantee: Gustavo Henrique Goldman
• Support type: Regular Research Grants
• FAPESP's process: 19/11929-7 - Molecular characterization of Aspergillus fumigatus GprJ, a G protein-coupled receptor involved in the control of melanin production
• Grantee: Guilherme Thomaz Pereira Brancini
• Support type: Scholarships in Brazil - Post-Doctorate
• FAPESP's process: 16/07870-9 - The influence of mitogen activated protein kinases (MAPK) on the expression of genetic determinants important for Aspergillus fumigatus virulence
• Grantee: Gustavo Henrique Goldman
• Support type: Research Projects - Thematic Grants
• FAPESP's process: 18/00715-3 - Molecular characterization of Aspergillus fumigatus transcription factors involved in calcium metabolism.
• Grantee: Clara Isabel Valero Fernández
• Support type: Scholarships in Brazil - Post-Doctorate
• FAPESP's process: 19/10727-1 - Molecular characterization of a receptor coupled to a g protein (GPCR) of Aspergillus fumigatus
• Grantee: Ailton Pereira da Costa Filho
• Support type: Scholarships in Brazil - Master
• 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 type: Scholarships in Brazil - Doctorate