Microbiota metabolism of intestinal amino acids impacts host nutrient homeostasis and physiology.

The intestine and liver are thought to metabolize dietary nutrients and regulate host nutrient homeostasis. Here, we find that the gut microbiota also reshapes the host amino acid (aa) landscape via efficiently metabolizing intestinal aa. To identify the responsible microbes/genes, we developed a metabolomics-based assay to screen 104 commensals and identified candidates that efficiently utilize aa. Using genetics, we identified multiple responsible metabolic genes in phylogenetically diverse microbes. By colonizing germ-free mice with the wild-type strain and their isogenic mutant deficient in individual aa-metabolizing genes, we found that these genes regulate the availability of gut and circulatory aa. Notably, microbiota genes for branched-chain amino acids (BCAAs) and tryptophan metabolism indirectly affect host glucose homeostasis via peripheral serotonin. Collectively, at single-gene level, this work characterizes a microbiota-encoded metabolic activity that affects host nutrient homeostasis and provides a roadmap to interrogate microbiota-dependent activity to improve human health.

Fungal microbiota sustains lasting immune activation of neutrophils and their progenitors in severe COVID-19.

Gastrointestinal fungal dysbiosis is a hallmark of several diseases marked by systemic immune activation. Whether persistent pathobiont colonization during immune alterations and impaired gut barrier function has a durable impact on host immunity is unknown. We found that elevated levels of Candida albicans immunoglobulin G (IgG) antibodies marked patients with severe COVID-19 (sCOVID-19) who had intestinal Candida overgrowth, mycobiota dysbiosis and systemic neutrophilia. Analysis of hematopoietic stem cell progenitors in sCOVID-19 revealed transcriptional changes in antifungal immunity pathways and reprogramming of granulocyte myeloid progenitors (GMPs) for up to a year. Mice colonized with C. albicans patient isolates experienced increased lung neutrophilia and pulmonary NETosis during severe acute respiratory syndrome coronavirus-2 infection, which were partially resolved with antifungal treatment or by interleukin-6 receptor blockade. sCOVID-19 patients treated with tocilizumab experienced sustained reductions in C. albicans IgG antibodies titers and GMP transcriptional changes. These findings suggest that gut fungal pathobionts may contribute to immune activation during inflammatory diseases, offering potential mycobiota-immune therapeutic strategies for sCOVID-19 with prolonged symptoms.

Selection of cross-reactive T cells by commensal and food-derived yeasts drives cytotoxic TH1 cell responses in Crohn's disease.

Aberrant CD4+ T cell reactivity against intestinal microorganisms is considered to drive mucosal inflammation in inflammatory bowel diseases. The disease-relevant microbial species and the corresponding microorganism-specific, pathogenic T cell phenotypes remain largely unknown. In the present study, we identified common gut commensal and food-derived yeasts, as direct activators of altered CD4+ T cell reactions in patients with Crohn's disease (CD). Yeast-responsive CD4+ T cells in CD display a cytotoxic T helper cell (TH1 cell) phenotype and show selective expansion of T cell clones that are highly cross-reactive to several commensal, as well as food-derived, fungal species. This indicates cross-reactive T cell selection by repeated encounter with conserved fungal antigens in the context of chronic intestinal disease. Our results highlighted a role of yeasts as drivers of aberrant CD4+ T cell reactivity in patients with CD and suggest that both gut-resident fungal commensals and daily dietary intake of yeasts might contribute to chronic activation of inflammatory CD4+ T cell responses in patients with CD.

Colibactin-induced genotoxicity and colorectal cancer exacerbation critically depends on adhesin-mediated epithelial binding.

Various bacteria are suggested to contribute to colorectal cancer (CRC) development, including pks+ E. coli which produce the genotoxin colibactin that induces characteristic mutational signatures in host epithelial cells. It remains unclear how the highly unstable colibactin molecule is able to access host epithelial cells and its DNA to cause harm. Using the microbiota-dependent ZEB2-transgenic mouse model of invasive CRC, we found that pks+ E. coli drives CRC exacerbation and tissue invasion in a colibactin-dependent manner. Using isogenic mutant strains, we further demonstrate that CRC exacerbation critically depends on expression of the E. coli type-1 pilus adhesin FimH and the F9-pilus adhesin FmlH. Blocking bacterial adhesion using a pharmacological FimH inhibitor attenuates colibactin-mediated genotoxicity and CRC exacerbation. Together, we show that the oncogenic potential of pks+ E. coli critically depends on bacterial adhesion to host epithelial cells and is critically mediated by specific bacterial adhesins. Adhesin-mediated epithelial binding subsequently allows production of the genotoxin colibactin in close proximity to host epithelial cells, which promotes DNA damage and drives CRC development. These findings present promising therapeutic avenues for the development of anti-adhesive therapies aiming at mitigating colibactin-induced DNA damage and inhibiting the initiation and progression of CRC, particularly in individuals at risk for developing CRC.

Gut epithelium modulates fungal pathogenesis.

Specialized epithelium secretes an antifungal peptide.

Inflammatory ER stress responses dictate the immunopathogenic progression of systemic candidiasis.

Recognition of pathogen-associated molecular patterns can trigger the inositol-requiring enzyme 1 α (IRE1α) arm of the endoplasmic reticulum (ER) stress response in innate immune cells. This process maintains ER homeostasis and also coordinates diverse immunomodulatory programs during bacterial and viral infections. However, the role of innate IRE1α signaling in response to fungal pathogens remains elusive. Here, we report that systemic infection with the human opportunistic fungal pathogen Candida albicans induced proinflammatory IRE1α hyperactivation in myeloid cells that led to fatal kidney immunopathology. Mechanistically, simultaneous activation of the TLR/IL-1R adaptor protein MyD88 and the C-type lectin receptor dectin-1 by C. albicans induced NADPH oxidase-driven generation of ROS, which caused ER stress and IRE1α-dependent overexpression of key inflammatory mediators such as IL-1ß, IL-6, chemokine (C-C motif) ligand 5 (CCL5), prostaglandin E2 (PGE2), and TNF-α. Selective ablation of IRE1α in leukocytes, or treatment with an IRE1α pharmacological inhibitor, mitigated kidney inflammation and prolonged the survival of mice with systemic C. albicans infection. Therefore, controlling IRE1α hyperactivation may be useful for impeding the immunopathogenic progression of disseminated candidiasis.

Immunoglobulins at the interface of the gut mycobiota and anti-fungal immunity.

The dynamic and complex community of microbes that colonizes the intestines is composed of bacteria, fungi, and viruses. At the mucosal surfaces, immunoglobulins play a key role in protection against bacterial and fungal pathogens, and their toxins. Secretory immunoglobulin A (sIgA) is the most abundantly produced antibody at the mucosal surfaces, while Immunoglobulin G (IgG) isotypes play a critical role in systemic protection. IgA and IgG antibodies with reactivity to commensal fungi play an important role in shaping the mycobiota and host antifungal immunity. In this article, we review the latest evidence that establishes a connection between commensal fungi and B cell-mediated antifungal immunity as an additional layer of protection against fungal infections and inflammation.

When IL-17 gets on your nerves.

Microbiota-induced IL-17 production mediates CNS processes and animal behavior. However, its role on the peripheral nervous system (PNS) remains largely unknown. Enamorado et al. demonstrate that commensal-specific Th17 cells are recalled following tissue injury to support local nerve regeneration, a process orchestrated by IL-17 signaling on peripheral neurons.

The future of fungi: threats and opportunities.

The fungal kingdom represents an extraordinary diversity of organisms with profound impacts across animal, plant, and ecosystem health. Fungi simultaneously support life, by forming beneficial symbioses with plants and producing life-saving medicines, and bring death, by causing devastating diseases in humans, plants, and animals. With climate change, increased antimicrobial resistance, global trade, environmental degradation, and novel viruses altering the impact of fungi on health and disease, developing new approaches is now more crucial than ever to combat the threats posed by fungi and to harness their extraordinary potential for applications in human health, food supply, and environmental remediation. To address this aim, the Canadian Institute for Advanced Research (CIFAR) and the Burroughs Wellcome Fund convened a workshop to unite leading experts on fungal biology from academia and industry to strategize innovative solutions to global challenges and fungal threats. This report provides recommendations to accelerate fungal research and highlights the major research advances and ideas discussed at the meeting pertaining to 5 major topics: (1) Connections between fungi and climate change and ways to avert climate catastrophe; (2) Fungal threats to humans and ways to mitigate them; (3) Fungal threats to agriculture and food security and approaches to ensure a robust global food supply; (4) Fungal threats to animals and approaches to avoid species collapse and extinction; and (5) Opportunities presented by the fungal kingdom, including novel medicines and enzymes.

A pan-cancer mycobiome analysis reveals fungal involvement in gastrointestinal and lung tumors.

Fungal microorganisms (mycobiota) comprise a small but immunoreactive component of the human microbiome, yet little is known about their role in human cancers. Pan-cancer analysis of multiple body sites revealed tumor-associated mycobiomes at up to 1 fungal cell per 104 tumor cells. In lung cancer, Blastomyces was associated with tumor tissues. In stomach cancers, high rates of Candida were linked to the expression of pro-inflammatory immune pathways, while in colon cancers Candida was predictive of metastatic disease and attenuated cellular adhesions. Across multiple GI sites, several Candida species were enriched in tumor samples and tumor-associated Candida DNA was predictive of decreased survival. The presence of Candida in human GI tumors was confirmed by external ITS sequencing of tumor samples and by culture-dependent analysis in an independent cohort. These data implicate the mycobiota in the pathogenesis of GI cancers and suggest that tumor-associated fungal DNA may serve as diagnostic or prognostic biomarkers.

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.

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.

Peritoneal Effluent Cell-Free DNA Sequencing in Peritoneal Dialysis Patients With and Without Peritonitis.

RATIONALE & OBJECTIVE: Conventional culture can be insensitive for the detection of rare infections and for the detection of common infections in the setting of recent antibiotic usage. Patients receiving peritoneal dialysis (PD) with suspected peritonitis have a significant proportion of negative conventional cultures. This study examines the utility of metagenomic sequencing of peritoneal effluent cell-free DNA (cfDNA) for evaluating the peritoneal effluent in PD patients with and without peritonitis. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: We prospectively characterized cfDNA in 68 peritoneal effluent samples obtained from 33 patients receiving PD at a single center from September 2016 to July 2018. OUTCOMES: Peritoneal effluent, microbial, and human cfDNA characteristics were evaluated in culture-confirmed peritonitis and culture-negative peritonitis. ANALYTICAL APPROACH: Descriptive statistics were analyzed and microbial cfDNA was detected in culture-confirmed peritonitis and culture-negative peritonitis. RESULTS: Metagenomic sequencing of cfDNA was able to detect and identify bacterial, viral, and eukaryotic pathogens in the peritoneal effluent from PD patients with culture-confirmed peritonitis, as well as patients with recent antibiotic usage and in cases of culture-negative peritonitis. LIMITATIONS: Parallel cultures were not obtained in all the peritoneal effluent specimens. CONCLUSIONS: Metagenomic cfDNA sequencing of the peritoneal effluent can identify pathogens in PD patients with peritonitis, including culture-negative peritonitis.

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.

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.

Effects of Intestinal Fungi and Viruses on Immune Responses and Inflammatory Bowel Diseases.

The intestinal microbiota comprises diverse fungal and viral components, in addition to bacteria. These microbes interact with the immune system and affect human physiology. Advances in metagenomics have associated inflammatory and autoimmune diseases with alterations in fungal and viral species in the gut. Studies of animal models have found that commensal fungi and viruses can activate host-protective immune pathways related to epithelial barrier integrity, but can also induce reactions that contribute to events associated with inflammatory bowel disease. Changes in our environment associated with modernization and the COVID-19 pandemic have exposed humans to new fungi and viruses, with unknown consequences. We review the lessons learned from studies of animal viruses and fungi commonly detected in the human gut and how these might affect health and intestinal disease.