N-Acyl Amides from Neisseria meningitidis and Their Role in Sphingosine Receptor Signaling.

Neisseria meningitidis is a Gram-negative opportunistic pathogen that is responsible for causing human diseases with high mortality, such as septicemia and meningitis. The molecular mechanisms N. meningitidis employ to manipulate the immune system, translocate the mucosal and blood-brain barriers, and exert virulence are largely unknown. Human-associated bacteria encode a variety of bioactive small molecules with growing evidence for N-acyl amides as being important signaling molecules. However, only a small fraction of these metabolites has been identified from the human microbiota thus far. Here, we heterologously expressed an N-acyltransferase encoded in the obligate human pathogen N. meningitidis and identified 30 N-acyl amides with representative members serving as agonists of the G-protein coupled receptor (GPCR) S1PR4. During this process, we also characterized two mammalian N-acyl amides derived from the bovine medium. Both groups of metabolites suppress anti-inflammatory interleukin-10 signaling in human macrophage cell types, but they also suppress the pro-inflammatory interleukin-17A+ population in TH 17-differentiated CD4+ T cells.

Algal p-coumaric acid induces oxidative stress and siderophore biosynthesis in the bacterial symbiont Phaeobacter inhibens.

The marine alpha-proteobacterium Phaeobacter inhibens engages in intermittent symbioses with microalgae. The symbiosis is biphasic and concludes in a parasitic phase, during which the bacteria release algaecidal metabolites in response to algal p-coumaric acid (pCA). The cell-wide effects of pCA on P. inhibens remain unknown. Herein, we report a microarray-based transcriptomic study and find that genes related to the oxidative stress response and secondary metabolism are upregulated most, while those associated with energy production and motility are downregulated in the presence of pCA. Among genes upregulated is a previously unannotated biosynthetic gene cluster and, using a combination of gene deletions and metabolic profiling, we show that it gives rise to an unreported siderophore, roseobactin. The simultaneous production of algaecides and roseobactin in the parasitic phase allows the bacteria to take up any iron that is released from dying algal cells, thereby securing a limited micronutrient.

Sulfamethoxazole drug stress upregulates antioxidant immunomodulatory metabolites in Escherichia coli.

Escherichia coli is an important model organism in microbiology and a prominent member of the human microbiota1. Environmental isolates readily colonize the gastrointestinal tract of humans and other animals, and they can serve diverse probiotic, commensal and pathogenic roles in the host2-4. Although certain strains have been associated with the severity of inflammatory bowel disease (IBD)2,5, the diverse immunomodulatory phenotypes remain largely unknown at the molecular level. Here, we decode a previously unknown E. coli metabolic pathway that produces a family of hybrid pterin-phenylpyruvate conjugates, which we named the colipterins. The metabolites are upregulated by subinhibitory levels of the antifolate sulfamethoxazole, which is used to treat infections including in patients with IBD6,7. The genes folX/M and aspC/tyrB involved in monapterin biosynthesis8-10 and aromatic amino acid transamination,11 respectively, were required to initiate the colipterin pathway. We show that the colipterins are antioxidants, harbour diverse immunological activities in primary human tissues, activate anti-inflammatory interleukin-10 and improve colitis symptoms in a colitis mouse model. Our study defines an antifolate stress response in E. coli and links its associated metabolites to a major immunological marker of IBD.

Cellular Stress Upregulates Indole Signaling Metabolites in Escherichia coli.

Escherichia coli broadly colonize the intestinal tract of humans and produce a variety of small molecule signals. However, many of these small molecules remain unknown. Here, we describe a family of widely distributed bacterial metabolites termed the "indolokines." In E. coli, the indolokines are upregulated in response to a redox stressor via aspC and tyrB transaminases. Although indolokine 1 represents a previously unreported metabolite, four of the indolokines (2-5) were previously shown to be derived from indole-3-carbonyl nitrile (ICN) in the plant pathogen defense response. We show that the indolokines are produced in a convergent evolutionary manner relative to plants, enhance E. coli persister cell formation, outperform ICN protection in an Arabidopsis thaliana-Pseudomonas syringae infection model, trigger a hallmark plant innate immune response, and activate distinct immunological responses in primary human tissues. Our molecular studies link a family of cellular stress-induced metabolites to defensive responses across bacteria, plants, and humans.

Characterization of Autoinducer-3 Structure and Biosynthesis in E. coli.

Escherichia coli is a common inhabitant of the human microbiota and a beacon model organism in biology. However, an understanding of its signaling systems that regulate population-level phenotypes known as quorum sensing remain incomplete. Here, we define the structure and biosynthesis of autoinducer-3 (AI-3), a metabolite of previously unknown structure involved in the pathogenesis of enterohemorrhagic E. coli (EHEC). We demonstrate that novel AI-3 analogs are derived from threonine dehydrogenase (Tdh) products and "abortive" tRNA synthetase reactions, and they are distributed across a variety of Gram-negative and Gram-positive bacterial pathogens. In addition to regulating virulence genes in EHEC, we show that the metabolites exert diverse immunological effects on primary human tissues. The discovery of AI-3 metabolites and their biochemical origins now provides a molecular foundation for investigating the diverse biological roles of these elusive yet widely distributed bacterial signaling molecules.

Bacterial Autoimmune Drug Metabolism Transforms an Immunomodulator into Structurally and Functionally Divergent Antibiotics.

Tapinarof is a stilbene drug that is used to treat psoriasis and atopic dermatitis, and is thought to function through regulation of the AhR and Nrf2 signaling pathways, which have also been linked to inflammatory bowel diseases. It is produced by the gammaproteobacterial Photorhabdus genus, which thus represents a model to probe tapinarof structural and functional transformations. We show that Photorhabdus transforms tapinarof into novel drug metabolism products that kill inflammatory bacteria, and that a cupin enzyme contributes to the conversion of tapinarof and related dietary stilbenes into novel dimers. One dimer has activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE), and another undergoes spontaneous cyclizations to a cyclopropane-bridge-containing hexacyclic framework that exhibits activity against Mycobacterium. These dimers lack efficacy in a colitis mouse model, whereas the monomer reduces disease symptoms.

Reporter-Guided Transposon Mutant Selection for Activation of Silent Gene Clusters in Burkholderia thailandensis.

Most natural product biosynthetic gene clusters that can be observed bioinformatically are silent. This insight has prompted the development of several methodologies for inducing their expression. One of the more recent methods, termed reporter-guided mutant selection (RGMS), entails creation of a library of mutants that is then screened for the desired phenotype via reporter gene expression. Herein, we apply a similar approach to Burkholderia thailandensis and, using transposon mutagenesis, mutagenize three strains, each carrying a fluorescent reporter in the malleilactone (mal), capistruin (cap), or an unidentified ribosomal peptide (tomm) gene cluster. We show that even a small library of <500 mutants can be used to induce expression of each cluster. We also explore the mechanism of activation and find that inhibition of pyrimidine biosynthesis is linked to the induction of the mal cluster. Both a transposon insertion into pyrF as well as small-molecule-mediated inhibition of PyrF trigger malleilactone biosynthesis. Our results pave the way toward the broad application of RGMS and related approaches to Burkholderia spp.

A deep learning genome-mining strategy for biosynthetic gene cluster prediction.

Natural products represent a rich reservoir of small molecule drug candidates utilized as antimicrobial drugs, anticancer therapies, and immunomodulatory agents. These molecules are microbial secondary metabolites synthesized by co-localized genes termed Biosynthetic Gene Clusters (BGCs). The increase in full microbial genomes and similar resources has led to development of BGC prediction algorithms, although their precision and ability to identify novel BGC classes could be improved. Here we present a deep learning strategy (DeepBGC) that offers reduced false positive rates in BGC identification and an improved ability to extrapolate and identify novel BGC classes compared to existing machine-learning tools. We supplemented this with random forest classifiers that accurately predicted BGC product classes and potential chemical activity. Application of DeepBGC to bacterial genomes uncovered previously undetectable putative BGCs that may code for natural products with novel biologic activities. The improved accuracy and classification ability of DeepBGC represents a major addition to in-silico BGC identification.

Roseochelin B, an Algaecidal Natural Product Synthesized by the Roseobacter Phaeobacter inhibens in Response to Algal Sinapic Acid.

The secondary metabolome of the representative Roseobacter, Phaeobacter inhibens, was examined in response to algal sinapic acid. In addition to roseobacticides, sinapic acid induced the production of two new natural products, roseochelin A and B, which were characterized by NMR and X-ray crystallography. Functional assays showed that roseochelin B binds iron and is algaecidal against the algal host Emiliania huxleyi. It appears to be produced by a rarely observed combination of nonenzymatic and enzymatic transformations.

Investigation of the Genetics and Biochemistry of Roseobacticide Production in the Roseobacter Clade Bacterium Phaeobacter inhibens.

Roseobacterclade bacteria are abundant in surface waters and are among the most metabolically diverse and ecologically significant species. This group includes opportunistic symbionts that associate with micro- and macroalgae. We have proposed that one representative member,Phaeobacter inhibens, engages in a dynamic symbiosis with the microalgaEmiliania huxleyi In one phase, mutualistically beneficial molecules are exchanged, including theRoseobacter-produced antibiotic tropodithietic acid (TDA), which is thought to protect the symbiotic interaction. In an alternative parasitic phase, triggered by algal senescence, the bacteria produce potent algaecides, the roseobacticides, which kill the algal host. Here, we employed genetic and biochemical screens to identify the roseobacticide biosynthetic gene cluster. By using a transposon mutagenesis approach, we found that genes required for TDA synthesis-thetdaoperon andpaacatabolon-are also necessary for roseobacticide production. Thus, in contrast to the one-cluster-one-compound paradigm, thetdagene cluster can generate two sets of molecules with distinct structures and bioactivities. We further show that roseobacticide production is quorum sensing regulated via anN-acyl homoserine lactone signal (3-OH-C10-HSL). To ensure tight regulation of algaecide production, and thus of a lifestyle switch from mutualism to parasitism, roseobacticide biosynthesis necessitates the presence of both an algal senescence molecule and a quorum sensing signal.IMPORTANCEMarineRoseobacterspecies are abundant in the oceans and engage in symbiotic interactions with microscopic algae. One member,P. inhibens, produces the antibiotic TDA and a growth hormone thought to protect and promote algal growth. However, in the presence of molecules released by senescing algae, the bacteria produce potent algaecides, the roseobacticides, which kill the host. We examined the regulatory networks and biosynthetic genes required for roseobacticide production. We found thatP. inhibensuses largely the same set of genes for production of both TDA and roseobacticides, thus providing a rare case in which one gene cluster synthesizes two structurally and functionally distinct molecules. Moreover, we found roseobacticide production to be regulated by quorum sensing. Thus, two small molecules, the algal metabolite and the quorum-sensing signal, ensure tight control in the production of roseobacticides. These results highlight the role of small molecules in regulating microbial symbioses.

Mode of action and resistance studies unveil new roles for tropodithietic acid as an anticancer agent and the γ-glutamyl cycle as a proton sink.

While we have come to appreciate the architectural complexity of microbially synthesized secondary metabolites, far less attention has been paid to linking their structural features with possible modes of action. This is certainly the case with tropodithietic acid (TDA), a broad-spectrum antibiotic generated by marine bacteria that engage in dynamic symbioses with microscopic algae. TDA promotes algal health by killing unwanted marine pathogens; however, its mode of action (MoA) and significance for the survival of an algal-bacterial miniecosystem remains unknown. Using cytological profiling, we herein determine the MoA of TDA and surprisingly find that it acts by a mechanism similar to polyether antibiotics, which are structurally highly divergent. We show that like polyether drugs, TDA collapses the proton motive force by a proton antiport mechanism, in which extracellular protons are exchanged for cytoplasmic cations. The α-carboxy-tropone substructure is ideal for this purpose as the proton can be carried on the carboxyl group, whereas the basicity of the tropylium ion facilitates cation export. Based on similarities to polyether anticancer agents we have further examined TDA's cytotoxicity and find it to exhibit potent, broad-spectrum anticancer activities. These results highlight the power of MoA-profiling technologies in repurposing old drugs for new targets. In addition, we identify an operon that confers TDA resistance to the producing marine bacteria. Bioinformatic and biochemical analyses of these genes lead to a previously unknown metabolic link between TDA/acid resistance and the γ-glutamyl cycle. The implications of this resistance mechanism in the context of the algal-bacterial symbiosis are discussed.

Hybrid biosynthesis of roseobacticides from algal and bacterial precursor molecules.

Roseobacticides regulate the symbiotic relationship between a marine bacterium (Phaeobacter inhibens) and a marine microalga (Emiliania huxleyi). This relationship can be mutualistic, when the algal host provides food for the bacteria and the bacteria produce growth hormones and antibiotics for the algae, or parasitic, when the algae senesce and release p-coumaric acid. The released p-coumaric acid causes the bacteria to synthesize roseobacticides, which are nM-µM toxins for the algae. We examined the biosynthesis of roseobacticides and report that all roseobacticide precursors play critical roles during the mutualist phase of the symbiosis. Roseobacticides are biosynthesized from the algal growth promoter, the major food molecule provided by the algal cells, and the algal senescence signal that initiates the mutualist-to-parasite switch. Thus, molecules that are beneficial during mutualism are diverted to the synthesis of toxins during parasitism. A plausible mechanism for assembling roseobacticides from these molecules is proposed.

Profiling the dynamic interfaces of fluorinated transcription complexes for ligand discovery and characterization.

The conformationally dynamic binding surfaces of transcription complexes present a particular challenge for ligand discovery and characterization. In the case of the KIX domain of the master coactivator CBP/p300, few small molecules have been reported that target its two allosterically regulated binding sites despite the important roles that KIX plays in processes ranging from memory formation to hematopoiesis. Taking advantage of the enrichment of aromatic amino acids at protein interfaces, here we show that the incorporation of six (19)F-labeled aromatic side chains within the KIX domain enables recapitulation of the differential binding footprints of three natural activator peptides (MLL, c-Myb, and pKID) in complex with KIX and effectively reports on allosteric changes upon binding using 1D NMR spectroscopy. Additionally, the examination of both the previously described KIX protein-protein interaction inhibitor Napthol-ASE-phosphate and newly discovered ligand 1-10 rapidly revealed both the binding sites and the affinities of these small molecules. Significantly, the utility of using fluorinated transcription factors for ligand discovery was demonstrated through a fragment screen leading to a new low molecular weight fragment ligand for CBP/p300, 1G7. Aromatic amino acids are enriched at protein-biomolecule interfaces; therefore, this quantitative and facile approach will be broadly useful for studying dynamic transcription complexes and screening campaigns complementing existing biophysical methods for studying these dynamic interfaces.