The Microbiota of Moon Snail Egg Collars is Shaped by Host-Specific Factors.

Moon Snails lay eggs using a mixture of mucus and sediment to form an egg mass commonly referred to as an egg collar. These collars do not appear to experience micro-biofouling or predation and this observation led us to hypothesize that the egg collars possess a chemically-rich microbiota that protect the egg collars from pathogens. Herein, we sought to gain an understanding of the bacterial composition of the egg collars by amplifying and sequencing the 16S rRNA gene from egg collar and sediment samples collected at four distinct geographical regions in SW Florida. Relative abundance and non-metric multidimensional scaling plots revealed distinct differences in the bacterial composition between the egg collar and sediment samples. In addition, the egg collars had a lower α-diversity than the sediment, with specific genera being significantly enriched in the egg collars. Analysis of microorganisms consistent across two seasons suggests that Flavobacteriaceae make up a large portion of the core microbiota (36 - 58% of 16S sequences). We also investigated the natural product potential of the egg collar microbiota by sequencing a core biosynthetic gene, the adenylation domains (AD), within the gene clusters of non-ribosomal peptide synthetase (NRPS). AD sequences matched multiple modules within known bioactive NRPs biosynthetic gene clusters, suggesting production is possible within the egg collar system and lays the foundation for future studies into the chemical and ecological role of this microbiota. IMPORTANCE: Animals commonly partner with microorganisms to accomplish essential tasks, including chemically defending the animal host from predation and/or infections. Understanding animal-microbe partnerships and the molecules used by the microbe to defend the animals from pathogens or predation have the potential to lead to new pharmaceutical agents. However, very few of these systems have been investigated. A particularly interesting system are nutrient rich marine egg collars, which often lack visible protections, and are hypothesized to harbor beneficial microbes that protect the eggs. In this study, we gained an understanding of the bacterial strains that form the core microbiota of Moon Snail egg collars and gained a preliminary understanding of their natural product potential. This work lays the foundation for future work to understand the ecological role of the core microbiome and to study the molecules involved in chemically defending the Moon Snail eggs.

The Chemistry of the Defensive Secretions of Three Species of Millipedes in the Genus Brachycybe.

Millipedes have long been known to produce a diverse array of chemical defense agents that deter predation. These compounds, or their precursors, are stored in high concentration within glands (ozadenes) and are released upon disturbance. The subterclass Colobognatha contains four orders of millipedes, all of which are known to produce terpenoid alkaloids-spare the Siphonophorida that produce terpenes. Although these compounds represent some of the most structurally-intriguing millipede-derived natural products, they are the least studied class of millipede defensive secretions. Here, we describe the chemistry of millipede defensive secretions from three species of Brachycybe: Brachycybe producta, Brachycybe petasata, and Brachycybe rosea. Chemical investigations using mass spectrometry-based metabolomics, chemical synthesis, and 2D NMR led to the identification of five alkaloids, three of which are new to the literature. All identified compounds are monoterpene alkaloids with the new compounds representing indolizidine (i.e. hydrogosodesmine) and quinolizidine alkaloids (i.e. homogosodesmine and homo-hydrogosodesmine). The chemical diversity of these compounds tracks the known species phylogeny of this genus, rather than the geographical proximity of the species. The indolizidines and quinolizidines are produced by non-sympatric sister species, B. producta and B. petasata, while deoxybuzonamine is produced by another set of non-sympatric sister species, B. rosea and Brachycybe lecontii. The fidelity between the chemical diversity and phylogeny strongly suggests that millipedes generate these complex defensive agents de novo and begins to provide insights into the evolution of their biochemical pathways.

Discovery of Biofilm Inhibitors from the Microbiota of Marine Egg Masses.

Biofilms commonly develop in immunocompromised patients, which leads to persistent infections that are difficult to treat. In the biofilm state, bacteria are protected against both antibiotics and the host's immune system; currently, there are no therapeutics that target biofilms. In this study, we screened a chemical fraction library representing the natural product capacity of the microbiota of marine egg masses, namely, the moon snail egg collars. This led to the identification of active fractions targeting both Pseudomonas aeruginosa and Staphylococcus aureus biofilms. Subsequent analysis revealed that a subset of these fractions were capable of eradicating preformed biofilms, all against S. aureus. Bioassay-guided isolation led us to identify pseudochelin A, a known siderophore, as a S. aureus biofilm inhibitor with an IC50 of 88.5 µM. Mass spectrometry-based metabolomic analyses revealed widespread production of pseudochelin A among fractions possessing S. aureus antibiofilm properties. In addition, a key biosynthetic gene involved in producing pseudochelin A was detected on 30% of the moon snail egg collars and pseudochelin A is capable of inhibiting the formation of biofilms (IC50 50.6 µM) produced by ecologically relevant bacterial strains. We propose that pseudochelin A may have a role in shaping the microbiome or protecting the egg collars from microbiofouling.

Design, Synthesis, and Antifungal Activity of 3-Substituted-2(5H)-Oxaboroles.

Next generation antimicrobial therapeutics are desperately needed as new pathogens with multiple resistance mechanisms continually emerge. Two oxaboroles, tavaborole and crisaborole, were recently approved as topical treatments for onychomycosis and atopic dermatitis, respectively, warranting further studies into this privileged structural class. Herein, we report the antimicrobial properties of 3-substituted-2(5H)-oxaboroles, an unstudied family of medicinally relevant oxaboroles. Our results revealed minimum inhibitory concentrations as low as 6.25 and 5.20 µg/mL against fungal (e.g., Penicillium chrysogenum) and yeast (Saccharomyces cerevisiae) pathogens, respectively. These oxaboroles were nonhemolytic and nontoxic to rat myoblast cells (H9c2). Structure-activity relationship studies suggest that planarity is important for antimicrobial activity, possibly due to the effects of extended conjugation between the oxaborole and benzene rings.

Lactiplantibacillus plantarum uses ecologically relevant, exogenous quinones for extracellular electron transfer.

IMPORTANCE: While quinones are essential for respiratory microorganisms, their importance for microbes that rely on fermentation metabolism is not understood. This gap in knowledge hinders our understanding of anaerobic microbial habitats, such in mammalian digestive tracts and fermented foods. We show that Lactiplantibacillus plantarum, a model fermentative lactic acid bacteria species abundant in human, animal, and insect microbiomes and fermented foods, uses multiple exogenous, environmental quinones as electron shuttles for a hybrid metabolism involving EET. Interestingly, quinones both stimulate this metabolism as well as cause oxidative stress when extracellular electron acceptors are absent. We also found that quinone-producing, lactic acid bacteria species commonly enriched together with L. plantarum in food fermentations accelerate L. plantarum growth and medium acidification through a mainly quinone- and EET-dependent mechanism. Thus, our work provides evidence of quinone cross-feeding as a key ecological feature of anaerobic microbial habitats.

Semisynthetic blasticidin S ester derivatives show enhanced antibiotic activity.

A rich potential source of new antibiotics are undeveloped natural product cytotoxins, provided they can be derivatized to restrict their activity to bacteria. In this work, we describe modification of one such candidate, the broad-spectrum, translation termination inhibitor, blasticidin S. By semisynthetically modifying blasticidin S, we produced a series of ester derivatives of this highly polar, zwitterionic compound in a single step. These derivatives showed a marked increase in activity against Gram-positive bacteria and an increase in selectivity index for pathogenic bacteria over human cells. The results of this study suggest that semisynthetic derivatization of blasticidin S and other neglected natural product antimicrobials has the potential to increase their activity against and selectivity for bacteria, an approach that can be leveraged for the development of leads against antimicrobial resistant pathogens.

Deoxybuzonamine Isomers from the Millipede Brachycybe lecontii (Platydesmida: Andrognathidae).

Millipedes (Diplopoda) are well known for their toxic or repellent defensive secretions. Here, we describe (6aR,10aS,10bR)-8,8-dimethyldodecahydropyrrolo[2,1-a]isoquinoline [trans-anti-trans-deoxybuzonamine (1a)] and (rel-6aR,10aR,10bR)-8,8-dimethyldodecahydropyrrolo[2,1-a]isoquinoline [trans-syn-cis-deoxybuzonamine (1b)], two isomers of deoxybuzonamine found in the chemical defense secretions of the millipede Brachycybe lecontii Wood (Colobognatha, Platydesmida, Andrognathidae). The carbon-nitrogen skeleton of these compounds was determined from their MS and GC-FTIR spectra obtained from the MeOH extract of whole millipedes, along with a subsequent selective synthesis. Their structures were established from their 1D (1H, 13C) and 2D NMR (COSY, NOESY, multiplicity-edited HSQC, HSQC-TOCSY, HMBC) spectra. Additionally, computational chemistry (DFT and DP4) was used to identify the relative configurations of 1a and 1b by comparing predicted 13C data to their experimental values, and the absolute configuration of 1a was determined by comparing its experimental specific rotation with that of the computationally calculated value. This is the first report of dodecahydropyrrolo[2,1-a]isoquinoline alkaloids from a platydesmidan millipede.

Luquilloamides, Cytotoxic Lipopeptides from a Puerto Rican Collection of the Filamentous Marine Cyanobacterium Oscillatoria sp.

Luquilloamides A-G (1-7) were isolated from a small environmental collection of a marine cyanobacterium found growing on eelgrass (Zostera sp.) near Luquillo, Puerto Rico. Structure elucidation of the luquilloamides was accomplished via detailed NMR and MS analyses, and absolute configurations were determined using a combination of advanced Mosher's method, J-based configuration analysis, semisynthetic fragment analysis derived from ozonolysis, methylation, Baeyer-Villiger oxidation, Mosher's esterification, specific rotations, and ECD data. Except for 2, the luquilloamides share a characteristic tert-butyl-containing polyketide fragment, ß-alanine, and a proposed highly modified polyketide extension. While compound 1 is a linear lipopeptide with two α-methyl branches and a vinyl chloride functionality in the polyketide portion, compounds 4, 6, and 7 possess a cyclohexanone structure with methylation on the α- or ß-positions of the polyketide as well as an acetyl group. Interestingly, the absolute configuration at C-5 and C-6 on the cyclohexanone unit in 7 is opposite to that of 4-6. Compound 3 was revealed to have a tert-butyl-containing polyketide, ß-alanine, and a PKS/NRPS-derived γ-isopropyl pyrrolinone. Compound 2 may be a hydrolysis product of 3. Of the seven new compounds, 1 showed the most potent cytotoxicity to human H-460 lung cancer cells.

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.

Bacterial diketopiperazines stimulate diatom growth and lipid accumulation.

Diatoms are photosynthetic microalgae that fix a significant fraction of the world's carbon. Because of their photosynthetic efficiency and high-lipid content, diatoms are priority candidates for biofuel production. Here, we report that sporulating Bacillus thuringiensis and other members of the Bacillus cereus group, when in co-culture with the marine diatom Phaeodactylum tricornutum, significantly increase diatom cell count. Bioassay-guided purification of the mother cell lysate of B. thuringiensis led to the identification of two diketopiperazines (DKPs) that stimulate both P. tricornutum growth and increase its lipid content. These findings may be exploited to enhance P. tricornutum growth and microalgae-based biofuel production. As increasing numbers of DKPs are isolated from marine microbes, the work gives potential clues to bacterial-produced growth factors for marine microalgae.

Chemical Exchanges between Multilateral Symbionts.

Herein is a report on the molecular exchange occurring between multilateral symbiosis partners-a tit-for-tat exchange that led to the characterization of two new metabolites, conocandin B (fungal-derived) and dentigerumycin F (bacterial-derived). The structures were determined by NMR, mass spectrometry, genomic analysis, and chemical derivatizations. Conocandin B exhibits antimicrobial activity against both the bacterial symbionts of fungus-growing ant and human pathogenic strains by selectively inhibiting FabH, thus disrupting fatty acid biosynthesis.

Research Tales from the Clardy Laboratory: Function-Driven Natural Product Discovery.

Over the past 70 years, the search for small molecules from nature has transformed biomedical research: natural products are the basis for half of all pharmaceuticals; the quest for total synthesis of natural products fueled development of methodologies for organic synthesis; and their biosynthesis presented unprecedented biochemical transformations, expanding our chemo-enzymatic toolkit. Initially, the discovery of small molecules was driven by bioactivity-guided fractionation. However, this approach yielded the frequent rediscovery of already known metabolites. As a result, focus shifted to identifying novel scaffolds through either structure-first methods or genome mining, relegating function as a secondary concern. Over the past two decades, the laboratory of Jon Clardy has taken an alternative route and focused on an ecology-driven, function-first approach in pursuit of uncovering bacterial small molecules with biological activity. In this review, we highlight several examples that showcase this ecology-first approach. Though the highlighted systems are diverse, unifying themes are (1) to understand how microbes interact with their host or environment, (2) to gain insights into the environmental roles of microbial metabolites, and (3) to explore pharmaceutical potential from these ecologically relevant metabolites.

Pyonitrins A-D: Chimeric Natural Products Produced by Pseudomonas protegens.

Bacterial symbionts frequently provide chemical defenses for their hosts, and such systems can provide discovery pathways to new antifungals and structurally intriguing metabolites. This report describes a small family of naturally occurring small molecules with chimeric structures and a mixed biosynthesis that features an unexpected but key nonenzymatic step. An insect-associated Pseudomonas protegens strain's activity in an in vivo murine candidiasis assay led to the discovery of a family of highly hydrogen-deficient metabolites. Bioactivity- and mass-guided fractionation led to the pyonitrins, highly complex aromatic metabolites in which 10 of the 20 carbons are quaternary, and 7 of them are contiguous. The P. protegens genome revealed that the production of the pyonitrins is the result of a spontaneous reaction between biosynthetic intermediates of two well-studied Pseudomonas metabolites, pyochelin and pyrrolnitrin. The combined discovery of the pyonitrins and identification of the responsible biosynthetic gene clusters revealed an unexpected biosynthetic route that would have prevented the discovery of these metabolites by bioinformatic analysis alone.

An elusive electron shuttle from a facultative anaerobe.

Some anaerobic bacteria use insoluble minerals as terminal electron acceptors and discovering the ways in which electrons move through the membrane barrier to the exterior acceptor forms an active field of research with implications for both bacterial physiology and bioenergy. A previous study suggested that Shewanella oneidensis MR-1 utilizes a small, polar, redox active molecule that serves as an electron shuttle between the bacteria and insoluble acceptors, but the shuttle itself has never been identified. Through isolation and synthesis, we identify it as ACNQ (2-amino-3-carboxy-1,4-naphthoquinone), a soluble analog of menaquinone. ACNQ is derived from DHNA (1,4-dihydroxy-2-naphthoic acid) in a non-enzymatic process that frustrated genetic approaches to identify the shuttle. Both ACNQ and DHNA restore reduction of AQDS under anaerobic growth in menaquinone-deficient mutants. Bioelectrochemistry analyses reveal that ACNQ (-0.32 VAg/AgCl) contributes to the extracellular electron transfer (EET) as an electron shuttle, without altering menaquinone generation or EET related cytochrome c expression.

A high-throughput, whole cell assay to identify compounds active against carbapenem-resistant Klebsiella pneumoniae.

Enteric Gram-negative rods (GNR), which are frequent causes of community-acquired and nosocomial infections, are increasingly resistant to the antibiotics in our current armamentarium. One solution to this medical dilemma is the development of novel classes of antimicrobial compounds. Here we report the development of a robust, whole cell-based, high-throughput metabolic assay that detects compounds with activity against carbapenem-resistant Klebsiella pneumoniae. We have used this assay to screen approximately 8,000 fungal extracts and 50,000 synthetic compounds with the goal of identifying extracts and compounds active against a highly resistant strain of Klebsiella pneumoniae. The primary screen identified 43 active fungal extracts and 144 active synthetic compounds. Patulin, a known fungal metabolite and inhibitor of bacterial quorum sensing and alanine racemase, was identified as the active component in the most potent fungal extracts. We did not study patulin further due to previously published evidence of toxicity. Three synthetic compounds termed O06, C17, and N08 were chosen for further study. Compound O06 did not have significant antibacterial activity but rather interfered with sugar metabolism, while compound C17 had only moderate activity against GNRs. Compound N08 was active against several resistant GNRs and showed minimal toxicity to mammalian cells. Preliminary studies suggested that it interferes with protein expression. However, its direct application may be limited by susceptibility to efflux and a tendency to form aggregates in aqueous media. Rapid screening of 58,000 test samples with identification of several compounds that act on CR-K. pneumoniae demonstrates the utility of this screen for the discovery of drugs active against this highly resistant GNR.

Amycomicin is a potent and specific antibiotic discovered with a targeted interaction screen.

The rapid emergence of antibiotic-resistant pathogenic bacteria has accelerated the search for new antibiotics. Many clinically used antibacterials were discovered through culturing a single microbial species under nutrient-rich conditions, but in the environment, bacteria constantly encounter poor nutrient conditions and interact with neighboring microbial species. In an effort to recapitulate this environment, we generated a nine-strain actinomycete community and used 16S rDNA sequencing to deconvolute the stochastic production of antimicrobial activity that was not observed from any of the axenic cultures. We subsequently simplified the community to just two strains and identified Amycolatopsis sp. AA4 as the producing strain and Streptomyces coelicolor M145 as an inducing strain. Bioassay-guided isolation identified amycomicin (AMY), a highly modified fatty acid containing an epoxide isonitrile warhead as a potent and specific inhibitor of Staphylococcus aureus Amycomicin targets an essential enzyme (FabH) in fatty acid biosynthesis and reduces S. aureus infection in a mouse skin-infection model. The discovery of AMY demonstrates the utility of screening complex communities against specific targets to discover small-molecule antibiotics.

A plant-responsive bacterial-signaling system senses an ethanolamine derivative.

Certain plant-associated Proteobacteria sense their host environment by detecting an unknown plant signal recognized by a member of a LuxR subfamily of transcription factors. This interkingdom communication is important for both mutualistic and pathogenic interactions. The Populus root endophyte Pseudomonas sp. GM79 possesses such a regulator, named PipR. In a previous study we reported that PipR activates an adjacent gene (pipA) coding for a proline iminopeptidase in response to Populus leaf macerates and peptides and that this activation is dependent on a putative ABC-type transporter [Schaefer AL, et al. (2016) mBio 7:e01101-16]. In this study we identify a chemical derived from ethanolamine that induces PipR activity at picomolar concentrations, and we present evidence that this is the active inducer present in plant leaf macerates. First, a screen of more than 750 compounds indicated ethanolamine was a potent inducer for the PipR-sensing system; however, ethanolamine failed to bind to the periplasmic-binding protein (PBP) required for the signal response. This led us to discover that a specific ethanolamine derivative, N-(2-hydroxyethyl)-2-(2-hydroxyethylamino) acetamide (HEHEAA), binds to the PBP and serves as a potent PipR-dependent inducer. We also show that a compound, which coelutes with HEHEAA in HPLC and induces pipA gene expression in a PipR-dependent manner, can be found in Populus leaf macerates. This work sheds light on how plant-associated bacteria can sense their environment and on the nature of inducers for a family of plant-responsive LuxR-like transcription factors found in plant-associated bacteria.

Discovery of (Dihydro)pyrazine N-Oxides via Genome Mining in Pseudomonas.

Overexpression of the Pseudomonas virulence factor ( pvf) biosynthetic operon led to the identification of a family of pyrazine N-oxides (PNOs), including a novel dihydropyrazine N,N'-dioxide (dPNO) metabolite. The nonribosomal peptide synthetase responsible for production of (d)PNOs was characterized, and a biosynthetic pathway for (d)PNOs was proposed. This work highlights the unique chemistry catalyzed by pvf-encoded enzymes and sets the stage for bioactivity studies of the metabolites produced by the virulence pathway.

Identification of Potent Ebola Virus Entry Inhibitors with Suitable Properties for in Vivo Studies.

Previous studies identified an adamantane dipeptide piperazine 3.47 that inhibits Ebola virus (EBOV) infection by targeting the essential receptor Niemann-Pick C1 (NPC1). The physicochemical properties of 3.47 limit its potential for testing in vivo. Optimization by improving potency, reducing hydrophobicity, and replacing labile moieties identified 3.47 derivatives with improved in vitro ADME properties that are also highly active against EBOV infection, including when tested in the presence of 50% normal human serum (NHS). In addition, 3A4 was identified as the major cytochrome P450 isoform that metabolizes these compounds, and accordingly, mouse microsome stability was significantly improved when tested in the presence of the CYP3A4 inhibitor ritonavir that is approved for clinical use as a booster of anti-HIV drugs. Oral administration of the EBOV inhibitors with ritonavir resulted in a pharmacokinetic profile that supports a b.i.d. dosing regimen for efficacy studies in mice.

Oridonin inhibits aberrant AKT activation in breast cancer.

Aberrant activation of phosphatidylinosito-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/AKT) signaling in cancer has led to pursuit of inhibitors for targeting this pathway. However, inhibitors of PI3K and AKT have failed to yield efficacious results without adverse effects. Here, we screened a library containing 441 authenticated traditional chinese medicine (TCM) plant extracts by examining their effect on cell viability of a human mammary epithelial cell line HMEC-PIK3CAH1047R, which expresses mutant PIK3CAH1047R and has constitutively active AKT signaling. We found that Oridonin, an extract from Rabdosia rubescens, reduced cell viability to the greatest extent. Oridonin binds to AKT1 and potentially functions as an ATP-competitive AKT inhibitor. Importantly, Oridonin selectively impaired tumor growth of human breast cancer cells with hyperactivation of PI3K/AKT signaling. Moreover, Oridonin prevented the initiation of mouse mammary tumors driven by PIK3CAH1047R. Our results suggest that Oridonin may serve as a potent and durable therapeutic agent for the treatment of breast cancers with hyperactivation of PI3K/AKT signaling.