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

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. fumigatus is 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 of gprM and gprJ causes 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 ΔgprM and ΔgprJ strains 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 ΔgprM and ΔgprJ strains, and that this regulation occurs, 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 signalling. Finally, we show that the ΔgprM and ΔgprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis. This work further contributes to unravelling functions of A. fumigatus GPCRs and shows that GprM and GprJ are essential for CWI, secondary metabolite production and virulence.G蛋白偶联受体（G-protein coupled receptors, GPCRs）是一类细胞外信号受体，可感知环境信号以协调生物响应。真菌主要通过GPCR介导的信号通路感知外界环境，该通路进而调控真菌的生长发育、代谢、致病性以及真菌毒素的生物合成。烟曲霉（A. fumigatus）是一种重要的人类病原菌，可引发曲霉病——一类具有多样临床表现的异质性疾病。本研究详细探究了GPCR家族成员GprM与GprJ在菌株生长及基因表达中的功能。研究发现，GprM与GprJ对于黑色素生成以及细胞壁完整性（cell wall integrity, CWI）通路的调控具有重要作用。与野生型（wild-type, WT）菌株相比，gprM与gprJ的过表达会使菌株生长速率分别降低20%与50%，同时提升其对细胞壁损伤剂的敏感性。与野生型菌株相比，ΔgprM与ΔgprJ缺失菌株中的CWI蛋白激酶MpkA磷酸化水平升高，而过表达突变体中的该磷酸化水平则显著降低。此外，上述菌株的细胞壁多糖含量与排布模式均存在明显差异。RNA测序（RNA-sequencing）结果显示，GprM与GprJ负调控次级代谢产物（secondary metabolites, SMs）编码基因的表达。质谱分析证实，在ΔgprM与ΔgprJ缺失菌株中，烟曲霉素、吡咯吡菌素、烟曲霉碱C、烟喹唑啉以及烟震颤素的合成量均有所降低，且该调控至少部分是通过激活MpkA实现的。过表达gprM还可调控大量转录因子的表达，其中AsgA被预测为GprM与MpkA信号通路的下游效应因子。最后，本研究在大蜡螟（Galleria mellonella）侵袭性曲霉病昆虫模型中发现，ΔgprM与ΔgprJ突变体的致病性显著降低。本研究进一步阐明了烟曲霉GPCR的功能，并证实GprM与GprJ对于细胞壁完整性、次级代谢产物合成以及病原菌致病性均至关重要。