Culture supernatant, further referred as CM was collected from a 5-day-old Bd (JEL423: highly virulent global panzootic lineage) or Bsal (AMFP13/1) culture grown at, respectively, 20°C and 15°C, containing both sporangia and zoospores (mid to late-exponential phase) (Supplementary Figure 1). The supernatant was collected from the flask and centrifuged at 3,000 rpm for 5 min to remove floating spores and sporangia, and the CM was stored at -20°C until further use. CM originating from cultures at different growth stages ranging from lag phase, early-late exponential phase, to stationary phase (Bd: varying from 1 to 14 days old and Bsal: varying from 5 to 8 days old) were screened for the presence of tryptophol, farnesol, tyrosol, and phenylethanol.

Bd spores were collected from a full-grown culture containing mature sporangia. Once the zoospores were released, the medium containing the zoospores was collected and passed over a sterile mesh filter with pore size 10 μm (Pluristrainer, PluriSelect). The flow-through was used as the zoospore fraction (>90% purity). To achieve maximal zoospore adherence to the wells, Bd spores (5 × 10⁵ spores per well) were seeded in TGhL medium (1.6% tryptone, 0.4% gelatin hydrolysate and 0.2% lactose in H₂O) supplemented with 50% H₂O in 24-well plates and incubated for 3 h. After the spores had attached to the bottom of the wells, the medium was replaced by 1 ml control TGhL medium, H₂O, CM or TGhL medium supplemented with different concentrations (20, 40, 60, or 80%) of H₂O or CM. After 5 days, total Bd counts were determined using quantitative PCR (Boyle et al., 2004). Results represent the means of three independent experiments conducted in triplicate.

We analyzed whether cell density is a determining factor that influences Bd growth. Therefore, we collected supernatants of 5-day-old Bd cultures grown at increasing density: (CM1) 1 ml of a mature Bd culture + 19 ml of TGhL broth; (CM2) 2 ml of a mature Bd culture + 18 ml of TGhL broth; (CM3) 3 ml of a mature Bd culture + 17 ml of TGhL broth; (CM4) 4 ml of a mature Bd culture + 16 ml of TGhL broth; and (CM5) 5 ml of a mature Bd culture + 15 ml of TGhL. Bd spores (5 × 10⁵ spores per well) were seeded in 24-well plates and after adhesion for 3 h, the spores were treated with TGhL broth supplemented with 40% of these different CM. As a control, TGhL medium was supplemented with 40% H₂O. After 5 days, total Bd counts were determined using quantitative PCR (Boyle et al., 2004). Results represent the means of three independent experiments conducted in triplicate.

Quorum sensing is a process that involves the production of small molecules (metabolites). In order to determine whether small metabolites are involved in the process of Bd growth regulation, we dialyzed the CM against fresh TGhL using Float-A-Lyzer^® G2 systems (Spectrum labs) with a pore size of 100–500 Da and 500–1,000 Da. Bd spores (5 × 10⁵ spores per well) were seeded in 24-well plates and after adhesion for 3 h the spores were treated with TGhL broth supplemented with 40% of the dialyzed CM (500 Da and 1,000 Da). This was compared to spores treated with TGhL and TGhL supplemented with 40% H₂O or 40% undialized CM. After 5 days, total Bd counts were determined using quantitative PCR (Boyle et al., 2004). Results represent the means of three independent experiments conducted in triplicate.

Bd (5 × 10⁵ spores per well) and Bsal (7,5 × 10⁵ spores per well) spores were seeded in 24-well plates and after adhesion for 3 h, the spores were treated with TGhL medium or TGhL medium supplemented with different concentration of tryptophol (10–500 μM). After 5 days, total Bd and Bsal counts were determined using quantitative PCR (Boyle et al., 2004; Blooi et al., 2013). Results represent the means of three independent experiments conducted in triplicate.

To test whether tryptophol is produced in a cell density-dependent way, Bd and Bsal spores were seeded in TGhL medium diluted 1:2 in H₂O, at a concentration of 5 × 10⁴, 10⁵, and 5 × 10⁵ spores for Bd and additionally 10⁶ spores for Bsal. Every condition was tested in sixfold. At day 1, 3, and 5 post seeding, tryptophol was determined in the supernatant and in order to determine the tryptophol production per zoospore genomic equivalent (GE), Bd and Bsal counts were determined using quantitative PCR (Boyle et al., 2004; Blooi et al., 2013). Therefore, Bd and Bsal fungi were scraped from the bottom of the well using a 1 ml pipette tip, transferred to an Eppendorf tube together with the supernatant of the well and centrifuged for 5 min at 3,000 rpm. The pellet was resuspended in 100 μl of Prepman Ultra reagent (Applied Biosystems), heated for 10 min at 100°C, and the samples were allowed to cool to room temperature for 2 min. Subsequently, the tubes were centrifuged for 2 min at 13,000 rpm and 50 μl of the supernatants was transferred to a new Eppendorf tube. Finally, each sample was diluted 1:10 in H₂O. Suspensions containing standardized numbers of Bd and Bsal zoospores were prepared as previously described (Boyle et al., 2004; Blooi et al., 2013) and 10-fold serial dilution series ranging from 1,000 to 0.01 GEs of zoospores per real-time PCR mixture were prepared for Bd and Bsal.

Tryptophol secretion can be linked to the expression of ARO8, ARO9, and ARO10, as shown in Saccharomyces cerevisiae (Avbelj et al., 2015). We now screened the genomes of Bd and Bsal for possible gene candidates involved in tryptophol production (ARO8, ARO9, ARO10). Therefore, we performed a BLASTP 2.2.30+ search of Bd JEL423 and Bsal proteins (Bioproject PRJNA13653 and PRJNA311566) against 198 UniProt ARO8 listed proteins, identifying OON05735.1 for Bsal and OAJ43843.1 for Bd as significant hits to ARO8. When setting the e-value cutoff to e^-50, BSLG_04451 (OON05735.1) and BDEG_27157 (variant OAJ43843.1) hit to 67 and 83 ARO8 proteins from the UniProt list, respectively (Supplementary Table 1). When considering the conserved domains using the NCBI’s Conserved Domains database, high similarity to ARO8 of Saccharomyces cerevisiae (Bioproject: PRJNA128, Accession: NP_011313) was observed with e-values of 4.17e^-97 and 1.39e^-85 for Bd and Bsal candidates, respectively (Supplementary Figure 2) (Altschul et al., 1997; Marchler-Bauer et al., 2017).

Using the RNeasy plant kit (Qiagen), total RNA was isolated from a 3-, 4-, 5-, and 7-day-old Bd and Bsal culture containing different life stages. We also extracted RNA from H₂O-collected Bd and Bsal spores at a density of approximately 5 × 10⁵ spores/ml and 5 × 10⁴ spores/ml, respectively (further called “spores low”). Expression levels in spores reaching a high density were analyzed by extracting RNA from PmTG-collected Bd and Bsal spores at a density of approximately 10⁷ spores/ml and 10⁶ spores/ml, respectively (further called “spores high”). Finally, we extracted RNA from skin tissue from Bd-infected Alytes obstetricans (10 mg per animal) (Table 2). The samples were homogenized in RLT buffer by beat beating (3 rounds of 1.30 min at 30 Hz) using a tissuelyser II (Qiagen). The RNA concentration was measured by absorbance at 260 nm using a NanoDrop spectrophotometer, and the quality of the RNA samples was checked using an Experion RNA StdSens Analysis kit (Bio-Rad). Total RNA (1 μg) was reverse transcribed to cDNA with the iScript cDNA synthesis kit (Bio-Rad). Expression levels were normalized to the expression of the three most stable reference genes (α-centractin, R6046, TEF1a or GAPDH), which were determined for each condition using geNorm (qBase, Biogazelle). The list of genes and sequences of the primers used for quantitative PCR analysis can be found in Table 3. Real-time quantitative PCR reactions were run in duplicate (technical replicate) and the reactions were performed in 10 μl volumes using the iQ SYBR Green Supermix (Bio-Rad) and 1 μl 1/5 diluted cDNA. The experimental protocol for PCR (40 cycles) was performed on a CFX384^TM RT-qPCR System with a C1000 Thermal Cycler (Bio-Rad, Hercules, CA, United States). The results were analyzed using the Bio-Rad CFX manager 3.1. Quantification cycle (Cq) values were obtained using auto baseline settings and they were applied per primer set. The threshold for maximum Cq difference between the technical replicates was set to 0.5. The results are shown as fold changes of mRNA expression of at least three biological replicates, which were calculated based on the CNRQ values obtained in qBase. Statistics were also performed on the CNRQ values.

A characteristic of tryptophol is that it has an autostimulatory mode of action. In Saccharomyces cerevisiae it has been shown that tryptophol activates the transcription factor Aro80p and, consequently, the expression of the ARO9 and ARO10 transaminase and decarboxylase genes, which results in a positive feedback loop (Chen and Fink, 2006). This implies that a high population density produces more tryptophol per cell than cells at a low density. To test whether this is also the case in chytrid cultures, Bd (5 × 10⁵ spores per well) and Bsal (7,5 × 10⁵ spores per well) spores were seeded in 24-well plates. After adhesion for 3 h, the spores were treated with control TGhL medium or tryptophol-supplemented medium (0,001–1 μM) and grown for 3 days at 20°C or 15°C. To determine the tryptophol background, we also included wells without spores, but with the respective control or tryptophol-supplemented media. The background tryptophol production was subtracted from the tryptophol production after 3 days. Subsequently, we determined the relative “extra” tryptophol production compared to the untreated control wells. Every condition was tested in triplicate for Bd and sixfold for Bsal.

The in vivo experiments were carried out in strict accordance with the recommendation in the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. The experimental setup and protocol was approved by the ethical committee of the Faculty of Veterinary Medicine (Ghent University 2016/120). All animals used were clinically healthy and free of Bd and Bsal as assessed by sampling the skin using cotton-tipped swabs and subsequent performing qPCR (Boyle et al., 2004; Blooi et al., 2013).

We analyzed whether BDEG_27157 is expressed during the later stages of infection, when high chytrid densities are reached. Six captive bred midwife toads (Alytes obstetricans) were exposed to 1 ml of 10⁶ Bd spores per ml water for 24 h at 15 ± 1 °C. As a negative control we included seven animals that were sham treated with 1 ml water for 24 h. Animals were followed up by clinical examination and the infection load was followed up weekly by taking swabs on which we performed qPCR. The animals were euthanized if clinical symptoms were observed (e.g., lethargy, loss of appetite, or weight reduction). A part of the skin (10 mg) was stored in RNA later buffer for 24 h and subsequently stored at -70°C. Skin samples for histopathology were stored in formalin. Histopathology confirmed Bd infection in all inoculated midwife toads.

Secondly, we investigated whether tryptophol is produced on the skin of chytrid negative animals. Therefore, the placebo inoculum (n = 7) was recovered and we quantified tryptophol production using U-HPLC-MS/MS. In addition, this was also tested in alpine newts (Ichthyosaurus alpestris belonging to the order of Chordata and family of Salamandridae) (n = 4).

All statistical analyses were performed using SPSS version 25 (SPSS Inc., Chicago, IL, United States). Normality of the data was assessed using a Kolmogorov–Smirnov and Shapiro–Wilk test. If normally distributed data showed equal variances (Levene’s test), they were analyzed using an unpaired Student’s t-test or one-way ANOVA with a Bonferroni post hoc test to address the significance of difference between mean values with significance set at p ≤ 0.05. If equal variances were not assessed or if the data were not normally distributed, they were analyzed using the non-parametric Kruskal–Wallis analysis with significance set at p ≤ 0.05. Multiple comparisons were assessed by a Kruskal –Wallis analysis followed by pairwise Mann–Whitney U-tests, with a Bonferroni-corrected p-value (p-value = 0.05/number of comparisons) (Supplementary Table 2).

To evaluate the presence of QS regulation in chytrid, we added CM from a 5-day-old Bd culture to fresh spores and analyzed the possible effect on Bd growth after 5 days (Figure 1A). Supplementation of TGhL with CM resulted in a significant (p < 0.01) and dose-dependent decrease in Bd growth compared to spores grown in unsupplemented TGhL. In order to determine whether a decrease in available nutrients caused the drop in Bd growth, or whether the production of possible QS molecules was involved, we also included TGhL medium supplemented with H₂O, showing no reduction in Bd growth.

Next, we prepared CM from Bd cultures with increasing density to analyze whether Bd regulates its growth in a cell density-dependent manner (Figure 1B). No significant effects were observed on Bd growth when supplementing its medium with CM derived from cultures with lower initial cell densities. CM originating from a dense culture did show a significant reducing effect (p < 0.05) in Bd growth, highlighting the cell density-dependent character of this inhibition mechanism.

Quorum sensing is a process that involves the production of small molecules (metabolites). In order to determine whether metabolites were involved in the process of Bd growth regulation, we analyzed the effect of CM following dialysis (500 and 1,000 Da) (Figure 1C). Supplementation with dialyzed CM did not cause a growth decrease, in contrast to a significant growth drop observed with undialyzed CM (p < 0.05). Both the CM originating from dialysis with a 500 and 1,000 Da filter reached growth levels similar to the control (unsupplemented TGhL), indicating that molecules with a molecular mass smaller than 500 Da are involved in the observed growth regulation by the CM.

Conditioned medium obtained from both Bd and Bsal cultures at different growth stages (lag, exponential, and stationary phase) were screened for the presence of the known fungal QS molecules tryptophol, farnesol, tyrosol, and phenylethanol. To this extent, we utilized U-HPLC-MS/MS analysis to quantify the above-mentioned metabolites. In both the CM of Bd and Bsal, tryptophol was detected but farnesol, tyrosol or phenylethanol were not (Figure 2A). The production of tryptophol depends on the growth stage of the fungus, reaching concentrations between 133–652 nM for Bd and 1,139–1,251 nM for Bsal.

To test whether tryptophol indeed can regulate chytrid growth, we analyzed the direct effect of this metabolite on Bd and Bsal growth (Figure 2B). Tryptophol caused a dose dependent growth reduction in both Bd and Bsal, reaching significance at concentrations ≥100 μM (p < 0.0083).

To determine the tryptophol kinetics, Bd and Bsal spores were seeded in 24-well plates at different concentrations and the tryptophol production was monitored at different days during its growth (Figure 3). Here, it can be observed that despite the difference in initial spore loads, the chytrid cultures reached similar densities. Although different initial starting concentrations were used, the GE loads after 5 days were very similar, with mean Log₁₀(GE/ml) values of 7.4–7.7 for Bd and 5.7–6.2 for Bsal. It can also be seen that relevant tryptophol concentrations in the medium were detected when a certain concentration of viable cells was reached (i.e., the quorum was achieved), despite the differences in growth curve. For Bd, the critical point for tryptophol secretion was around 2.5 × 10⁷ GE/ml or a Log₁₀(GE/ml) value of 7.4, whereas for Bsal at least a concentration of 3.5 × 10⁵ GE/ml or a Log₁₀(GE/ml) value of 5.5 had to be reached. This indicates that in cultures with a higher initial start concentration of spores, tryptophol will be produced earlier in the growth process to regulate its own growth. For Bsal seeded at a density of 10⁶ GE/ml, even a small decrease in growth was observed after 1 day, corresponding with high production rates of tryptophol per GE (Figure 4).

To determine whether tryptophol demonstrated an autostimulatory mode of action, as described for Saccharomyces cerevisiae (Chen and Fink, 2006), we analyzed if stimulation of Bd and Bsal with tryptophol results in an increased tryptophol production. The tryptophol production in 3-day-old cultures after tryptophol stimulation was determined, and we defined the “extra” tryptophol production by subtracting the background tryptophol (tryptophol media without spores). We then compared this to non-stimulated control cultures (Figure 5A). Autostimulation was observed in both Bd and Bsal. A concentration-dependent increase in “extra” tryptophol production was noticed, which reached significance (p < 0.0125) in both chytrid cultures challenged with 1 μM tryptophol. This indicates that cultures with high cell densities produced more tryptophol per cell than cells at a low density, confirming the results shown in Figure 4.

We first optimized an RT-qPCR in Bd and Bsal targeting BDEG_27157 and BSLG_04451, respectively. These targets hit to ARO8 (Supplementary Table 1 and Supplementary Figure 2), being aminotransferase I which is involved in the production of tryptophol (Hazelwood et al., 2008). In Saccharomyces cerevisiae, ARO8 expression was shown to be directly linked to pathogen density and tryptophol concentrations produced by this organism (Avbelj et al., 2015). Tryptophol production in chytrid cultures depends on the density of the culture (Figures 3, 5A), suggesting that BDEG_27157 and BSLG_04451 expression will vary during chytrid growth and that BDEG_27157 and BSLG_04451 will be upregulated in high-density cultures. We now show that in both Bd and Bsal, these genes indeed are upregulated in high-density spore samples compared to low-density spore samples (p < 0.05) and that the expression differs depending on the growth stage of the chytrid culture (Figure 5B and Supplementary Figure 3).

By screening Bd-infected tissue of midwife toads for BDEG_27157 expression, we analyzed whether tryptophol also plays a role during chytridiomycosis in amphibians, or whether its effect is limited to the in vitro situation. Results show that BDEG_27157 was expressed in all the samples where fungal RNA was detected (five from six midwife toads) (Table 2). A significantly higher expression (p < 0.05) of this gene compared to H₂O-induced Bd spores was observed, with an upregulation of 4.0 ± 0.70 (Figure 5C).

We analyzed whether the U-HPLC-MS/MS method can be used as a screening method for tryptophol in skin washes (Figure 5D). Both in skin washes of midwife toads and alpine newts, tryptophol was found. In four out of seven midwife toads tryptophol was detected at levels ranging from 10.2 to 43.9 nM. In alpine newts (four out of four), slightly higher concentrations were observed, ranging from 20.6 to 60.6 nM.