Bacteria use small molecules to mediate their relationships with nearby microbes, and these molecules represent both a promising source of therapeutic agents and a model system for the evolution and dissemination of molecular diversity. This study deals with one such molecule, selvamicin, which is produced by ant-associated bacteria. These bacteria protect the ants’ nests against fungal pathogens. Selvamicin is an atypical member of a clinically important class of antifungal agents, and it appears to have both better therapeutic properties and a different mechanism of action. Further, the genes for producing it are found on the bacteria’s chromosome in one ant nest but on a plasmid in another, illustrating the likely path by which it has spread.

The bacteria harbored by fungus-growing ants produce a variety of small molecules that help maintain a complex multilateral symbiosis. In a survey of antifungal compounds from these bacteria, we discovered selvamicin, an unusual antifungal polyene macrolide, in bacterial isolates from two neighboring ant nests. Selvamicin resembles the clinically important antifungals nystatin A 1 and amphotericin B, but it has several distinctive structural features: a noncationic 6-deoxymannose sugar at the canonical glycosylation site and a second sugar, an unusual 4-O-methyldigitoxose, at the opposite end of selvamicin’s shortened polyene macrolide. It also lacks some of the pharmacokinetic liabilities of the clinical agents and appears to have a different target. Whole genome sequencing revealed the putative type I polyketide gene cluster responsible for selvamicin’s biosynthesis including a subcluster of genes consistent with selvamicin’s 4-O-methyldigitoxose sugar. Although the selvamicin biosynthetic cluster is virtually identical in both bacterial producers, in one it is on the chromosome, in the other it is on a plasmid. These alternative genomic contexts illustrate the biosynthetic gene cluster mobility that underlies the diversity and distribution of chemical defenses by the specialized bacteria in this multilateral symbiosis.

Fungus-growing ants range from central Argentina to New York State, and in tropical regions they are the dominant herbivores. The complex web of interactions involving these ants, their fungal crops, specialized pathogens, and symbiotic bacteria has become both a model system for chemical ecology and a productive source of naturally occurring small molecules (1⇓⇓–4).

Fungus-growing ants collect plant material to feed their fungal crop, which metabolizes the plant matter to provide nutrients for the ants. Pathogenic ascomycetous fungi, especially members of the genus Escovopsis, threaten the fungal crop. In response, the ants maintain antibiotic-producing Actinobacteria (genus Pseudonocardia) to provide chemical defenses (5). These bacteria produce antifungal and/or antibacterial agents, including representatives of both nonribosomal peptide synthetase and polyketide synthase (PKS) biosynthetic pathways (6⇓⇓–9).

As part of a systematic study of Pseudonocardia isolates derived from the basal fungus-growing ant genus Apterostigma, we discovered an unusual antifungal polyene, which we have named selvamicin (Fig. 1). Selvamicin was found in two bacterial isolates from nearby ant nests. Selvamicin shares features with the clinically important antifungal agents amphotericin B and nystatin A 1 , both of which are on the World Health Organization’s List of Essential Medicines (10), and with the food preservative and topical antifungal natamycin (Fig. 1). Amphotericin B and nystatin A 1 have serious liabilities including high toxicity and negligible oral bioavailability, and their continued use reflects the lack of better alternatives (11, 12).

Several features distinguish selvamicin from nystatin A 1 and amphotericin B: a second sugar, a truncated macrocyclic core, and missing carboxylate and ammonium groups. The biosynthetic gene cluster for selvamicin, which includes a subcluster of biosynthetic genes for the distinctive second sugar, was identified bioinformatically. Whole genome sequencing revealed that in one isolate the cluster resides on the chromosome while in the other it resides on a plasmid. These divergent genomic contexts highlight the gene cluster mobility that introduces and disseminates molecular diversity in this system.

Results and Discussion

Discovery and Structure Elucidation. We examined two Pseudonocardia isolates from ants in the genus Apterostigma collected at La Selva Biological Station, Costa Rica, HH130629-09 and HH130630-07 (hereafter LS1 and LS2, respectively). We evaluated antifungal activity of organic-soluble extracts of both cultured strains against the common human fungal pathogen Candida albicans. The LS1 extract was active and we used activity-guided fractionation through a C 18 cartridge followed by reverse-phase HPLC to trace this activity to a molecule with a previously unreported molecular formula of C 47 H 76 O 18 [high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) [M+Na]+ calcd 951.4924, expt 951.4928]. We examined the LS2 extract by high-resolution liquid chromatography–mass spectrometry (LC-MS) and observed the same compound, although at approximately fivefold lower abundance. The active compound’s UV-vis spectrum is characteristic of a polyene, with three prominent peaks (319, 334, 352 nm) consistent with a chromophore of five conjugated double bonds (SI Appendix, Fig. S1). Subsequent NMR analysis using a variety of 2D methods (COSY, TOCSY, HMBC, H2BC, and ROESY) revealed this compound to be an unreported polyene macrolide, which we have named selvamicin after the site of our original collection. Selvamicin production can be greatly up-regulated by adding high concentrations of sodium butyrate (150 mM) to the culture medium (SI Appendix, Fig. S7), consistent with reports that butyrate can regulate secondary metabolism (13). We observed 13C labeling of selvamicin when [1-13C] sodium butyrate was used, indicating that butyrate can also act as a metabolic precursor, and similar results were obtained with [1-13C] propionate (SI Appendix, Fig. S8). COSY and TOCSY correlations allowed us to construct two major fragments of the selvamicin macrolide: one from C2–C8 and another from C13 across the pentaene to the molecule’s terminus at C31 (overlap of the polyene resonances prevented definitive assignments of C19–C24, Fig. 2). HMBC couplings link the C2–C8 fragment to quaternary carbons at either end: an ester carbonyl at C1 (172.7 ppm) and a hemiketal at C9 (97.3 ppm). The hemiketal forms a six-membered ring established by a series of HMBC couplings from the hemiketal OH at C9, a tertiary alcohol and methyl substituent at C12, and the other bridgehead carbon at C13. H2BC correlations support the placement of substituents along the macrolide core of selvamicin (SI Appendix, Fig. S2). A series of ROESY correlations establishes an extended geometry for the C2–C8 aliphatic chain and a chair conformation for the hemiketal ring (SI Appendix, Fig. S2). These correlations, corroborated by available scalar coupling constants, allowed the assignment of relative stereochemistry from C4 to C13. Fig. 2. Key NMR correlations establishing the planar structure of selvamicin. Our NMR analysis also revealed two sugars in the structure of selvamicin. COSY and HMBC couplings revealed their planar structures as 6-deoxy and 2,6-dideoxy hexoses, as shown in Fig. 2. To better resolve the crowded sugar CH signals and reveal additional peak fine structure, we reacted selvamicin with acetic anhydride to modify its free hydroxyl groups. In the acetylation product, the hemiketal at C9 was instead observed as a ketone, and with the exception of the tertiary alcohol at C12, all OH groups were acetylated (SI Appendix, Fig. S3). Scalar couplings and ROESY correlations allowed the acetylated sugars in this product to be assigned as (Ac) 3 -β-6-deoxymannose and Ac-α-4-O-methyldigitoxose (SI Appendix, Fig. S4). The absolute configurations of the sugars were not determined. A clear HMBC coupling from the anomeric proton of the β-6-deoxymannose places this sugar at C15 of selvamicin (Fig. 2). Whereas no HMBC couplings were observed for the anomeric proton of 4-O-methyldigitoxose, a series of ROESY correlations (1′′-H/27-H, 1′′-H/33-H, 5′′-H/34′′-H) locates this sugar on the opposite side of the macrolide at C27. The 1H and 13C chemical shifts of the CH at position 27 support an oxygen substituent linking this sugar. From C25–C31, we observed broadened 1H and 13C resonances, which obscured the couplings needed to establish relative stereochemistry in this region. This peak broadening could reflect conformational flexibility near the 4-O-methyldigitoxose attachment. Selvamicin’s structure diverges from the antifungal polyenes amphotericin B, nystatin A 1 , and natamycin in several key respects. Its 30-membered polyene macrolide core is intermediate between that of the smaller antifungal natamycin and those of amphotericin B and nystatin A 1 . Selvamicin’s unusual glycosylation is also noteworthy. The 6-deoxymannose replaces the mycosamine sugar common to most antifungal polyenes, and a second glycosylation, observed here at C27, is also unusual. Although uncommon, several diglycosylated antifungal polyenes have been reported in the literature, some derived from Pseudonocardia. A diglycosylated nystatin analog named NPP was isolated from Pseudonocardia autotrophica, although the additional sugar, an N-acetylglucosamine, is appended to the 4′ position of the mycosamine (14). A yet-unidentified nystatin analog from the ant-associated Pseudonocardia strain P1 also appears to have an additional sugar appendage at this same 4′ position (15). A second glycosylation located instead on the opposite end of the macrolide, as in selvamicin, has been observed among the minor fermentation products of the nystatin A 1 producer Streptomyces noursei (nystatin A 3 , Fig. 1, and NYST1070), and the candidin producer Streptomyces viridoflavus (candidoin), with the second sugar located at C35, the position corresponding to selvamicin’s 4-O-methyldigitoxose attachment (16⇓–18). Whereas structurally distinct from 4-O-methyldigitoxose, these are also 2,6-dideoxy sugars (digitoxose, mycarose, and 2,6-dideoxy-l-erythro-hexopyranos-3-ulose, respectively). Notably, in contrast to fermentations of S. noursei and S. viridoflavus, we observe the diglycosylated polyene selvamicin as the major polyene species, and neither monoglycosylated analog is detectable by LC-MS in extracts of LS1 or LS2. The presence of 4-deoxymannose in place of mycosamine represents the only example to our knowledge of a noncationic sugar at that position in a glycosylated polyene natural product. Correspondingly, the usual paired carboxylate substituent (C16 in nystatin and amphotericin B and C12 in natamycin) is absent in selvamicin. Instead, C12 bears a methyl group and a tertiary alcohol.

Antifungal Activity and Solubility. Liquid broth-based activity testing confirmed selvamicin’s antifungal activity against C. albicans (minimum inhibitory concentration, MIC, = 23 μM), with similar activity observed across a panel of fungi (Saccharomyces cerevisiae, Aspergillus fumigatus, and Trichoderma harzianum, Fig. 3 and SI Appendix, Table S3). No activity was detected against either Gram-negative (Escherichia coli) or Gram-positive (Bacillus subtilis, Micrococcus luteus) bacteria. We note that selvamicin has more modest antifungal activity than clinically used antifungal polyenes such as nystatin A 1 (MIC = 1.0 μM against C. albicans). However, its improved aqueous solubility (2.3 mM compared with 0.3 mM for nystatin A 1 ) addresses a major limitation of clinically available antifungal polyenes. Selvamicin’s improved solubility, despite its lack of charged carboxylate and ammonium groups, is probably contributed by its second sugar moiety. Indeed, glycosylation has been reported to improve solubility dramatically in analogs of nystatin; NPP, a diglycosylated analog bearing N-acetylglucosamine, has more than 300-fold greater aqueous solubility than nystatin A 1 (14). Fig. 3. Growth inhibition of C. albicans, S. cerevisiae, T. harzianum, and A. fumigatus by selvamicin. The activity of known antifungal polyenes derives from interactions with ergosterol, the primary sterol of fungal plasma membranes. Such interactions can compromise membrane integrity and inhibit the function of membrane proteins (19, 20). Recent studies suggest that ergosterol sequestration into extracellular aggregates may be the dominant mechanism of action (21, 22), although several polyenes, including nystatin and amphotericin B, have also long been known to permeabilize membranes by the formation of ergosterol-dependent transmembrane channels (23). The presumed geometry of these channels situates the charged end of the molecule at the lipid–water interface, with the polyene and polyol interacting with ergosterol within the plasma membrane. The dramatically different electrostatic nature of selvamicin would likely preclude channel formation, with a hydrophilic yet uncharged sugar at each end of the molecule. We probed for an interaction with ergosterol using an established isothermal calorimetry assay for binding to liposome-embedded ergosterol (21, 24). These experiments showed no evidence for binding, in stark contrast to control experiments using nystatin A 1 , suggesting that this interaction is much attenuated if present at all (SI Appendix, Fig. S9). Further investigation of selvamicin’s mechanism of action is currently underway.

Biosynthetic Gene Cluster. To understand the genetic origins of selvamicin biosynthesis, we turned to the genomes of Pseudonocardia isolates LS1 (7) and LS2, which were sequenced using PacBio technology (25, 26). We readily identified a large type I PKS gene cluster in both genomes that matches the biosynthetic requirements for selvamicin (Fig. 4). The 109-kbp selvamicin biosynthetic gene clusters (BGC) from each isolate share perfect synteny and 98.4% nucleotide identity over their length. In contrast, the whole genomes differ more substantially. The average nucleotide identity (27) calculated across conserved replicons on both chromosomes is only 83% and a comparison of housekeeping gene sequences places LS1 and LS2 into distinct clades previously established for ant-associated Pseudonocardia (28, 29). Overall, the two BGCs are much more similar to one another than are their bacterial hosts. Fig. 4. (A) Genomes of Pseudonocardia isolates LS1 and LS2. The selvamicin BGC in each is marked with a red box. (B) Selvamicin BGCs from LS1 and LS2. Mobile genetic element genes flanking the selvamicin clusters are shown as red arrows. Surprisingly, the selvamicin BGC is situated in completely different genomic contexts in the two selvamicin producers; in LS1 it resides on the 6.1 Mbp circular chromosome, whereas in LS2 it is on a 376-kbp plasmid, pLS2-1 (Fig. 4A). The presence of an identical BGC in two divergent Pseudonocardia isolates, and in different genomic contexts, points to horizontal transfer. In keeping with BGC transfer, numerous mobile genetic elements including transposases and integrases flank it in both genomes (Fig. 4B). Mobile genetic elements are prominent features of both genomes. On the pLS2-1 plasmid containing the selvamicin BGC, an impressive 24% of all RAST-annotated genes are mobile genetic elements. Selvamicin provides the most striking example yet for the emerging theme that plasmids drive the genetic, chemical, and functional diversity found in Pseudonocardia symbionts. Plasmid-derived BGCs for an antibacterial rebeccamycin analog and for the gerumycin depsipeptides feature in other ant-associated Pseudonocardia (7, 8). A rearranged variant of the gerumycin BGC also appears on the LS1 chromosome, suggesting that plasmid-mediated exchange also links the two gerumycin BGCs (7), but at greater evolutionary distance than for the virtually identical selvamicin BGCs.