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.

中文翻译：

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烟曲霉G蛋白偶联受体GprM和GprJ对于调节细胞壁完整性途径，次级代谢产物的产生和毒力很重要

G蛋白偶联受体（GPCR）是细胞外信号传导受体，可感知环境线索以协调生物反应。真菌主要通过GPCR介导的信号通路来感知其环境，该信号通路进而调节真菌的发育，代谢，毒力和霉菌毒素的生物合成。烟曲霉是引起曲霉病的重要人类病原体，曲霉病是一种异质性疾病，具有广泛的临床表现。在这里，我们详细研究了GPCR GprM和GprJ在生长和基因表达中的作用。GprM和GprJ对于黑色素的产生和细胞壁完整性（CWI）通路的调节很重要。与野生型（WT）菌株相比，gprM和gprJ的过度表达导致生长率降低20％和50％，并增加了对细胞壁破坏剂的敏感性。与WT菌株相比，CWI蛋白激酶MpkA的磷酸化在ΔgprM和ΔgprJ菌株中增加，而在过表达突变体中降低。此外，在这些菌株中观察到细胞壁多糖浓度和组织的差异。RNA测序表明GprM和GprJ负调控编码次级代谢产物（SMs）的基因。质谱分析证实，在ΔgprM和ΔgprJ菌株中，烟曲霉素，吡啶并戊二烯，烟熏锁骨C，烟灭喹唑啉和烟曲霉菌素的产生减少，并且该调节至少部分地通过MpkA的激活而发生。grpM的过度表达还导致许多转录因子的调控，AsgA预计在GprM和MpkA信号传导下游起作用。最后，我们表明在侵袭性曲霉病的马勒种虫昆虫模型中，ΔgprM和ΔgprJ突变体的毒力降低。这项工作进一步有助于揭开烟曲霉GPCR的功能，并表明GprM和GprJ对于CWI，次级代谢产物的产生和毒力至关重要。