Redox metabolites signal polymicrobial biofilm development via the NapA oxidative stress cascade in Aspergillus

Abstract

Background: Filamentous fungi and bacteria form mixed-species biofilms in nature and diverse clinical contexts. They secrete a wealth of redox-active small molecule secondary metabolites, which are traditionally viewed as toxins that inhibit growth of competing microbes.

Results: Here, we report that these "toxins" can act as interspecies signals, affecting filamentous fungal development via oxidative stress regulation. Specifically, in coculture biofilms, Pseudomonas aeruginosa phenazine-derived metabolites differentially modulated Aspergillus fumigatus development, shifting from weak vegetative growth to induced asexual sporulation (conidiation) along a decreasing phenazine gradient. The A. fumigatus morphological shift correlated with the production of phenazine radicals and concomitant reactive oxygen species (ROS) production generated by phenazine redox cycling. Phenazine conidiation signaling was conserved in the genetic model A. nidulans and mediated by NapA, a homolog of AP-1-like bZIP transcription factor, which is essential for the response to oxidative stress in humans, yeast, and filamentous fungi. Expression profiling showed phenazine treatment induced a NapA-dependent response of the global oxidative stress metabolome, including the thioredoxin, glutathione, and NADPH-oxidase systems. Conidiation induction in A. nidulans by another microbial redox-active secondary metabolite, gliotoxin, also required NapA.

Conclusions: This work highlights that microbial redox metabolites are key signals for sporulation in filamentous fungi, which are communicated through an evolutionarily conserved eukaryotic stress response pathway. It provides a foundation for interspecies signaling in environmental and clinical biofilms involving bacteria and filamentous fungi.

Figure 1

Phenazine production modulates P. aeruginosa–A. fumigatus interaction phenotype in co-culture biofilms. (A) Scanning images of co-culture plates following development of PA14 (phenazine-producing DKN370, wild-type and phzM::TnM, and phenazine-null Δphz) colonies and AF293 (wild-type A. fumigatus) lawns over 7 days. Scale bar, 2.5 cm. (B) Surface coverage of PA14 colonies in co-cultures (left) and axenic control cultures (right). (C) Microscopic images at day 6 showing: spatially dependent AF293 conidiation within an operationally defined co-culture interaction zone from the edge of PA14 colonies (left to right); homogeneous AF293 conidiation in its axenic control cultures. Scale bar, 0.5 mm. (D) Quantification of AF293 conidiation in the co-culture interaction zone and in axenic control cultures. (E) Quantification of phenazines secreted by co-cultures with the phenazine producing PA14 strains over 7 days. Results are representative of four biological replicate experiments. Error bars indicate SD of four replicates. See also Figure S1, S2, and S3; Table S1.

Figure 2

5-Me-PCA and PMS at high concentrations inhibit growth but at moderate concentrations enhance conidiation in A. fumigatus (AF293 wild-type). (A–C) 5-Me-PCA at moderate concentrations is the primary phenazine responsible for the enhanced conidiation. Green conidial pigmentation (A) and number of conidia (B) in the region immediately surrounding exogenous application measured for the hole-diffusion assay, after treating AF293 lawns for 6 days with organic and aqueous fractions of extracts prepared from co-cultures of AF293 with DKN370 and wild-type PA14, and AF293 axenic cultures collected periodically throughout incubation. Letters indicate p < 0.001 using a one-way ANOVA test for statistical significance with SigmaPlot, version 12.0. (C) Concentrations of 5-Me-PCA and PYO in the treatment extracts prepared from co-cultures of AF293 with DKN370. (D) 5-Me-PCA and PMS can elicit the switch between inhibiting growth and enhancing conidiation along concentration gradients. Green conidial pigmentation and growth inhibition are imaged and graphically represented for the hole-diffusion assay, after treating AF293 lawns for 4 days with 5-Me-PCA and PMS at different concentrations (from left to right): 315 μM, 36 μM, and 2 μM for 5-Me-PCA; 800 μM, 200 μM, and 40 μM for PMS. See also Table S2 for all phenazine species concentrations in crude extracts. Images (A and D) are representative of biological triplicate plates; scale bars, 2.5 cm. Error bars indicate SD of biological triplicates.

Figure 3

5-Me-PCA, PMS, and PYO modulate A. fumigatus (AF293 wild-type) conidiation via formation of radical intermediates. (A) Phenazines modulate AF293 conidiation and growth via E_1/2-dependent redox activity. See also Figure S5A. (B) Adding a radical scavenging solvent (10 % ethanol, methanol or DMSO), or (C) increasing the assay pH to 8.0, or adding ascorbic acid (AA, 10 mM) but not H₂O₂ (10 mM) significantly represses the enhanced conidiation caused by 5-Me-PCA (36 μM) or PMS (200 μM), as reflected by green conidial pigmentation surrounding the treatment hole imaged in biological triplicate plates at day 4. Scale bars, 2.5 cm. See also Table S2 for all phenazine species concentrations in crude extracts. (D) Decreasing the assay pH to 2.6 helps PYO to enhance AF293 conidiation, as quantified at day 6 for the region immediately surrounding the treatment hole. Error bars indicate SD of biological triplicates. (E) Representative cyclic voltammograms (CVs) of 200 μM PYO in aqueous electrolytes buffered at pH 2.6 versus 4.2. Scan rate, 20 mV/s. See also Table S3.

Figure 4

PMS and PYO can induce A. nidulans conidiation through NapA oxidative stress regulation. To quantify the conidiation in A. nidulans strains we operationally defined “Inner” region immediately next to and “Outer” region away from exogenous application in the hole-diffusion assay. (A and B) wild type and napA deletion (ΔnapA) strains, (C and D) wild type and overexpression (OE::napA) strain supplemented with 200 μg/l pyridoxine (auxotrophic marker) were quantified after 3.5 days of treatment with 40 μM PMS or 100 μM PYO. Error bars indicate SD of biological triplicates. Asterisk refers to statistical significance that measured with a student t test of significance using Excel 2007. *, 0.01 < p < 0.05; **, 0.001 < p < 0.01; ***, p < 0.001. (E) Gene expression analysis of A. nidulans strains, wild type (WT, RDIT9.32) and ΔnapA (RWY10.3) grown in 20 mL liquid GMM at 37 °C with shaking at 225 rpm for 18 h followed by further incubation for 30 min after adding PMS in the cultures at the concentrations of 5μM and 100μM. Ethidium bromide-stained rRNA and gpdA expression are indicated for loading.

Figure 5

Gliotoxin can induce A. nidulans conidiation under mildly reducing conditions through NapA oxidative stress regulation. Effects of gliotoxin alone or together with 100 mM ascorbic acid (AA) on fungal conidiation quantified for each A. nidulans wild-type and napA deletion (ΔnapA) (Left) or wild type and overexpression (OE::napA) strain supplemented with 200 μg/l pyridoxine (Right) after 4 days of treatment. Error bars indicate SD of biological triplicates. See also Figure S6. Asterisk refers to statistical significance that measured with a student T-test of significance using Excel 2007. *, 0.01 < p < 0.05; **, 0.001 < p < 0.01; ***, p < 0.001.

Figure 6

Model of oxidative stress (OS) hormesis mediated by “toxic” microbial redox metabolites in filamentous fungal development. Phenazines and gliotoxin, through inducing differential levels of oxidative stress controlled by metabolite redox properties and their environment-dependent activities, play dual roles as a toxin (high levels) and as a conidiation signal in Aspergillus development (moderate levels), which is fine-tuned by the NapA oxidative stress response pathway.