Glucose and HODEs regulate Aspergillus ochraceus quorum sensing through the GprC-AcyA pathway

Abstract

Aspergillus ochraceus is the traditional ochratoxin A (OTA)-producing fungus with density-dependent behaviors, which is known as quorum sensing (QS) that is mediated by signaling molecules. Individual cells trend to adapt environmental changes in a "whole" flora through communications, allowing fungus to occupy an important ecological niche. Signals perception, transmission, and feedback are all rely on a signal network that constituted by membrane receptors and intracellular effectors. However, the interference of density information in signal transduction, which regulates most life activities of Aspergillus, have yet to be elucidated. Here we show that the G protein-coupled receptor (GPCR) to cAMP pathway is responsible for transmitting density information, and regulates the key point in life cycle of A. ochraceus. Firstly, the quorum sensing phenomenon of A. ochraceus is confirmed, and identified the density threshold is 10³ spores/mL, which represents the low density that produces the most OTA in a series quorum density. Moreover, the GprC that classified as sugar sensor, and intracellular adenylate cyclase (AcyA)-cAMP-PKA pathway that in response to ligands glucose and HODEs are verified. Furthermore, GprC and AcyA regulate the primary metabolism as well as secondary metabolism, and further affects the growth of A. ochraceus during the entire life cycle. These studies highlight a crucial G protein signaling pathway for cell communication that is mediated by carbohydrate and oxylipins, and clarified a comprehensive effect of fungal development, which include the direct gene regulation and indirect substrate or energy supply. Our work revealed more signal molecules that mediated density information and connected effects on important adaptive behaviors of Aspergillus ochraceus, hoping to achieve comprehensive prevention and control of mycotoxin pollution from interrupting cell communication.

Keywords: Cell communication; G protein–coupled receptor; Mycotoxin; Oxylipin; Second messenger.

Fig. 1

Density-dependent activities of A. ochraceus. a Growth in solid PDA (upper) and liquid PDB (bottom) medium. b Germination rate and the expression of polocyte forming gene spaA after culturing 14 h in PDB. c Diameter of colony and clump pellet, and d OTA production as well as the expression of pks and bZIP located in the OTA biosynthetic gene cluster after culturing 72 h in PDA and PDB. e The quantity of spores and the expression of sporulation regulatory factor gene brlA after culturing 120 h in PDA. Dashed lines represent genes, and columns represent physical substances

Fig. 2

Mechanism analysis of A. ochraceus responding density information. a Statistics of specific expression genes between representative low-density (experimental group, 10³ spores/mL) versus high-density (control group, 10⁶ spores/mL). b The density dependence of cAMP-cAMP-PKA pathway, including the intracellular cAMP level as well as expression of acyA and pkaA. c Gene expression difference of GPCR family. d The density dependent expression of gprC. e Molecular docking simulation of the receptor GprC with the ligand glucose and 9-HODE, with the binding amino acids (upper) and chemical structure (bottom) zoomed on the right

Fig. 3

Transcriptomes pairwise aligned of wild type, ΔgprC, and ΔacyA after culturing 120 h in PDA. a Veen intersection of differentially expressed genes. b Bubble chart of representational enrichment pathways. c Gene expression heatmap of the OTA biosynthetic gene cluster that containing structural genes (pks, cyc, nrps, p450, hal), cluster-specific regulator (bZIP), and co-regulator (otaR2), as well as the central pathway that regulate sporulation, in which brlA is the only differently expressed gene. d DEGs statistics of KEGG carbohydrate metabolism pathways, and e heatmap of these DEGs

Fig. 4

Effects of the GprC to AcyA pathway on glucose activating spore germination of A. ochraceus. a Density dependent transport and metabolism of glucose as the carbon source. b Relative expression of gprC, rasA, ganB, and acyA of WT, ΔgprC and ΔacyA in minimum medium (MM) and with glucose as the only carbon source. The (*) denotes the significant difference in gene expression between the same strain in MM and glucose medium, and different colors represent different strains. c G protein signaling pathways in germination. Glucose binding with membrane receptor to activate intracellular heterotrimer G protein (Gαβγ) or small G protein Ras, the Gα subunit-AcyA-cAMP pathway and Ras pathway are relatively independent, and affect spore germination by initiating gene transcription at different stages, including ①hypopus absorbency swelling and isotropic expansion at the early stage, ②bud tube germination at the middle stage, ③polarized extension at the late stage. d Spore germination related genes in three stages, including the trehalase encoding gene TreB that affects absorbency swelling during the isotropic growth phase, the polocyte forming gene SpaA that affects bud tube germination during the polar growth, and the septin forming gene Spe during the hyphae extension stage. The black (*) denotes the significance of the difference in gene expression in the same strain with or without cAMP supplementation. e Without or f with cAMP supplementation, the transmission electron microscopy (upper, magnification 4000) and light microscopy (bottom, magnification 400) observations of three strains

Fig. 5

The role of G protein pathways in the utilization of carbon sources during the life cycle of A. ochraceus. a Final colony morphology of WT, ΔacyA, and ΔgprC in the absence and presence of supplementary cAMP. Without or with cAMP supplementation, the b, c germination rates at 14 h, d, e growth rates from 24 to 36 h, f, g stable colony diameter after culturing 120 h, h, i OTA production after culturing 120 h. Colored lowercase letters indicate the significance of differences in growth of the same strain in different carbon sources, P < 0.05

Fig. 6

GprC-AcyA pathway transmit intra- and interspecific communication signals as well as the effect of standard oxylipins on A. ochraceus. a The concentration of HODEs (histogram), expression of lox (dotted line), and proportion of 9-HODE (embedded line graph) of WT in serial density systems after culturing 72 h in PDB. b Experimental flow diagram of the effect of autocrine signaling molecule systems. c Effect of autocrine signaling molecules on OTA production in WT, ΔacyA, and ΔgprC. d Scanning electron microscopy of the asexual propagation structure of three strains, each scale indicates 50 μm (upper) and 10 μm (bottom). e The varying degrees of three strains infect maize, soybean, and peanut seeds. f The concentration of HODEs (histogram) and proportion of 9-HODE (line graph) in crop seeds (upper) and WT A. ochraceus-plant infection system (bottom) after culturing 72 h. The g OTA production and h spore quantity in fungi-plant infection system after culturing 72 h. The black asterisk represents the difference between three strains in the same seed, the colored letters represent the differences of the same strain in three seeds, different colors indicate different strains. i Scanning electron microscopy of WT affected by PGE₂, 9-HODE, and 13-HODE. j intracellular cAMP levels of three strains affected by standard oxylipins. k OTA biosynthesis and l spore generation with or without additional cAMP in WT, ΔacyA, and ΔgprC. The colored asterisk denotes the significance of difference in response to different oxylipins in the same strain compared to the ethanol control (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (ns) non-significant

Fig. 7

The hypothesized comprehensive mechanism of carbon source as well as QSM influence on A. ochraceus behaviours

Fig. 8

The GprC-AcyA pathway senses a wide range of signals and regulates the life cycle of Aspergillus. (1, 2, 3, 4) representing different stages of life cycle, facing environmental stresses, and the G protein pathway, consisting of ligand, receptor, effector, second messenger, and regulatory factors, can respond to various cues and achieve adaptive regulation of behavior