Gut microbiome and metabolome profiling in Framingham heart study reveals cholesterol-metabolizing bacteria.

Accumulating evidence suggests that cardiovascular disease (CVD) is associated with an altered gut microbiome. Our understanding of the underlying mechanisms has been hindered by lack of matched multi-omic data with diagnostic biomarkers. To comprehensively profile gut microbiome contributions to CVD, we generated stool metagenomics and metabolomics from 1,429 Framingham Heart Study participants. We identified blood lipids and cardiovascular health measurements associated with microbiome and metabolome composition. Integrated analysis revealed microbial pathways implicated in CVD, including flavonoid, γ-butyrobetaine, and cholesterol metabolism. Species from the Oscillibacter genus were associated with decreased fecal and plasma cholesterol levels. Using functional prediction and in vitro characterization of multiple representative human gut Oscillibacter isolates, we uncovered conserved cholesterol-metabolizing capabilities, including glycosylation and dehydrogenation. These findings suggest that cholesterol metabolism is a broad property of phylogenetically diverse Oscillibacter spp., with potential benefits for lipid homeostasis and cardiovascular health.

Discovery of bioactive microbial gene products in inflammatory bowel disease.

Microbial communities and their associated bioactive compounds1-3 are often disrupted in conditions such as the inflammatory bowel diseases (IBD)4. However, even in well-characterized environments (for example, the human gastrointestinal tract), more than one-third of microbial proteins are uncharacterized and often expected to be bioactive5-7. Here we systematically identified more than 340,000 protein families as potentially bioactive with respect to gut inflammation during IBD, about half of which have not to our knowledge been functionally characterized previously on the basis of homology or experiment. To validate prioritized microbial proteins, we used a combination of metagenomics, metatranscriptomics and metaproteomics to provide evidence of bioactivity for a subset of proteins that are involved in host and microbial cell-cell communication in the microbiome; for example, proteins associated with adherence or invasion processes, and extracellular von Willebrand-like factors. Predictions from high-throughput data were validated using targeted experiments that revealed the differential immunogenicity of prioritized Enterobacteriaceae pilins and the contribution of homologues of von Willebrand factors to the formation of Bacteroides biofilms in a manner dependent on mucin levels. This methodology, which we term MetaWIBELE (workflow to identify novel bioactive elements in the microbiome), is generalizable to other environmental communities and human phenotypes. The prioritized results provide thousands of candidate microbial proteins that are likely to interact with the host immune system in IBD, thus expanding our understanding of potentially bioactive gene products in chronic disease states and offering a rational compendium of possible therapeutic compounds and targets.

Integrated annotation prioritizes metabolites with bioactivity in inflammatory bowel disease.

Microbial biochemistry is central to the pathophysiology of inflammatory bowel diseases (IBD). Improved knowledge of microbial metabolites and their immunomodulatory roles is thus necessary for diagnosis and management. Here, we systematically analyzed the chemical, ecological, and epidemiological properties of ~82k metabolic features in 546 Integrative Human Microbiome Project (iHMP/HMP2) metabolomes, using a newly developed methodology for bioactive compound prioritization from microbial communities. This suggested >1000 metabolic features as potentially bioactive in IBD and associated ~43% of prevalent, unannotated features with at least one well-characterized metabolite, thereby providing initial information for further characterization of a significant portion of the fecal metabolome. Prioritized features included known IBD-linked chemical families such as bile acids and short-chain fatty acids, and less-explored bilirubin, polyamine, and vitamin derivatives, and other microbial products. One of these, nicotinamide riboside, reduced colitis scores in DSS-treated mice. The method, MACARRoN, is generalizable with the potential to improve microbial community characterization and provide therapeutic candidates.

Absence of a pancreatic microbiome in intraductal papillary mucinous neoplasm.

OBJECTIVE: This study aims to validate the existence of a microbiome within intraductal papillary mucinous neoplasm (IPMN) that can be differentiated from the taxonomically diverse DNA background of next-generation sequencing procedures. DESIGN: We generated 16S rRNA amplicon sequencing data to analyse 338 cyst fluid samples from 190 patients and 19 negative controls, the latter collected directly from sterile syringes in the operating room. A subset of samples (n=20) and blanks (n=5) were spiked with known concentrations of bacterial cells alien to the human microbiome to infer absolute abundances of microbial traces. All cyst fluid samples were obtained intraoperatively and included IPMNs with various degrees of dysplasia as well as other cystic neoplasms. Follow-up culturing experiments were conducted to assess bacterial growth for microbiologically significant signals. RESULTS: Microbiome signatures of cyst fluid samples were inseparable from those of negative controls, with no difference in taxonomic diversity, and microbial community composition. In a patient subgroup that had recently undergone invasive procedures, a bacterial signal was evident. This outlier signal was not characterised by higher taxonomic diversity but by an increased dominance index of a gut-associated microbe, leading to lower taxonomic evenness compared with the background signal. CONCLUSION: The 'microbiome' of IPMNs and other pancreatic cystic neoplasms does not deviate from the background signature of negative controls, supporting the concept of a sterile environment. Outlier signals may appear in a small fraction of patients following recent invasive endoscopic procedures. No associations between microbial patterns and clinical or cyst parameters were apparent.

On the possible ecological roles of antimicrobials.

The Introduction of antibiotics into the clinical use in the middle of the 20th century had a profound impact on modern medicine and human wellbeing. The contribution of these wonder molecules to public health and science is hard to overestimate. Much research has informed our understanding of antibiotic mechanisms of action and resistance at inhibitory concentrations in the lab and in the clinic. Antibiotics, however, are not a human invention as most of them are either natural products produced by soil microorganisms or semisynthetic derivatives of natural products. Because we use antibiotics to inhibit the bacterial growth, it is generally assumed that growth inhibition is also their primary ecological function in the environment. Nevertheless, multiple studies point to diverse nonlethal effects that are exhibited at lower levels of antibiotics. Here we review accumulating evidence of antibiosis and of alternative functions of antibiotics exhibited at subinhibitory concentrations. We also speculate on how these effects might alter phenotypes, fitness, and community composition of microbes in the context of the environment and suggest directions for future research.

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.

IsdB-dependent hemoglobin binding is required for acquisition of heme by Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive pathogen responsible for tremendous morbidity and mortality. As with most bacteria, S. aureus requires iron to cause disease, and it can acquire iron from host hemoglobin. The current model for staphylococcal hemoglobin-iron acquisition proposes that S. aureus binds hemoglobin through the surface-exposed hemoglobin receptor IsdB. IsdB removes heme from bound hemoglobin and transfers this cofactor to other proteins of the Isd system, which import and degrade heme to release iron in the cytoplasm. Here we demonstrate that the individual components of the Isd system are required for growth on low nanomolar concentrations of hemoglobin as a sole source of iron. An in-depth study of hemoglobin binding by IsdB revealed key residues that are required for hemoglobin binding. Further, we show that these residues are necessary for heme extraction from hemoglobin and growth on hemoglobin as a sole iron source. These processes are found to contribute to the pathogenicity of S. aureus in a murine model of infection. Together these results build on the model for Isd-mediated hemoglobin binding and heme-iron acquisition during the pathogenesis of S. aureus infection.

Lectin-Seq: A method to profile lectin-microbe interactions in native communities.

Soluble human lectins are critical components of innate immunity. Genetic models suggest that lectins influence host-resident microbiota, but their specificity for commensal and mutualist species is understudied. Elucidating lectins' roles in regulating microbiota requires an understanding of which microbial species they bind within native communities. To profile human lectin recognition, we developed Lectin-Seq. We apply Lectin-Seq to human fecal microbiota using the soluble mannose-binding lectin (MBL) and intelectin-1 (hItln1). Although each lectin binds a substantial percentage of the samples (10 to 20%), the microbial interactomes of MBL and hItln1 differ markedly in composition and diversity. MBL binding is highly selective for a small subset of species commonly associated with humans. In contrast, hItln1's interaction profile encompasses a broad range of lower-abundance species. Our data uncover stark differences in the commensal recognition properties of human lectins.

Structural basis for hemoglobin capture by Staphylococcus aureus cell-surface protein, IsdH.

Pathogens must steal iron from their hosts to establish infection. In mammals, hemoglobin (Hb) represents the largest reservoir of iron, and pathogens express Hb-binding proteins to access this source. Here, we show how one of the commonest and most significant human pathogens, Staphylococcus aureus, captures Hb as the first step of an iron-scavenging pathway. The x-ray crystal structure of Hb bound to a domain from the Isd (iron-regulated surface determinant) protein, IsdH, is the first structure of a Hb capture complex to be determined. Surface mutations in Hb that reduce binding to the Hb-receptor limit the capacity of S. aureus to utilize Hb as an iron source, suggesting that Hb sequence is a factor in host susceptibility to infection. The demonstration that pathogens make highly specific recognition complexes with Hb raises the possibility of developing inhibitors of Hb binding as antibacterial agents.

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.

Applying microbial ecology to antimicrobial discovery.

Introduction of antibiotics into clinical use has contributed to some of the greatest improvements to public health in the 20th century. Most antibiotics are based on antimicrobials that were isolated from environmental microorganisms over 50 years ago, but emerging resistance requires discovery of new molecules and development of these molecules into therapeutics. Bioinformatic analyses of microbial genomes indicate that many more microbial bioactive molecules remain undiscovered. Understanding when, where, and why these molecules are produced informs efforts to tap into the hidden unexplored chemical diversity. Expanding the search to undersampled ecological niches and improving culturing techniques will ensure discovery of new antibiotics.

The Microbiomes of Seven Lichen Genera Reveal Host Specificity, a Reduced Core Community and Potential as Source of Antimicrobials.

The High Andean Paramo ecosystem is a unique neotropical mountain biome considered a diversity and evolutionary hotspot. Lichens, which are complex symbiotic structures that contain diverse commensal microbial communities, are prevalent in Paramos. There they play vital roles in soil formation and mineral fixation. In this study we analyzed the microbiomes of seven lichen genera in Colombian Paramos using 16S rRNA gene amplicon sequencing and provide the first description of the bacterial communities associated with Cora and Hypotrachyna lichens. Paramo lichen microbiomes varied in diversity indexes and number of OTUs, but were composed predominantly by the phyla Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Proteobacteria, and Verrucomicrobia. In the case of Cora and Cladonia, the microbiomes were distinguished based on the identity of the lichen host. While the majority of the lichen-associated microorganisms were not present in all lichens sampled, sixteen taxa shared among this diverse group of lichens suggest a core lichen microbiome that broadens our concept of these symbiotic structures. Additionally, we identified strains producing compounds active against clinically relevant microbial strains. These results indicate that lichen microbiomes from the Paramo ecosystem are diverse and host-specific but share a taxonomic core and can be a source of new bacterial taxa and antimicrobials.

A Small-Molecule Modulator of Metal Homeostasis in Gram-Positive Pathogens.

Metals are essential nutrients that all living organisms acquire from their environment. While metals are necessary for life, excess metal uptake can be toxic; therefore, intracellular metal levels are tightly regulated in bacterial cells. Staphylococcus aureus, a Gram-positive bacterium, relies on metal uptake and metabolism to colonize vertebrates. Thus, we hypothesized that an expanded understanding of metal homeostasis in S. aureus will lead to the discovery of pathways that can be targeted with future antimicrobials. We sought to identify small molecules that inhibit S. aureus growth in a metal-dependent manner as a strategy to uncover pathways that maintain metal homeostasis. Here, we demonstrate that VU0026921 kills S. aureus through disruption of metal homeostasis. VU0026921 activity was characterized through cell culture assays, transcriptional sequencing, compound structure-activity relationship, reactive oxygen species (ROS) generation assays, metal binding assays, and metal level analyses. VU0026921 disrupts metal homeostasis in S. aureus, increasing intracellular accumulation of metals and leading to toxicity through mismetalation of enzymes, generation of reactive oxygen species, or disruption of other cellular processes. Antioxidants partially protect S. aureus from VU0026921 killing, emphasizing the role of reactive oxygen species in the mechanism of killing, but VU0026921 also kills S. aureus anaerobically, indicating that the observed toxicity is not solely oxygen dependent. VU0026921 disrupts metal homeostasis in multiple Gram-positive bacteria, leading to increased reactive oxygen species and cell death, demonstrating the broad applicability of these findings. Further, this study validates VU0026921 as a probe to further decipher mechanisms required to maintain metal homeostasis in Gram-positive bacteria.IMPORTANCEStaphylococcus aureus is a leading agent of antibiotic-resistant bacterial infections in the world. S. aureus tightly controls metal homeostasis during infection, and disruption of metal uptake systems impairs staphylococcal virulence. We identified small molecules that interfere with metal handling in S. aureus to develop chemical probes to investigate metallobiology in this organism. Compound VU0026921 was identified as a small molecule that kills S. aureus both aerobically and anaerobically. The activity of VU0026921 is modulated by metal supplementation, is enhanced by genetic inactivation of Mn homeostasis genes, and correlates with increased cellular reactive oxygen species. Treatment with VU0026921 causes accumulation of multiple metals within S. aureus cells and concomitant upregulation of genes involved in metal detoxification. This work defines a small-molecule probe for further defining the role of metal toxicity in S. aureus and validates future antibiotic development targeting metal toxicity pathways.

Subcellular localization of the Staphylococcus aureus heme iron transport components IsdA and IsdB.

Staphylococcus aureus is a human pathogen that represents a tremendous threat to global public health. An important aspect of S. aureus pathogenicity is the ability to acquire iron from its host during infection. In vertebrates, iron is sequestered predominantly within heme, the majority of which is bound by hemoglobin. To acquire iron, S. aureus binds hemoglobin, removes heme, and transports it into the cytoplasm, where heme is degraded. This process is carried out by the iron-regulated surface determinant system (Isd); however, the mechanism by which hemoglobin recognition occurs is not completely understood. Here we report that the surface receptor components of the Isd system, IsdA and IsdB, physically interact with each other and are anchored to a discrete location within the cell wall. This organized localization pattern is dependent upon the iron status of the bacterium. Furthermore, we have found that hemoglobin colocalizes with IsdB at discrete sites within the cell wall. Virulence studies revealed that IsdB is required for the efficient colonization of the heart and that IsdB is differentially expressed within infected organs, suggesting that S. aureus experiences various degrees of iron starvation depending on the site of infection. These findings significantly expand our understanding of hemoglobin iron acquisition and demonstrate an orchestrated pattern of regulation and localization for the S. aureus heme iron acquisition system.

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.

Microbial community modulates growth of symbiotic fungus required for stingless bee metamorphosis.

The Brazilian stingless bee Scaptotrigona depilis requires the brood cells-associated fungus Zygosaccharomyces sp. as steroid source for metamorphosis. Besides the presence of Zygosaccharomyces sp., other fungi inhabit S. depilis brood cells, but their biological functions are unknown. Here we show that Candida sp. and Monascus ruber, isolated from cerumen of S. depilis brood provisions, interact with Zygosaccharomyces sp. and modulate its growth. Candida sp. produces volatile organic compounds (VOCs) that stimulate Zygosacchromyces sp. development. Monascus ruber inhibits Zygosacchromyces sp. growth by producing lovastatin, which blocks steroid biosynthesis. We also observed that in co-cultures M. ruber inhibits Candida sp. through the production of monascin. The modulation of Zygosaccharomyces sp. growth by brood cell-associated fungi suggests their involvement in S. depilis larval development. This tripartite fungal community opens new perspectives in the research of microbial interactions with bees.

Staphylococcus aureus redirects central metabolism to increase iron availability.

Staphylococcus aureus pathogenesis is significantly influenced by the iron status of the host. However, the regulatory impact of host iron sources on S. aureus gene expression remains unknown. In this study, we combine multivariable difference gel electrophoresis and mass spectrometry with multivariate statistical analyses to systematically cluster cellular protein response across distinct iron-exposure conditions. Quadruplicate samples were simultaneously analyzed for alterations in protein abundance and/or post-translational modification state in response to environmental (iron chelation, hemin treatment) or genetic (Deltafur) alterations in bacterial iron exposure. We identified 120 proteins representing several coordinated biochemical pathways that are affected by changes in iron-exposure status. Highlighted in these experiments is the identification of the heme-regulated transport system (HrtAB), a novel transport system which plays a critical role in staphylococcal heme metabolism. Further, we show that regulated overproduction of acidic end-products brought on by iron starvation decreases local pH resulting in the release of iron from the host iron-sequestering protein transferrin. These findings reveal novel strategies used by S. aureus to acquire scarce nutrients in the hostile host environment and begin to define the iron and heme-dependent regulons of S. aureus.

Stingless Bee Larvae Require Fungal Steroid to Pupate.

The larval stage of the stingless bee Scaptotrigona depilis must consume a specific brood cell fungus in order to continue development. Here we show that this fungus is a member of the genus Zygosaccharomyces and provides essential steroid precursors to the developing bee. Insect pupation requires ecdysteroid hormones, and as insects cannot synthesize sterols de novo, they must obtain steroids in their diet. Larval in vitro culturing assays demonstrated that consuming ergosterol recapitulates the developmental effects on S. depilis as ingestion of Zygosaccharomyces sp. cells. Thus, we determined the molecular underpinning of this intimate mutualistic symbiosis. Phylogenetic analyses showed that similar cases of bee-Zygosaccharomyces symbiosis may exist. This unprecedented case of bee-fungus symbiosis driven by steroid requirement brings new perspectives regarding pollinator-microbiota interaction and preservation.