Amycolatopsis from Desert Specialist Fungus-Growing Ants Suppresses Contaminant Fungi Using the Antibiotic ECO-0501.

Symbiotic Actinobacteria help fungus-growing ants suppress fungal pathogens through the production of antifungal compounds. Trachymyrmex ants of the southwest desert of the United States inhabit a unique niche far from the tropical rainforests in which most fungus-growing ant species are found. These ants may not encounter the specialist fungal pathogen Escovopsis known to threaten colonies of other fungus-growing ants. It is unknown whether Actinobacteria associated with these ants antagonize contaminant fungi and, if so, what the chemical basis of such antagonism is. We find that Pseudonocardia and Amycolatopsis strains isolated from three desert specialist Trachymyrmex species do antagonize diverse contaminant fungi isolated from field-collected ant colonies. We did not isolate the specialist fungal pathogen Escovopsis in our sampling. We trace strong antifungal activity from Amycolatopsis isolates to the molecule ECO-0501, an antibiotic that was previously under preclinical development as an antibacterial agent. In addition to suppression of contaminant fungi, we find that this molecule has strong activity against ant-associated Actinobacteria and may also play a role in bacterial competition in this niche. By studying interspecies interactions in a previously unexplored niche, we have uncovered novel bioactivity for a structurally unique antibiotic. IMPORTANCE Animal hosts often benefit from chemical defenses provided by microbes. These molecular defenses are a potential source of novel antibiotics and offer opportunities for understanding how antibiotics are used in ecological contexts with defined interspecies interactions. Here, we recover contaminant fungi from nests of Trachymyrmex fungus-growing ants of the southwest desert of the United States and find that they are suppressed by Actinobacteria isolated from these ants. The antibiotic ECO-0501 is an antifungal agent used by some of these Amycolatopsis bacterial isolates. This antibiotic was previously investigated in preclinical studies and known only for antibacterial activity.

Bacterial Associates of a Desert Specialist Fungus-Growing Ant Antagonize Competitors with a Nocamycin Analog.

Fungus-growing ants are defended by antibiotic-producing bacterial symbionts in the genus Pseudonocardia. Nutrients provisioned by the ants support these symbionts but also invite colonization and competition from other bacteria. As an arena for chemically mediated bacterial competition, this niche offers a window into ecological antibiotic function with well-defined competing organisms. From multiple colonies of the desert specialist ant Trachymyrmex smithi, we isolated Amycolatopsis bacteria that inhibit the growth of Pseudonocardia symbionts under laboratory conditions. Using bioassay-guided fractionation, we discovered a novel analog of the antibiotic nocamycin that is responsible for this antagonism. We identified the biosynthetic gene cluster for this antibiotic, which has a suite of oxidative enzymes consistent with this molecule's more extensive oxidative tailoring relative to similar tetramic acid antibiotics. High genetic similarity to globally distributed soil Amycolatopsis isolates suggest that this ant-derived Amycolatopsis strain may be an opportunistic soil strain whose antibiotic production allows for competition in this specialized niche. This nocamycin analog adds to the catalog of novel bioactive molecules isolated from bacterial associates of fungus-growing ants, and its activity against ant symbionts represents, to our knowledge, the first putative ecological function for the widely distributed enoyl tetramic acid family of antibiotics.

Specialized Metabolites Reveal Evolutionary History and Geographic Dispersion of a Multilateral Symbiosis.

Fungus-growing ants engage in a multilateral symbiosis: they cultivate a fungal garden as their primary food source and host symbiotic actinobacteria (Pseudonocardia spp.) that provide chemical defenses. The bacterial symbionts produce small specialized metabolites that protect the fungal garden from specific fungal pathogens (Escovopsis spp.), and in return, they are fed by the ant hosts. Multiple studies on the molecules underlying this symbiotic system have led to the discovery of a large number of structurally diverse antifungal molecules, but somewhat surprisingly no shared structural theme emerged from these studies. A large systematic study of Brazilian nests led to the discovery of the widespread production of a potent but overlooked antifungal agent, which we named attinimicin, by nearly two-thirds of all Pseudonocardia strains from multiple sites in Brazil. Here we report the structure of attinimicin, its putative biosynthetic gene cluster, and the evolutionary relationship between attinimicin and two related peptides, oxachelin A and cahuitamycin A. All three nonribosomal peptides are structural isomers with different primary peptide sequences. Attinimicin shows iron-dependent antifungal activity against specific environmental fungal parasites but no activity against the fungal cultivar. Attinimicin showed potent in vivo activity in a mouse Candida albicans infection model comparable to clinically used azole-containing antifungals. In situ detection of attinimicin in both ant nests and on worker ants supports an ecological role for attinimicin in protecting the fungal cultivar from pathogens. The geographic spread of the attinimicin biosynthetic gene cluster in Brazilian Pseudonocardia spp. marks attinimicin as the first specialized metabolite from ant-associated bacteria with broad geographic distribution.

Thiopeptide Defense by an Ant's Bacterial Symbiont.

Fungus-growing ants and their microbial symbionts have emerged as a model system for understanding antibiotic deployment in an ecological context. Here we establish that bacterial symbionts of the ant Trachymyrmex septentrionalis antagonize their most likely competitors, other strains of ant-associated bacteria, using the thiopeptide antibiotic GE37468. Genomic analysis suggests that these symbionts acquired the GE37468 gene cluster from soil bacteria. This antibiotic, with known activity against human pathogens, was previously identified in a biochemical screen but had no known ecological role. GE37468's host-associated defense role in this insect niche intriguingly parallels the function of similar thiopeptides in the human microbiome.

Defense contracts: molecular protection in insect-microbe symbioses.

Insects cope with environmental threats using a broad array of strategies. A key strategy, widespread among insects but unappreciated until recently, is the use of molecular defenses from symbiotic microbes. Insect-microbe defensive symbioses span the diversity of insect lineages and microbial partners and use molecules ranging from reactive oxygen species to small molecules to protein toxins to defend against predators, parasites, and microbial pathogens. These systems have a strong initial track record as sources of novel biologically active compounds with therapeutic potential. This review surveys the molecular basis for insect-microbe defensive symbioses with a focus on the ecological contexts for defense and on emerging lessons about molecular diversity from bacterial genomes.

Selvamicin, an atypical antifungal polyene from two alternative genomic contexts.

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 A1 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.

A Rebeccamycin Analog Provides Plasmid-Encoded Niche Defense.

Bacterial symbionts of fungus-growing ants occupy a highly specialized ecological niche and face the constant existential threat of displacement by another strain of ant-adapted bacteria. As part of a systematic study of the small molecules underlying this fraternal competition, we discovered an analog of the antitumor agent rebeccamycin, a member of the increasingly important indolocarbazole family. While several gene clusters consistent with this molecule's newly reported modification had previously been identified in metagenomic studies, the metabolite itself has been cryptic. The biosynthetic gene cluster for 9-methoxyrebeccamycin is encoded on a plasmid in a manner reminiscent of plasmid-derived peptide antimicrobials that commonly mediate antagonism among closely related Gram-negative bacteria.

Variable genetic architectures produce virtually identical molecules in bacterial symbionts of fungus-growing ants.

Small molecules produced by Actinobacteria have played a prominent role in both drug discovery and organic chemistry. As part of a larger study of the actinobacterial symbionts of fungus-growing ants, we discovered a small family of three previously unreported piperazic acid-containing cyclic depsipeptides, gerumycins A-C. The gerumycins are slightly smaller versions of dentigerumycin, a cyclic depsipeptide that selectively inhibits a common fungal pathogen, Escovopsis. We had previously identified this molecule from a Pseudonocardia associated with Apterostigma dentigerum, and now we report the molecule from an associate of the more highly derived ant Trachymyrmex cornetzi. The three previously unidentified compounds, gerumycins A-C, have essentially identical structures and were produced by two different symbiotic Pseudonocardia spp. from ants in the genus Apterostigma found in both Panama and Costa Rica. To understand the similarities and differences in the biosynthetic pathways that produced these closely related molecules, the genomes of the three producing Pseudonocardia were sequenced and the biosynthetic gene clusters identified. This analysis revealed that dramatically different biosynthetic architectures, including genomic islands, a plasmid, and the use of spatially separated genetic loci, can lead to molecules with virtually identical core structures. A plausible evolutionary model that unifies these disparate architectures is presented.

In vivo incorporation of non-canonical amino acids by using the chemical aminoacylation strategy: a broadly applicable mechanistic tool.

We describe a strategy for incorporating non-canonical amino acids site-specifically into proteins expressed in living cells, involving organic synthesis to chemically aminoacylate a suppressor tRNA, protein expression in Xenopus oocytes, and monitoring protein function, primarily by electrophysiology. With this protocol, a very wide range of non-canonical amino acids can be employed, allowing both systematic structure-function studies and the incorporation of reactive functionalities. Here, we present an overview of the methodology and examples meant to illustrate the versatility and power of the method as a tool for investigating protein structure and function.

Functional probes of drug-receptor interactions implicated by structural studies: Cys-loop receptors provide a fertile testing ground.

Structures of integral membrane receptors provide valuable models for drug-receptor interactions across many important classes of drug targets and have become much more widely available in recent years. However, it remains to be determined to what extent these images are relevant to human receptors in their biological context and how subtle issues such as subtype selectivity can be informed by them. The high precision structural modifications enabled by unnatural amino acid mutagenesis on mammalian receptors expressed in vertebrate cells allow detailed tests of predictions from structural studies. Using the Cys-loop superfamily of ligand-gated ion channels, we show that functional studies lead to detailed binding models that, at times, are significantly at odds with the structural studies on related invertebrate proteins. Importantly, broad variations in binding interactions are seen for very closely related receptor subtypes and for varying drugs at a given binding site. These studies highlight the essential interplay between structural studies and functional studies that can guide efforts to develop new pharmaceuticals.

An unusual pattern of ligand-receptor interactions for the α7 nicotinic acetylcholine receptor, with implications for the binding of varenicline.

The α7 nicotinic acetylcholine receptor shows broad pharmacology, complicating the development of subtype-specific nicotinic receptor agonists. Here we use unnatural amino acid mutagenesis to characterize binding to α7 by the smoking cessation drug varenicline (Chantix; Pfizer, Groton, CT), an α4ß2-targeted agonist that shows full efficacy and modest potency at the α7 receptor. We find that unlike binding to its target receptor, varenicline does not form a cation-π interaction with TrpB, further supporting a unique binding mode for the cationic amine of nicotinic agonists at the α7 receptor. We also evaluate binding to the complementary face of the receptor's binding site by varenicline, the endogenous agonist acetylcholine, and the potent nicotine analog epibatidine. Interestingly, we find no evidence for functionally important interactions involving backbone NH and CO groups thought to bind the canonical agonist hydrogen bond acceptor of the nicotinic pharmacophore, perhaps reflecting a lesser importance of this pharmacophore element for α7 binding. We also show that the Trp55 and Leu119 side chains of the binding site's complementary face are important for the binding of the larger agonists epibatidine and varenicline, but dispensable for binding of the smaller, endogenous agonist acetylcholine.

Binding interactions with the complementary subunit of nicotinic receptors.

The agonist-binding site of nicotinic acetylcholine receptors (nAChRs) spans an interface between two subunits of the pentameric receptor. The principal component of this binding site is contributed by an α subunit, and it binds the cationic moiety of the nicotinic pharmacophore. The other part of the pharmacophore, a hydrogen bond acceptor, has recently been shown to bind to the complementary non-α subunit via the backbone NH of a conserved Leu. This interaction was predicted by studies of ACh-binding proteins and confirmed by functional studies of the neuronal (CNS) nAChR, α4ß2. The ACh-binding protein structures further suggested that the hydrogen bond to the backbone NH is mediated by a water molecule and that a second hydrogen bonding interaction occurs between the water molecule and the backbone CO of a conserved Asn, also on the non-α subunit. Here, we provide new insights into the nature of the interactions between the hydrogen bond acceptor of nicotinic agonists and the complementary subunit backbone. We studied both the nAChR of the neuromuscular junction (muscle-type) and a neuronal subtype, (α4)2(ß4)3. In the muscle-type receptor, both ACh and nicotine showed a strong interaction with the Leu NH, but the potent nicotine analog epibatidine did not. This interaction was much attenuated in the α4ß4 receptor. Surprisingly, we found no evidence for a functionally significant interaction with the backbone carbonyl of the relevant Asn in either receptor with an array of agonists.

Dissecting the functions of conserved prolines within transmembrane helices of the D2 dopamine receptor.

G protein-coupled receptors (GPCRs) contain a number of conserved proline residues in their transmembrane helices, and it is generally assumed these play important functional and/or structural roles. Here we use unnatural amino acid mutagenesis, employing α-hydroxy acids and proline analogues, to examine the functional roles of five proline residues in the transmembrane helices of the D2 dopamine receptor. The well-known tendency of proline to disrupt helical structure is important at all sites, while we find no evidence for a functional role for backbone amide cis-trans isomerization, another feature associated with proline. At most proline sites, the loss of the backbone NH is sufficient to explain the role of the proline. However, at one site, P210(5.50), a substituent on the backbone N appears to be essential for proper function. Interestingly, the pattern in functional consequences that we see is mirrored in the pattern of structural distortions seen in recent GPCR crystal structures.