Characterization of gprK Encoding a Putative Hybrid G-Protein-Coupled Receptor in Aspergillus fumigatus

Abstract

The G-protein-coupled receptor (GPCR) family represents the largest and most varied collection of membrane embedded proteins that are sensitized by ligand binding and interact with heterotrimeric G proteins. Despite their presumed critical roles in fungal biology, the functions of the GPCR family members in the opportunistic human pathogen Aspergillus fumigatus are largely unknown, as only two (GprC and GprD) of the 15 predicted GPCRs have been studied. Here, we characterize the gprK gene, which is predicted to encode a hybrid GPCR with both 7-transmembrane and regulator of G-protein signaling (RGS) domains. The deletion of gprK causes severely impaired asexual development coupled with reduced expression of key developmental activators. Moreover, ΔgprK results in hyper-activation of germination even in the absence of carbon source, and elevated expression and activity of the protein kinase A PkaC1. Furthermore, proliferation of the ΔgprK mutant is restricted on the medium when pentose is the sole carbon source, suggesting that GprK may function in external carbon source sensing. Notably, the absence of gprK results in reduced tolerance to oxidative stress and significantly lowered mRNA levels of the stress-response associated genes sakA and atfA. Activities of catalases and SODs are severely decreased in the ΔgprK mutant, indicating that GprK may function in proper activation of general stress response. The ΔgprK mutant is also defective in gliotoxin (GT) production and slightly less virulent toward the greater wax moth, Galleria mellonella. Transcriptomic studies reveal that a majority of transporters are down-regulated by ΔgprK. In summary, GprK is necessary for proper development, GT production, and oxidative stress response, and functions in down-regulating the PKA-germination pathway.

Fig 1. The role of GprK in asexual development.

(A) Colony photographs of WT (AF293), ΔgprK, and complemented (C') strain point-inoculated on solid MMG with 0.1% YE and grown for 3 days (Top: left; Bottom: middle panels). The enlarged photographs from the plate (indicated by the white box) are shown in the right panels with the bar indicating 1 mm. (B) Conidia numbers produced by each strain per plate. Student’s t-test: *p < 0.05, **p < 0.01. (C) mRNA levels of the asexual developmental regulator genes in WT, ΔgprK, and C' strains determined by quantitative PCR (qRT-PCR). Cultures were incubated in liquid MMY and mRNA levels were normalized using the ef1α gene, according to the ΔΔCt method. Data are expressed as the mean ± standard deviation from three independent experiments. Student’s t-test: *p < 0.05, **p < 0.01. (D) Photographs of colonies of WT, ΔgprK, C', and ΔgprK:gpcr⁺ strains point-inoculated on solid MMG and grown for 3 days (Top: left; Bottom: right panels).

Fig 2. The role of GprK in spore germination and PKA activity.

(A) Kinetics of germ outgrowth in A. fumigatus strains when inoculated in liquid MMG at 37°C in the presence or absence (“no C”) of glucose. (B) Accumulation of pkaC1 mRNA in WT, ΔgprK, and C' strains analyzed by qRT-PCR. Student’s t-test: **p < 0.01. (C) PKA activity of A. fumigatus strains as monitored by gel electrophoresis. A phosphorylated substrate migrates toward the cathode (+). Each strain was grown in MMG for 24 h at 37°C, at which time a mycelial extract was analyzed. Note that the expression of pkaC1 mRNA and PKA activity were significantly increased in the ΔgprK mutant strain compared to WT and C' strains.

Fig 3. Nutrient sensing and stress responses mediated by GprK.

(A) Effect of various carbon sources on the growth of WT, ΔgprK, and C' strains. Note that the growth of the mutant was severely restricted on arabinose, ribose, and xylose compared to WT and C' strains. (B) Radial growth of WT, ΔgprK, and C' strains in presence of oxidative stressors H₂O₂, menadione (MD), or paraquat (PQ) at indicated concentrations following incubation at 37°C for 48 h. (C) Levels of oxidative stress-related genes’ mRNA in WT, ΔgprK, and C' strains analyzed by qRT-PCR. Statistical significance was determined by a Student’s t-test: *p < 0.05 and **p < 0.01. (D) Conidial catalases and SODs activities of WT, ΔgprK, and C' strains shown in non-denaturing polyacrylamide gels.

Fig 4. The role of GprK in GT production.

(A) qRT-PCR analysis of four GT-related genes in WT, ΔgprK, and C' strains. Statistical differences between WT and mutant strains were evaluated with Student's unpaired t-test. *p < 0.05 and **p < 0.01. (B) Determination of GT production in WT, ΔgprK, and C' strains. The culture supernatant of each strain was extracted with chloroform and subjected to TLC. The arrow indicates the migration position for GT.

Fig 5. The role of GprK in virulence.

(A) Conidia invasion index in type II human alveolar A549 cells inoculated with the ΔgprK strain was significantly lower than that of WT. (B) There were no significant differences between WT, ΔgprK, and complemented strains as measured by Kaplan-Meier curve survival analysis and log-rank test. (C) Histological analysis of larval tissues infected by WT and ΔgprK strains was performed using H&E and PAS staining 24 h post inoculation. Arrows indicated fungal hyphae in infected larval tissues.

Fig 6. Genome-wide expression correlation between WT and ΔgprK strains.

(A) Heat map illustration of expression level changes between WT and ΔgprK strains. (B) Linear fitted model showing the correlation between overall gene expression for WT and ΔgprK strains. The correlation coefficient R is indicated.