Abstract

The regulator of G-protein signaling (RGS) proteins play an important role in upstream control of heterotrimeric G-protein signaling pathways. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus, six RGS protein-encoding genes are present. To characterize the rgsA gene predicted to encode a protein with an RGS domain, we generated an rgsA null mutant and observed the phenotypes of the mutant. The deletion (Δ) of rgsA resulted in increased radial growth and enhanced asexual sporulation in both solid and liquid culture conditions. Accordingly, transcripts levels of the key asexual developmental regulators abaA, brlA, and wetA are elevated in the ΔrgsA mutant. Moreover, ΔrgsA resulted in elevated spore germination rates in the absence of a carbon source. The activity of cAMP-dependent protein kinase A (PKA) and mRNA levels of genes encoding PKA signaling elements are elevated by ΔrgsA. In addition, mRNA levels of genes associated with stress-response signaling increased with the lack of rgsA, and the ΔrgsA spores showed enhanced tolerance against oxidative stressors. Comparative transcriptomic analyses revealed that the ΔrgsA mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes. Accordingly, the rgsA null mutant exhibited increased production of GT and elevated virulence in the mouse. Conversely, the majority of genes encoding glucan degrading enzymes were down-regulated by ΔrgsA, and endoglucanase activities were reduced. In summary, RgsA plays multiple roles, governing growth, development, stress responses, virulence, and external polymer degradation—likely by attenuating PKA signaling.

Highlights

  • Aspergillus fumigatus is a widespread saprophytic fungus in nature, typically found in soil and decaying vegetation [1,2]

  • We compared with RgsA-like proteins in other aspergilli (Supplementary Figure S2B)

  • Canonical regulator of G protein signaling (RGS) proteins accelerate the hydrolysis of GTP bound to Gα, leading the formation of the inactive hetero-trimer and turning off G protein signaling [9]

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Summary

Introduction

Aspergillus fumigatus is a widespread saprophytic fungus in nature, typically found in soil and decaying vegetation [1,2]. This ubiquitous fungus is the most prevalent airborne fungal pathogen, causing a multitude of diseases in humans, such as allergic bronchopulmonary aspergillosis, aspergilloma, and invasive aspergillosis [3]. Characterizations of the roles of RGS proteins may serve as the bases for the identification of novel targets to control human pathogenic fungi. The deletion of rgsA in Aspergillus nidulans resulted in elevated mycelial and conidial pigmentation, and increased the resistance of conidia and vegetative hyphae to oxidative and thermal stresses [20]. This study presents a thorough functional characterization of RgsA’s governing of various key biological processes in A. fumigatus

Results
RgsA Attenuates Hyphal Growth and Asexual Development
RgsA Down-regulates GT Production and Virulence
RgsA Plays a Positive Role in Endoglucanase Activity
Materials and Methods
Nucleic Acid Isolation and Manipulation
Phenotypic Analyses
Enzyme Assay
Murine Virulence Assay
Fungal Burden in Mice
RNA-seq Experiment and Analyses
4.10. Data Analysis
Conclusions
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