In this review, we summarize the recent findings on the gut mycobiome and its major interactions with the host and the other digestive microorganisms in order to decipher both the existence and the role of a mycobiome-gut-brain axis. Finally, we review the existing literature assessing the links between fungi, the digestive ecosystem, and neurological or neuropsychiatric disorders.

The gut microbiome is a rich and complex ecosystem composed of bacteria, archaea, viruses, fungi, protists, and (sometimes) helminths. The essential role of this ecosystem in host homeostasis, including metabolic and immune functions, is now well demonstrated, as is its involvement in the pathophysiology of digestive and extra-digestive disorders [ 7 8 ]. The development of culture-independent techniques for identifying microorganisms, such as next-generation sequencing (NGS), has improved our knowledge on the composition and dynamics of this ecosystem. However, most studies have focused exclusively on the bacterial component, the dominant domain, neglecting fungi and other minority kingdoms [ 9 ]. GBA illustrates this trend well since few studies integrate fungal analysis. No review to date has been devoted to the role of intestinal fungi—also named gut mycobiome—in the microbiome-GBA, despite the key role conferred to fungi in digestive diseases [ 10 ].

It has long been accepted that the central nervous system (CNS) and the intestine are closely connected, as suggested by satiety sensations or visceral pains. However, the concept of “microbiome-gut-brain axis (-GBA)” has emerged very recently as a bidirectional communication system in which the digestive microbial flora, also known as the gut microbiome, play a key role [ 1 ]. Indeed, accumulated evidence suggests that the intestinal microbiome may modulate CNS activities, which may, in turn, have an impact on the intestinal microbiome [ 2 ]. Several studies on mice have illustrated this mutual dialogue well between the gut microbiome and the brain. On the one hand, mice elevated in a sterile environment have an increased anxiety-like behavior that can be reversed after gut colonization with a commensal microbiome [ 3 4 ]. On the other hand, the diversity of the gut microbiome is diminished in rodent maternal separation, a model of depression [ 5 ]. Lastly, the clinical efficacy of specific probiotic strains in human neuropsychiatric pathologies, such as anxiety or depression, strengthens the concept of the microbiome-GBA [ 6 ].

Even if the fungal component is a limited part of the gut ecosystem, it appears to be an essential player of the human microbiome. The increasing interest in gut mycobiome, its dysbiosis, and its role in the GBA is driven by recent data supporting its interactions with the host and the bacterial microbiome.

Despite a recent increased number of published data on the gut mycobiome, defining the healthy gut mycobiome is still difficult, especially regarding the high inter- and intra-volunteer variability of the mycobiome. In contrast with gut-associated bacteria, several studies have found a lack of stability in the gut mycobiome over time and low abundance and diversity [ 13 34 ]. To date, there is no consensus on the mycobiome “normobiosis,” a term referring to a balanced composition of gut flora in healthy individuals (by contrast, a disruption of this balanced microbial composition of gut flora is named “dysbiosis”). In most studies, Ascomycota is by far the most prevalent fungus phylum in the gut, followed by Zygomycota (corresponding at the previous phylogenetic classification, now distributed among Glomeromycota and several subphyla incertae sedis, including Mucoromycotina, Entomophthoromycotina, Kickxellomycotina, and Zoopagomycotina) and Basidiomycota phyla [ 33 36 ]. Hallen-Adams and colleagues [ 37 ] have sequenced stool samples from 45 subjects and observed solely 72 operational taxonomic units (OTUs) assigned as fungal sequences, which is clearly less than bacterial abundance. These OTUs were distributed in two phyla (Ascomycota and Basidiomycota) and in ten classes of micromycetes. The most abundant fungi wereand yeasts belonging to Dipodascaceae. Interestingly, gut fungi observed in this study included known human symbionts (, andspp.), environmental fungi (sp.), and food-associated fungi () [ 37 ]. These data reinforce the wide exposure of humans to molds throughout a person’s life. Another NGS study identified 66 fungal genera within 96 stool samples collected from 50 patients, of which 12 were healthy control patients [ 33 ].corresponded to the most prevalent genus followed byand. A third study observed 75 fungal genera with, andbeing the most prevalent [ 38 ]. Recently, for the gut mycobiome, Nash and colleagues sequenced 317 stool samples from the American HMP project [ 13 ]. Gut-associated fungi in this healthy cohort were mainly composed of a high prevalence of, and, with, andbeing found in 96.8%, 88.3%, and 80.8% of the samples, respectively. Taking together these studies confirms the lower diversity of gut mycobiome in healthy subjects compared to the gut bacterial microbiome [ 13 38 ].

Since fungi are ubiquitous in our environment—present in the air we breathe, in the food we eat, such as bread, cheese, beer or even in antibiotics—nobody is fungus-free [ 14 27 ]. Therefore, fungi have been recognized as an integral part of our commensal flora at different body sites (skin, lung, vagina, oral tract, and gut) [ 28 29 ]. In the digestive tract, fungi seem to colonize the gut shortly after birth [ 30 31 ]. Briefly, the fungal composition of gut flora is influenced by several factors such as age, host genetics, host immunity, diet, and medication [ 32 33 ], as well as the bacterial microbiome that also impacts the mycobiome through inter-kingdom interactions [ 33 ].

Unlike the bacteria that inhabit our digestive tract, the human gut mycobiome has been poorly studied and characterized in healthy as well as in diseased individuals. Initially, the large-scale projects such as the National Institutes of Health’s Human Microbiome Project (HMP) and Metagenomics of the Human Intestinal Tract (MetaHIT) Project were focused exclusively on the bacterial flora to characterize their composition and impact on human health and diseases [ 11 12 ]. Bacteria represent huge quantities of microorganisms that inhabit the intestinal mucosa whereas fungi represent a tiny part, estimated at less than 0.01% to 0.1% of genes in stool samples [ 13 14 ]. Furthermore, a large part of these fungi are difficult to culture in vitro or are uncultivable [ 14 ]. However, the NGS development has been valuable in revealing this poorly understood compartment of our whole microbiome [ 15 ]. The main steps of NGS mycobiome analysis are summarized in Table 1 25 ].

3. Mycobiome Interactions within the Gut Ecosystem

Similar to gut bacteria, the gut mycobiome contributes to physiological functions and homeostasis throughout a host’s lifetime. The effect of the whole microbiome on host health is highlighted by disruptions observed in germ-free mice models [ 39 ]. Here we summarize both experimental and clinical data focusing on mycobiome interactions that may be involved in mycobiome-GBA communication through immune and non-immune mediated crosstalk systems, similar to those described in the microbiome-GBA [ 40 ].

Saccharomyces boulardii (the most common probiotic, isolated from fruit) against Clostridium difficile colitis. In mice, a prior administration of S. boulardii increases the production of immunoglobulin A (IgA), particularly of intestinal anti-toxin IgA [ S. boulardii has also been investigated by Thomas and colleagues [ S. boulardii cultures inhibit the inflammatory response of patients with inflammatory bowel disease (IBD) by inhibiting the activation of T and dendritic cells. The secretion of key pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin(IL)-6 are also reduced [ S. boulardii promotes IL-10, an anti-inflammatory cytokine, and epithelial growth factor production [ S. cerevisiae and C. albicans also seem to participate in immune system maturation, inducing functional reprogramming of monocytes and leading to enhanced cytokine production [ C. albicans is able to block monocyte nitric oxide production [47, d -glucan (a major polysaccharide motif of fungal cell walls) are associated with severe colitis in mice [ S. cerevisiae antibodies (usually named ASCA) are found to be significantly associated with Crohn disease (CD) in patients [ A first example illustrating the dialogue between fungi and the host immune system is the protective effect of(the most common probiotic, isolated from fruit) againstcolitis. In mice, a prior administration ofincreases the production of immunoglobulin A (IgA), particularly of intestinal anti-toxin IgA [ 41 ]. Modulation of the host immune response byhas also been investigated by Thomas and colleagues [ 42 ] who demonstrated that supernatants fromcultures inhibit the inflammatory response of patients with inflammatory bowel disease (IBD) by inhibiting the activation of T and dendritic cells. The secretion of key pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin(IL)-6 are also reduced [ 42 ]. In addition,promotes IL-10, an anti-inflammatory cytokine, and epithelial growth factor production [ 42 ].andalso seem to participate in immune system maturation, inducing functional reprogramming of monocytes and leading to enhanced cytokine production [ 43 44 ]. Furthermore,is able to block monocyte nitric oxide production [ 44 ]. This “trained immunity” could be a key factor in the gut immune homeostasis by modulating both the interaction of the host immune system with commensal microorganisms and the host defense against pathogens [ 45 ]. Another illustration of this close link between fungi and the immune system is the fungal dysbiosis observed in IBD, an intestinal inflammatory disorder considered as an inappropriate immune reaction against the gut microbiome [ 46 ]. Several teams have studied the role of fungal dysbiosis in the pathogenesis of IBD [ 10 48 ]. To illustrate the fungal impact on IBD, Wheeler and colleagues increased the colitis severity of mice after antifungal administration [ 48 ]. Of note, increased plasma levels of (1,3)-β--glucan (a major polysaccharide motif of fungal cell walls) are associated with severe colitis in mice [ 49 ]. In addition, anti-antibodies (usually named ASCA) are found to be significantly associated with Crohn disease (CD) in patients [ 50 ], which reinforces the concept that fungi are implicated in the inflammatory immune disorder of IBD.

These data highlight the crucial dialogue between the host’s innate immune system and the mycobiome, involving many actors (for review see [ 51 ]). Among them, Dectin-1 is one of the most important pattern recognition receptors (PRRs) expressed by immune cells that interact with β-glucan [ 52 ]. Dectin-1 knockout mice have more severe colitis compared to wild-type; furthermore, polymorphisms of Dectin-1 gene are associated with increased severity of disease in patients with ulcerative colitis (UC) [ 53 ].

Aspergillus amstelodami , Epicoccum nigrum , and Wallemia sebi and a decrease of Penicillium brevicompactum and C. tropicalis . In parallel, this fungal dysbiosis is clinically associated with a significant increase in allergic airway disease occurrence, which was confirmed by an increased infiltration of inflammatory cells (mainly eosinophils) into animal lungs [ A. amstelodami , E. nigrum , and W. sebi , in order to reproduce the observed post-antifungal dysbiosis) replicated effects similar to allergic airway disease occurrence [ Interactions between the gut mycobiome and the host system also influence extra-intestinal immune responses. In mice for example, an antifungal administration induces a disruption of the gut mycobiome, characterized by an expansion of, andand a decrease ofand. In parallel, this fungal dysbiosis is clinically associated with a significant increase in allergic airway disease occurrence, which was confirmed by an increased infiltration of inflammatory cells (mainly eosinophils) into animal lungs [ 48 ]. Moreover, fungal supplementation in normobiosis mice with these post-antifungal increased strains (, and, in order to reproduce the observed post-antifungal dysbiosis) replicated effects similar to allergic airway disease occurrence [ 48 ]. Taken together, these results indicate that the commensal mycobiome may be a crucial factor in gut and systemic immunological disorders, based on systemic diffusion of either cytokines, fungal products or metabolites, or micromycetous translocation [ 49 ].

S. cerevisiae and Penicillium chrysogenum can produce high concentrations of norepinephrine [ C. albicans is able to produce histamine, another neuromediator involved in appetite regulation, sleep–wake rhythm, and cognitive activity [ C. albicans [ C. albicans virulence [ On the non-immune mediated crosstalk side and focusing on GBA, fungi are able to synthesize and release neurotransmitters, similar to many bacteria.andcan produce high concentrations of norepinephrine [ 54 ], which is involved in brain activation. This neuromediator increases locomotor activity and aggressive behavior and decreases anxiety reactions. In addition,is able to produce histamine, another neuromediator involved in appetite regulation, sleep–wake rhythm, and cognitive activity [ 55 ]. The direct impact of these mycobiome-produced neuromediators is not entirely clear yet. Even if these neurotransmitters seem unlikely to directly modulate CNS, they could locally act on the enteric nervous system (ENS). Conversely, neuromediators may have an impact on gut fungi. For example, gamma-aminobutyric acid (GABA) is able to increase virulence and germ tube formation of 56 ], while serotonin attenuates thevirulence [ 57 ].

C. albicans or S. cerevisiae can have the same protective benefits as intestinal bacteria in terms of immune system modulation and prevention of mucosal tissue injuries [ S. boulardii is able to secrete enzymes, such as proteases or phosphatases, which can inactivate toxins produced by highly inflammatory intestinal pathogens such as C. difficile and E. coli [59, C. albicans , Salmonella typhimurium , and Yersinia enterocolitica [ E. coli abundance in animal stools [ E. coli and other bacteria, which in turn supports inter-kingdom interactions. On the other hand, C. albicans germination is modulated by fatty acids produced locally by bacterial flora [ Finally, inter-kingdom interactions between fungi and bacteria at the gut site may also be implicated in the mycobiome-GBA. While gut bacteria are a known essential actor of the microbiome-GBA [ 2 ], mycobiome equilibrium has also been demonstrated as being critical for microbiome stability in a mice model of colitis [ 49 ]. In this model, an antifungal exposition induced a fungal diversity decrease along with an increased bacterial diversity, aggravating colitis inflammation and severity [ 49 ]. In healthy subjects, Hoffmann and colleagues uncovered specific and significant fungal-bacterial correlations in gut flora [ 33 ]. In addition, in the case of bacterial intestinal dysbiosis, such as after antibiotic exposure, commensal fungi or mono-fungal supplementation withorcan have the same protective benefits as intestinal bacteria in terms of immune system modulation and prevention of mucosal tissue injuries [ 58 ]. At molecular levels,is able to secrete enzymes, such as proteases or phosphatases, which can inactivate toxins produced by highly inflammatory intestinal pathogens such asand 60 ]. This yeast also directly inhibits the growth and dissemination of several intestinal pathogens, such as, and 61 ]. Additionally, β-glucan decreasesabundance in animal stools [ 62 ], a result suggesting a notable influence of this major fungus wall component on the intestinal growth ofand other bacteria, which in turn supports inter-kingdom interactions. On the other hand,germination is modulated by fatty acids produced locally by bacterial flora [ 63 ]. Therefore, we may consider the possibility that the impact of fungi on GBA is due to the local interplay between bacteria and fungi, even though no study has yet focused specifically on this aspect.

These interactions clearly suggest a potential implication of the mycobiome in GBA and, therefore, in various psychiatric and neurological diseases. In the next section, we review evidence of the digestive and neurological aspects of mycobiome influence.