Aspergillus fumigatusG-protein coupled receptors GprM and GprJ are important for the regulation of the cell wall integrity pathway, secondary metabolite production, and virulence
Authors: da Costa Filho Aílton Pereira , Pereira Brancini Guilherme Thomaz , de Castro Patrícia Alves , Ferreira Jaire Alves , Silva Lilian Pereira , Rocha Marina Campos , Malavazi Iran , de Moraes Pontes João Guilherme , Fill Taícia , Silva Roberto Nascimento , Almeida Fausto , Steenwyk Jacob L. , Rokas Antonis , dos Reis Thaila F. , Ries Laure N.A. , Goldman Gustavo H. 
Published: August 2020

G-protein coupled receptors (GPCRs) are extracellular signalling receptors that sense environmental cues to coordinate a biological response. Fungi sense their environment primarily through GPCR-mediated signalling pathways, which in turn regulate fungal development, metabolism, virulence and mycotoxin biosynthesis.A. fumigatusis an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that presents 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 ofgprMandgprJcauses a 20 and 50% reduction in growth rate when 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 ΔgprMand ΔgprJstrains and decreased in the overexpression mutants when compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. RNA-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 ΔgprMand ΔgprJstrains, and that this regulation occurs, at least partially, through the activation of MpkA. Overexpression ofgrpMalso resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signalling. Finally, we show that the ΔgprMand ΔgprJmutants are reduced in virulence in theGalleria mellonellainsect model of invasive aspergillosis. This work further contributes to unravelling functions ofA. fumigatusGPCRs and shows that GprM and GprJ are essential for CWI, secondary metabolite production and virulence.

Author summary

A. fumigatusis the main ethiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immuno-compromised humans. Withstanding the host environment is essential forA. fumigatusvirulence 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. TheA. fumigatusgenome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GPCRs 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 modelGalleria mellonella. This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial forA. fumigatusvirulence.