Upregulation of Escovopsis virulence factors

The pathogenesis of Escovopsis on the fungal cultivar L. gongylophorus was studied by co-culturing with E. weberi strain G (Supplementary Table 1) on potato glucose agar (PGA) plates and comparing the secondary metabolome to that of axenic cultures of E. weberi. Chemical profiling of extracts taken from the resulting plates, using ultra performance liquid chromatography (UPLC) coupled with high-resolution mass spectrometry (HRMS), revealed the presence of two major, and a range of minor, metabolites produced during pathogenesis. The major metabolites had signals at m/z 729.0937 ([M+H]+) for compound 1 and 600.3340 ([M+H]+) for compound 2 (Fig. 2). Using liquid chromatography (ultraviolet)-mass spectrometry (LC(UV)MS) analysis calibrated using isolated standards (see Supplementary Notes 1 and 2), we confirmed that 1 and 2 were elevated significantly (3.4-fold for 1 and 8.9-fold for 2) when compared to axenic cultures of E. weberi (Fig. 2). Similar results were obtained for the pathogenesis of E. weberi strain A when carried out independently, with upregulation of compounds 1 (2.2-fold) and 2 (2.9-fold) during pathogenesis, and we further confirmed that 1 and 2 were absent in extracts of the healthy food fungus (Supplementary Fig. 1).

Fig. 2 Escovopsis pathogenesis involves upregulation of small molecule virulence factors. HPLC profiles (λ = 254 nm) of ethyl acetate extracts of a L. gongylophorus infected with E. weberi strain G; b E. weberi strain G axenic culture. Melinacidin IV, 1; shearinine D, 2 Full size image

Identification of the Escovopsis virulence factors

For structure elucidation we isolated 1 and 2 from large-scale axenic cultures of E. weberi strain A grown on PGA plates. Ethyl acetate extraction, silica-based column chromatography and semi-preparative high-performance liquid chromatography (HPLC) yielded pure samples of 1 (2.0 mg) and 2 (1.4 mg). HRMS analysis of 1 showed an intense M+2 signal, indicating a number of incorporated sulfur atoms. Only half the expected number of signals could be observed in the 1H and 13C nuclear magnetic resonance (NMR) spectrum, indicating a symmetrical compound. Combining these data, a molecular formula of C 30 H 28 N 6 O 8 S 4 was derived. Analysis of 2D NMR data (heteronuclear single-quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC)) pointed to a 1,2-substituted phenyl-ring as part of an indole moiety, while the second part of the molecule was clearly comprised of a diketopiperazine. Comparison of all analytical data, including optical rotation with literature values35, finally confirmed 1 as the epipolythiodiketopiperazine (ETP) antibiotic melinacidin IV, originally isolated from the fungus Acrostalagmus cinnabarinus var. melinacidinus36 (Fig. 3; Supplementary Note 1).

Fig. 3 Chemical structures of the major metabolites produced by Escovopsis strains Full size image

The 1H NMR spectrum of 2 showed 4 aromatic protons, 14 aliphatic protons and 24 protons that could be assigned to 8 methyl groups. Further, we determined 16 quaternary carbon atoms, including 1 carbonyl group by analysis of the 13C NMR spectrum. Accordingly, we could assign a molecular formula of C 37 H 45 NO 6 , featuring 16 double bond equivalents. Structure elucidation based on 2D NMR data (HSQC and HMBC) revealed an indole moiety as part of a highly condensed carbon backbone and a Michael acceptor system in proximity to an acetal unit. These features along with the comparison of the chemical shift of all NMR signals with literature data, nuclear Overhauser effect spectroscopy (NOESY) analysis, and the measurement of the optical rotation unambiguously confirmed the identity of 2 as shearinine D, a terpene-indole alkaloid previously isolated from the endophytic fungus Penicillium janthinellum (Fig. 3; Supplementary Fig. 2; Supplementary Note 2)37. Predicted BGCs for terpene-indole alkaloid and ETP metabolites are present in the published E. weberi genome of strain G38 (Supplementary Figs 3 and 4; Supplementary Tables 2-7), and shearinines D, F and J have been identified using imaging mass spectrometry of the Escovopsis strain TZ49 isolated from Trachymyrmex zeteki nests from the same Panamanian field site39. No function was assigned to these compounds in the previous study and the species of Escovopsis was not identified39.

Metabolomics and molecular networking

Having identified two major classes of secondary metabolites upregulated during E. weberi infection of the fungal cultivar, we clarified their distribution among Escovopsis lineages from different attine hosts. We tested five additional Escovopsis strains isolated from the nests of the leafcutter ants Atta colombica (strain C), Acromyrmex echinatior (strains B, E and F), and the higher, non-leafcutter attine Trachymyrmex cornetzi (strain D) (Supplementary Table 1). We cultivated five replicates of each strain, plus strain A as a control, and performed UPLC-MS/MS-based profiling. Data were uploaded to the Global Natural Product Social Molecular Networking (GNPS) web platform and used to perform a molecular networking analysis approach40. Molecular networking captures the similarity of analytes by the comparison of their MS/MS spectra and this allowed us to identify a number of metabolite families being produced by the Escovopsis strains (Supplementary Fig. 5). Molecular networking revealed that additional congeners of 1 are present in all six strains (Figs. 4 and 5). We observed compound 3, putatively annotated as melinacidin III (m/z 713.0969 [M+H]+, molecular formula of C 30 H 28 N 6 O 7 S 4 ; Supplementary Note 3) and a further derivative of melinacidin III, missing one methyl group (m/z 699.0866 [M+H]+, molecular formula of C 29 H 26 N 6 O 7 S 4 ). Two related signals could be annotated as the ETPs chetracin B (4; m/z 761.0670 [M+H]+, molecular formula of C 30 H 28 N 6 O 8 S 5 ; Supplementary Note 4) and chetracin C (5; m/z 793.0639 [M+H]+, molecular formula of C 30 H 28 N 6 O 8 S 6 ; Supplementary Note 5) which was verified by NMR41. ETPs 4 and 5 feature polysulfide bridges of their diketopiperazine moiety of various lengths and are present in lower amounts in the extracts42 (Fig. 3).

Fig. 4 Molecular network and chemical structures of ETPs being produced by Escovopsis strains A–F Full size image

Fig. 5 List of selected signals assigned to ETPs and shearinine-like terpene-indole alkaloid metabolites. Includes a heat map of their average ion count in logarithmic representation. *Metabolite putatively annotated; **metabolite identified and authenticated against isolated sample Full size image

We also identified a complex network of shearinine-like terpene-indole alkaloid congeners (Fig. 5; Supplementary Fig. 6). Production levels were sufficiently high to allow isolation of several compounds from a scaled-up culture of strain C which resulted in 2 (3.5 mg), along with 6 (shearinine A; 1.0 mg) and 7 (22,23-dehydro-shearinine A; 1.8 mg), in addition to the polyketide metabolite emodin (8; 0.85 mg) (Fig. 3). Compound 6 showed an HRMS signal of m/z = 584.3379 corresponding to a molecular formula of C 37 H 45 NO 5 . Comparison of 1H NMR, MS2 data and the retention time of 6 with an authentic reference confirmed its identity as shearinine A (Supplementary Note 6). Compound 7 featured an HRMS signal of m/z = 582.3209 [M+H]+ (corresponding to a molecular formula of C 37 H 43 NO 5 ) and could be identified as 22,23-dehydro-shearinine A by the comparison of 1H and 13C NMR data (Supplementary Note 7). Additionally, we observed the presence of likely pathway intermediates such as putative shearinine J and penitrem D (Fig. 5). The discovery of 8 (Supplementary Note 8) as a secondary metabolite produced by an Escovopsis sp. is highly intriguing. Anthraquinones have a long history as a feeding deterrent for ants43,44,45 and 8 in particular has proven to be a broad-spectrum insecticide comprising activity against three different mosquito species46, the white fly Bemisia tabaci47 and caterpillar larvae48. Analysis of the sequenced genomes (see below) showed that all the Escovopsis strains contain the biosynthetic genes required for production of 8 (Supplementary Fig. 7; Supplementary Tables 8-10).

To determine the distribution and relative levels of the annotated metabolites among the different Escovopsis strains, we used the software Profiling Solutions (Shimadzu) for data analysis, peak picking, data alignment and filtering of metabolic profiling data. A heat map was generated showing the abundance of each ion in the MS profile (Fig. 5). The metabolomics data clearly showed that ETPs are produced by all six Escovopsis strains A–F. In contrast, shearinines are only produced by the five strains isolated from leafcutter ant nests (Atta and Acromyrmex species) but not by Escovopsis isolated from the higher, non-leafcutter attine ant T. cornetzi (strain D) (Fig. 5). We validated our metabolomics data through genome sequencing of strains A–F, which verified that a shearinine-like BGC is present in strains A–C and E–F (Supplementary Fig. 3; Supplementary Tables 2-4); however the BGC was absent from strain D.

Genome sequencing and analysis of Escovopsis strains

To gain deeper insight into the secondary metabolism of the fungal pathogen, we sequenced the genomes of the six Escovopsis strains A–F used in this study. The genome characteristics of these Escovopsis strains are shown in Supplementary Table 1. As a reference, we used the published genome of an E. weberi strain (strain G) isolated from Atta cephalotes collected in Gamboa, Panama; the same field site used to isolate strains A–F and for the collection of ant colonies38. According to Meirelles et al.49, there are nine clades of Escovopsis, with five classified species. Phylogenetic analysis (Supplementary Fig. 8, Supplementary Table 11) using the tef1 gene and internal transcriber spacer (ITS) DNA sequences shows that strains A and C most closely resemble E. weberi species from clade I, and strains B, E and F most closely resemble E. weberi species from clade II. Strain D is distantly related to the E. weberi strains and most closely resembles Escovopsis aspergilloides and aligns to clade VII49. We analyzed all the sequenced Escovopsis genomes using fungiSMASH, an online resource for the rapid identification of natural product BGCs from fungi50. We detected 20–23 putative BGCs for each strain and these mainly comprise terpene, type 1 polyketide synthase and non-ribosomal peptide synthetase clusters. Based on the reported BGC for 2 in P. janthinellum51, we identified homologous BGCs in strains A–C and E–G (Supplementary Fig. 3; Supplementary Tables 2-4). This is consistent with the fact that these strains all make shearinine-like terpene-indole alkaloids, but strain D does not. BGCs for the biosynthesis of ETPs like 1 are present in all seven genomes (Supplementary Fig. 4; Supplementary Tables 5-7) and resemble the BGC responsible for production of the ETP chaetocin from Chaetomium virescens52.

Virulence factors 1 and 2 inhibit Pseudonocardia mutualists

Melinacidins are known to have potent activity against Gram-positive bacteria and we reasoned it would benefit the parasite to inhibit the Pseudonocardia mutualists36. As in some more basal attine branches[16], the Acromyrmex have a tight association with these bacteria as the ants have specialized crypts with small glands for housing and feeding the Pseudonocardia mutualist15. To test our hypothesis, we determined the minimum inhibitory concentration (MIC) of compounds 1 and 2 against representative strains from the two lineages of Pseudonocardia associated with A. echinatior colonies collected in Gamboa, Panama. Strain Ae707 belongs to the Ps1 lineage (P. octospinosus) and strain Ae706 belongs to the Ps2 lineage (P. echinatior)27. Liquid-based colorimetric assays were performed in microtiter plates to determine the MIC. Ciprofloxacin was used as a positive control and all the assays were done in triplicate. The results show that compounds 1 and 2 are active against both tested Pseudonocardia mutualist strains. Compound 1 has an MIC of 10 µg ml−1 against Ae706 and 0.5 µg ml−1 against Ae707, while compound 2 has an MIC of 5 µg ml−1 against both Ae706 and Ae707 (Fig. 6).

Fig. 6 Growth of Pseudonocardia mutualists is inhibited by Escovopsis small molecule virulence factors. Microplate assay of 1 (top) and 2 (bottom) against Pseudonocardia strains Ae706 and Ae707. *PC is the positive control (50 µg ml−1 ciprofloxacin). **NC is the negative control (no inoculation). ***MC, the bacteria grown in LB with 5% of methanol. The pink color indicates viable bacterial cells Full size image

Effects of dietary intake of 2 on A. echinatior worker ants

Members of the terpene-indole alkaloid family such as the paspalines53 and the janthitrems54,55,56 are potent mycotoxins and exhibit insecticidal and tremorgenic activity57. The structurally related penitrems additionally act as feeding deterrents and modulators of ion channels in various insects58,59,60. The tremorgenic terpene-indole alkaloid 2 has been shown to strongly inhibit the ion channel BK Ca , indicating a potent effect on insect nervous systems37. The enhanced production levels of 2 during E. weberi infection of the fungal cultivar suggested that it could be an important feature in the pathogenesis of E. weberi. Even though the pathogen infects the fungal cultivar rather than the ants themselves, we postulated that 2 is a virulence factor primarily affecting the worker ants. Fungal virulence factors are highly diverse but the most well-known example is produced by the fungus Ophiocordyceps unilateralis, which causes infected ‘zombie’ ants to adopt highly unusual behaviors including random walking, repeated convulsions and final death grips underneath leaves5. The identity of the virulence factor(s) is not known but transcriptomics data indicate that O. unilateralis genes with high similarity to those for the biosynthesis of terpene-indole alkaloids such as penitrems, close structural analogs of the shearinines, are upregulated during manipulated biting behavior of infected carpenter ants34.

To characterize the effect of 2 on A. echinatior ants, we established viable sub-populations of five worker ants in a single Petri dish. These were each supplied with a glucose solution supplemented with concentrations of up to 2 mM of 2 dissolved in 50% methanol (Supplementary Fig. 9 Supplementary Movies 1 and 2) or methanol only as a control. After 10 days, the percentage mortality of the worker ants was significantly affected by the concentration of dietary 2 (Supplementary Fig. 10, Kruskall–Wallis test: H = 12.6431 DF=3 , P = 0.027) with greater levels of mortality occurring at higher concentrations. Survival analysis also demonstrated that the probability of survival over time was significantly reduced at increasing concentrations of 2, even after controlling for different runs of the experiment (Supplementary Fig. 11, Cox’s mixed effects model: hazard ratio = 3.83, z = 0.289, P < 0.001). Supplementation with 50% methanol alone had no effect on ant mortality and all the ants survived the experiment. Additionally, in a control experiment, all ants survived 10 days of exposure to reduced glucose concentrations (dietary concentrations were reduced from 5% to 3% glucose) confirming that mortality was not due to starvation caused by a reduced sugar content at higher concentrations of 2. In addition to the effects on mortality, we also observed reduced mobility, disorientated movement and loss of balance; ants could no longer successfully traverse the sides and lid of the petri dishes as the level of 2 increased. Time-lapse videos showed that mobility decreased as the concentration of 2 increased, and that each ant spent a longer amount of time sitting stationary on the cotton wool. For example, individual ants in the highest concentration treatment group (2 mM) were stationary for a significantly longer period of time compared to the control treatment group; an average ( ± SE) of 62.8 ± 1.74 s out of the total 65 s of film, compared to an average of 8.8 ± 2.1 s in the control group, respectively (t (7.73) = 19.74, P ≤ 0.001, Welch's t-test) (Supplementary Fig. 12, Supplementary Movies 1 and 2).

To determine the amount of 2 ingested during these experiments, we developed a multiple reaction monitoring (MRM) MS-based method. An authentic reference of 2 was used along with the internal standard yohimbine to scan for selective MS/MS mass transitions. This confirmed that significantly higher concentrations of 2 are present in the worker ant tissues compared to those in control worker ants (P = 0.001, Dunn’s test) (Fig. 7), and we observed large differences in the tolerated dose of up to 70 ng mg−1 of bodyweight (Supplementary Fig. 13). Effects such as unstable movements and reduced mobility were observed in ants from a minimum level of approximately 10 ng of ingested 2 per mg of bodyweight. We also quantified the levels of 2 in worker ants taken from A. echinatior sub-colony infection experiments, and from ants in a captive A. colombica colony that naturally suffered an uncontrolled outbreak of Escovopsis infection (Fig. 7). This confirmed that ants in infected colonies had ingested large amounts of 2. The levels of 2 in ants during infection with strain E, and also in ants from naturally infected nests, were not significantly different from levels observed in ants that were fed a 5% glucose solution containing 0.1 mM of 2 (Dunn’s test: P = 0.4844 in both cases respectively) (Fig. 7). Worker ants from control colonies fed 5% glucose solution or from colonies infected with strain D isolated from T. cornetzi were indistinguishable (Dunn’ test: P = 0.397), consistent with the lack of shearinine production by strain D (Fig. 7).