Mucosal fungi promote gut barrier function and social behavior via Type 17 immunity.

Fungal communities (the mycobiota) are an integral part of the gut microbiota, and the disruption of their integrity contributes to local and gut-distal pathologies. Yet, the mechanisms by which intestinal fungi promote homeostasis remain unclear. We characterized the mycobiota biogeography along the gastrointestinal tract and identified a subset of fungi associated with the intestinal mucosa of mice and humans. Mucosa-associated fungi (MAF) reinforced intestinal epithelial function and protected mice against intestinal injury and bacterial infection. Notably, intestinal colonization with a defined consortium of MAF promoted social behavior in mice. The gut-local effects on barrier function were dependent on IL-22 production by CD4+ T helper cells, whereas the effects on social behavior were mediated through IL-17R-dependent signaling in neurons. Thus, the spatial organization of the gut mycobiota is associated with host-protective immunity and epithelial barrier function and might be a driver of the neuroimmune modulation of mouse behavior through complementary Type 17 immune mechanisms.

Human gut mycobiota tune immunity via CARD9-dependent induction of anti-fungal IgG antibodies.

Antibodies mediate natural and vaccine-induced immunity against viral and bacterial pathogens, whereas fungi represent a widespread kingdom of pathogenic species for which neither vaccine nor neutralizing antibody therapies are clinically available. Here, using a multi-kingdom antibody profiling (multiKAP) approach, we explore the human antibody repertoires against gut commensal fungi (mycobiota). We identify species preferentially targeted by systemic antibodies in humans, with Candida albicans being the major inducer of antifungal immunoglobulin G (IgG). Fungal colonization of the gut induces germinal center (GC)-dependent B cell expansion in extraintestinal lymphoid tissues and generates systemic antibodies that confer protection against disseminated C. albicans or C. auris infection. Antifungal IgG production depends on the innate immunity regulator CARD9 and CARD9+CX3CR1+ macrophages. In individuals with invasive candidiasis, loss-of-function mutations in CARD9 are associated with impaired antifungal IgG responses. These results reveal an important role of gut commensal fungi in shaping the human antibody repertoire through CARD9-dependent induction of host-protective antifungal IgG.

Immune regulation by fungal strain diversity in inflammatory bowel disease.

The fungal microbiota (mycobiota) is an integral part of the complex multikingdom microbial community colonizing the mammalian gastrointestinal tract and has an important role in immune regulation1-6. Although aberrant changes in the mycobiota have been linked to several diseases, including inflammatory bowel disease3-9, it is currently unknown whether fungal species captured by deep sequencing represent living organisms and whether specific fungi have functional consequences for disease development in affected individuals. Here we developed a translational platform for the functional analysis of the mycobiome at the fungal-strain- and patient-specific level. Combining high-resolution mycobiota sequencing, fungal culturomics and genomics, a CRISPR-Cas9-based fungal strain editing system, in vitro functional immunoreactivity assays and in vivo models, this platform enables the examination of host-fungal crosstalk in the human gut. We discovered a rich genetic diversity of opportunistic Candida albicans strains that dominate the colonic mucosa of patients with inflammatory bowel disease. Among these human-gut-derived isolates, strains with high immune-cell-damaging capacity (HD strains) reflect the disease features of individual patients with ulcerative colitis and aggravated intestinal inflammation in vivo through IL-1ß-dependent mechanisms. Niche-specific inflammatory immunity and interleukin-17A-producing T helper cell (TH17 cell) antifungal responses by HD strains in the gut were dependent on the C. albicans-secreted peptide toxin candidalysin during the transition from a benign commensal to a pathobiont state. These findings reveal the strain-specific nature of host-fungal interactions in the human gut and highlight new diagnostic and therapeutic targets for diseases of inflammatory origin.

From Birth and Throughout Life: Fungal Microbiota in Nutrition and Metabolic Health.

The human gastrointestinal tract is home to a vibrant, diverse ecosystem of prokaryotic and eukaryotic microorganisms. The gut fungi (mycobiota) have recently risen to prominence due to their ability to modulate host immunity. Colonization of the gut occurs through a combination of vertical transmission from the maternal mycobiota and environmental and dietary exposure. Data from human and animal studies demonstrate that nutrition strongly affects the mycobiota composition and that changes in the fungal communities can aggravate metabolic diseases. The mechanisms pertaining to the mycobiota's influence on host health, pathology, and resident gastrointestinal communities through intrakingdom, transkingdom, and immune cross talk are beginning to come into focus, setting the stage for a new chapter in microbiota-host interactions. Herein, we examine the inception, maturation, and dietary modulation of gastrointestinal and nutritional fungal communities and inspect their impact on metabolic diseases in humans.

Synthesis of Transition-State Inhibitors of Chorismate Utilizing Enzymes from Bromobenzene cis-1,2-Dihydrodiol.

In order to survive in a mammalian host, Mycobacterium tuberculosis (Mtb) produces aryl-capped siderophores known as the mycobactins for iron acquisition. Salicylic acid is a key building block of the mycobactin core and is synthesized by the bifunctional enzyme MbtI, which converts chorismate into isochorismate via a SN2″ reaction followed by further transformation into salicylate through a [3,3]-sigmatropic rearrangement. MbtI belongs to a family of chorismate-utilizing enzymes (CUEs) that have conserved topology and active site residues. The transition-state inhibitor 1 described by Bartlett, Kozlowski, and co-workers is the most potent reported inhibitor to date of CUEs. Herein, we disclose a concise asymmetric synthesis and the accompanying biochemical characterization of 1 along with three closely related analogues beginning from bromobenzene cis-1S,2S-dihydrodiol produced through microbial oxidation that features a series of regio- and stereoselective transformations for introduction of the C-4 hydroxy and C-6 amino substituents. The flexible synthesis enables late-stage introduction of the carboxy group and other bioisosteres at the C-1 position as well as installation of the enol-pyruvate side chain at the C-5 position.

Vinylogous Dehydration by a Polyketide Dehydratase Domain in Curacin Biosynthesis.

Polyketide synthase (PKS) enzymes continue to hold great promise as synthetic biology platforms for the production of novel therapeutic agents, biofuels, and commodity chemicals. Dehydratase (DH) catalytic domains play an important role during polyketide biosynthesis through the dehydration of the nascent polyketide intermediate to provide olefins. Our understanding of the detailed mechanistic and structural underpinning of DH domains that control substrate specificity and selectivity remains limited, thus hindering our efforts to rationally re-engineer PKSs. The curacin pathway houses a rare plurality of possible double bond permutations containing conjugated olefins as well as both cis- and trans-olefins, providing an unrivaled model system for polyketide dehydration. All four DH domains implicated in curacin biosynthesis were characterized in vitro using synthetic substrates, and activity was measured by LC-MS/MS analysis. These studies resulted in complete kinetic characterization of the all-trans-trienoate-forming CurK-DH, whose kcat of 72 s-1 is more than 3 orders of magnitude greater than that of any previously reported PKS DH domain. A novel stereospecific mechanism for diene formation involving a vinylogous enolate intermediate is proposed for the CurJ and CurH DHs on the basis of incubation studies with truncated substrates. A synthetic substrate was co-crystallized with a catalytically inactive Phe substitution in the His-Asp catalytic dyad of CurJ-DH to elucidate substrate-enzyme interactions. The resulting complex suggested the structural basis for dienoate formation and provided the first glimpse into the enzyme-substrate interactions essential for the formation of olefins in polyketide natural products. This examination of both canonical and non-canonical dehydration mechanisms reveals hidden catalytic activity inherent in some DH domains that may be leveraged for future applications in synthetic biology.

Functional Characterization of a Dehydratase Domain from the Pikromycin Polyketide Synthase.

Metabolic engineering of polyketide synthase (PKS) pathways represents a promising approach to natural products discovery. The dehydratase (DH) domains of PKSs, which generate an α,ß-unsaturated bond through a dehydration reaction, have been poorly studied compared with other domains, likely because of the simple nature of the chemical reaction they catalyze and the lack of a convenient assay to measure substrate turnover. Herein we report the first steady-state kinetic analysis of a PKS DH domain employing LC-MS/MS analysis for product quantitation. PikDH2 was selected as a model DH domain. Its substrate specificity and mechanism were interrogated with a systematic series of synthetic triketide substrates containing a nonhydrolyzable thioether linkage as well as by site-directed mutagenesis, evaluation of the pH dependence of the catalytic efficiency (V(max)/K(M)), and kinetic characterization of a mechanism-based inhibitor. These studies revealed that PikDH2 converts d-alcohol substrates to trans-olefin products. The reaction is reversible with equilibrium constants ranging from 1.2 to 2. Moreover, the enzyme activity is robust, and PikDH2 was used on a preparative scale for the chemoenzymatic synthesis of unsaturated triketide products. PikDH2 was shown to possess remarkably strict substrate specificity and is unable to turn over substrates that are epimeric at the ß-, γ-, or δ-position. We also demonstrated that PikDH2 has a key ionizable group with a pK(a) of 7.0 and can be irreversibly inactivated through covalent modification by a mechanism-based inhibitor, which provides a foundation for future structural studies to elucidate substrate-protein interactions.

Zafirlukast Is a Promising Scaffold for Selectively Inhibiting TNFR1 Signaling.

Tumor necrosis factor (TNF) plays an important role in the pathogenesis of inflammatory and autoimmune diseases such as rheumatoid arthritis and Crohn's disease. The biological effects of TNF are mediated by binding to TNF receptors, TNF receptor 1 (TNFR1), or TNF receptor 2 (TNFR2), and this coupling makes TNFR1-specific inhibition by small-molecule therapies essential to avoid deleterious side effects. Recently, we engineered a time-resolved fluorescence resonance energy transfer biosensor for high-throughput screening of small molecules that modulate TNFR1 conformational states and identified zafirlukast as a compound that inhibits receptor activation, albeit at low potency. Here, we synthesized 16 analogues of zafirlukast and tested their potency and specificity for TNFR1 signaling. Using cell-based functional assays, we identified three analogues with significantly improved efficacy and potency, each of which induces a conformational change in the receptor (as measured by fluorescence resonance energy transfer (FRET) in cells). The best analogue decreased NF-κB activation by 2.2-fold, IκBα efficiency by 3.3-fold, and relative potency by two orders of magnitude. Importantly, we showed that the analogues do not block TNF binding to TNFR1 and that binding to the receptor's extracellular domain is strongly cooperative. Despite these improvements, the best candidate's maximum inhibition of NF-κB is only 63%, leaving room for further improvements to the zafirlukast scaffold to achieve full inhibition and prove its potential as a therapeutic lead. Interestingly, while we find that the analogues also bind to TNFR2 in vitro, they do not inhibit TNFR2 function in cells or cause any conformational changes upon binding. Thus, these lead compounds should also be used as reagents to study conformational-dependent activation of TNF receptors.

Mycobiota-induced IgA antibodies regulate fungal commensalism in the gut and are dysregulated in Crohn's disease.

Secretory immunoglobulin A (sIgA) plays an important role in gut barrier protection by shaping the resident microbiota community, restricting the growth of bacterial pathogens and enhancing host protective immunity via immunological exclusion. Here, we found that a portion of the microbiota-driven sIgA response is induced by and directed towards intestinal fungi. Analysis of the human gut mycobiota bound by sIgA revealed a preference for hyphae, a fungal morphotype associated with virulence. Candida albicans was a potent inducer of IgA class-switch recombination among plasma cells, via an interaction dependent on intestinal phagocytes and hyphal programming. Characterization of sIgA affinity and polyreactivity showed that hyphae-associated virulence factors were bound by these antibodies and that sIgA influenced C. albicans morphotypes in the murine gut. Furthermore, an increase in granular hyphal morphologies in patients with Crohn's disease compared with healthy controls correlated with a decrease in antifungal sIgA antibody titre with affinity to two hyphae-associated virulence factors. Thus, in addition to its importance in gut bacterial regulation, sIgA targets the uniquely fungal phenomenon of hyphal formation. Our findings indicate that antifungal sIgA produced in the gut can play a role in regulating intestinal fungal commensalism by coating fungal morphotypes linked to virulence, thereby providing a protective mechanism that might be dysregulated in patients with Crohn's disease.

Gut mycobiota under scrutiny: fungal symbionts or environmental transients?

The human gastrointestinal tract is home to a thriving community of microbes including the fungal 'mycobiota'. Although sequencing methodology has enumerated diverse fungal genera within this niche, discerning persistent symbiotic residents from contaminants and purely environmental transients remains a challenge. Recent advances in culturomics and sequencing employing metagenomics, metatranscriptomics and longitudinal studies have begun to reveal a human symbiont 'core mycobiome' that may contribute to human health and disease. Trans-kingdom interactions between the bacterial microbiota and evolution within the niche have defined C. albicans as a true symbiont, setting a bar for defining other fungi. Additionally, elegant investigations of mammalian antifungal immunity have examined mononuclear phagocytes, neutrophils, antigen-specific recognition by T cells and other mechanisms important for local and systemic effects on the host, providing further evidence supporting gut persistence. In this review we discuss current research aimed at investigating the symbiotic mycobiota and propose four criteria aiding in the differentiation of fungal symbionts from environmental transients.

Response to Fungal Dysbiosis by Gut-Resident CX3CR1+ Mononuclear Phagocytes Aggravates Allergic Airway Disease.

Sensing of the gut microbiota, including fungi, regulates mucosal immunity. Whether fungal sensing in the gut can influence immunity at other body sites is unknown. Here we show that fluconazole-induced gut fungal dysbiosis has persistent effects on allergic airway disease in a house dust mite challenge model. Mice with a defined community of bacteria, but lacking intestinal fungi were not susceptible to fluconazole-induced dysbiosis, while colonization with a fungal mixture recapitulated the detrimental effects. Gut-resident mononuclear phagocytes (MNPs) expressing the fractalkine receptor CX3CR1 were essential for the effect of gut fungal dysbiosis on peripheral immunity. Depletion of CX3CR1+ MNPs or selective inhibition of Syk signaling downstream of fungal sensing in these cells ameliorated lung allergy. These results indicate that disruption of intestinal fungal communities can have persistent effects on peripheral immunity and aggravate disease severity through fungal sensing by gut-resident CX3CR1+ MNPs.

Structural Basis of Polyketide Synthase O-Methylation.

Modular type I polyketide synthases (PKSs) produce some of the most chemically complex metabolites in nature through a series of multienzyme modules. Each module contains a variety of catalytic domains to selectively tailor the growing molecule. PKS O-methyltransferases ( O-MTs) are predicted to methylate ß-hydroxyl or ß-keto groups, but their activity and structure have not been reported. We determined the domain boundaries and characterized the catalytic activity and structure of the StiD and StiE O-MTs, which methylate opposite ß-hydroxyl stereocenters in the myxobacterial stigmatellin biosynthetic pathway. Substrate stereospecificity was demonstrated for the StiD O-MT. Key catalytic residues were identified in the crystal structures and investigated in StiE O-MT via site-directed mutagenesis and further validated with the cyanobacterial CurL O-MT from the curacin biosynthetic pathway. Initial structural and biochemical analysis of PKS O-MTs supplies a new chemoenzymatic tool, with the unique ability to selectively modify hydroxyl groups during polyketide biosynthesis.

Teixobactin and Its Analogues: A New Hope in Antibiotic Discovery.

Increasing bacterial resistance against current antibiotics and lack of new molecules to combat bacterial resistance are key challenges to global health. There is, therefore, a continuing need to develop new antibiotics. Teixobactin, a cyclic undecapeptide, displays excellent antibacterial activities against a range of pathogenic bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis. Interestingly, it operates by multiple modes of actions and is bactericidal toward S. aureus without detectable resistance. This unique combination of wide Gram-positive activity coupled with its inability to elicit resistance make teixobactin a very attractive molecule for antimicrobial therapeutic development. This Viewpoint discusses teixobactin, its analogues, and the challenges and opportunities associated with their future development.

Domain Organization and Active Site Architecture of a Polyketide Synthase C-methyltransferase.

Polyketide metabolites produced by modular type I polyketide synthases (PKS) acquire their chemical diversity through the variety of catalytic domains within modules of the pathway. Methyltransferases are among the least characterized of the catalytic domains common to PKS systems. We determined the domain boundaries and characterized the activity of a PKS C-methyltransferase (C-MT) from the curacin A biosynthetic pathway. The C-MT catalyzes S-adenosylmethionine-dependent methyl transfer to the α-position of ß-ketoacyl substrates linked to acyl carrier protein (ACP) or a small-molecule analog but does not act on ß-hydroxyacyl substrates or malonyl-ACP. Key catalytic residues conserved in both bacterial and fungal PKS C-MTs were identified in a 2 Å crystal structure and validated biochemically. Analysis of the structure and the sequences bordering the C-MT provides insight into the positioning of this domain within complete PKS modules.

Tylosin polyketide synthase module 3: stereospecificity, stereoselectivity and steady-state kinetic analysis of ß-processing domains via diffusible, synthetic substrates.

Polyketide synthase (PKS) ß-processing domains are responsible for much of the stereochemical complexity of polyketide natural products. Although the importance of ß-processing domains has been well noted and significantly explored, key stereochemical details pertaining to cryptic stereochemistry and the impact of remote stereogenic centers have yet to be fully discerned. To uncover the inner workings of ketoreductases (KR) and dehydratases (DH) from the tylosin pathway a didomain composed of TylDH3-KR3 was recombinantly expressed and interrogated with full-length tetraketide substrates to probe the impact of vicinal and distal stereochemistry. In vitro product isolation analysis revealed the products of the cryptic KR as d-alcohols and of the DH as trans-olefins. Steady-state kinetic analysis of the dehydration reaction demonstrated a strict stereochemical tolerance at the ß-position as d-configured substrates were processed more than 100 times more efficiently than l-alcohols. Unexpectedly, the kcat/KM values were diminished 14- to 45-fold upon inversion of remote ε- and ζ-stereocenters. This stereochemical discrimination is predicted to be driven by a combination of allylic A1,3 strain that likely disfavors binding of the ε-epimer and a loss of electrostatic interactions with the ζ-epimer. Our results strongly suggest that dehydratases may play a role in refining the stereochemical outcomes of preceding modules through their substrate stereospecificity, honing the configurational purity of the final PKS product.

Polyketide intermediate mimics as probes for revealing cryptic stereochemistry of ketoreductase domains.

Among natural product families, polyketides have shown the most promise for combinatorial biosynthesis of natural product-like libraries. Though recent research in the area has provided many mechanistic revelations, a basic-level understanding of kinetic and substrate tolerability is still needed before the full potential of combinatorial biosynthesis can be realized. We have developed a novel set of chemical probes for the study of ketoreductase domains of polyketide synthases. This chemical tool-based approach was validated using the ketoreductase of pikromycin module 2 (PikKR2) as a model system. Triketide substrate mimics 12 and 13 were designed to increase stability (incorporating a nonhydrolyzable thioether linkage) and minimize nonessential functionality (truncating the phosphopantetheinyl arm). PikKR2 reduction product identities as well as steady-state kinetic parameters were determined by a combination of LC-MS/MS analysis of synthetic standards and a NADPH consumption assay. The d-hydroxyl product is consistent with bioinformatic analysis and results from a complementary biochemical and molecular biological approach. When compared to widely employed substrates in previous studies, diketide 63 and trans-decalone 64, substrates 12 and 13 showed 2-10 fold lower K(M) values (2.4 ± 0.8 and 7.8 ± 2.7 mM, respectively), indicating molecular recognition of intermediate-like substrates. Due to an abundance of the nonreducable enol-tautomer, the k(cat) values were attenuated by as much as 15-336 fold relative to known substrates. This study reveals the high stereoselectivity of PikKR2 in the face of gross substrate permutation, highlighting the utility of a chemical probe-based approach in the study of polyketide ketoreductases.