Cultivating fungal research

Fungi cover the epithelial surfaces of the human body, engaging in many mutualistic interactions with the host and other microbiota such as the more prevalent bacteria. These interactions are shaped by host physiology and immunity, nutrient competition, and multiple other factors. Beneficial fungal effects on hosts include colonization resistance against pathogens and tuning the immune system. Within microbial communities, bacteria and fungi inhabit the same niches and can affect each other as well – epitomized by the fungal Penicillium spp. producing the antibacterial molecule penicillin and vulvovaginal candidiasis developing after systemic antibiotics. While health benefits continue to be explored, recent studies have revealed expanded roles of fungi in human disease, including inflammatory disorders and specific cancers. The global burden of fungal infections also is expanding with increased numbers of at-risk patients, and increased resistance to a limited set of antifungal drugs. More fungal research is needed.

Fungi exist as single-celled yeast, multi-cellular molds, or dimorphic species occurring as both yeast and filamentous cells. Saccharomyces cerevisiae, Cryptococcus neoformans, Candida and Malassezia spp. are frequently studied yeast, while Penicillium, Mucor, and Aspergillus are well known molds. As with many microbiota, fungi are classically recognized for their roles as human-associated pathogens. Candidal bloodstream infections are the most common form of fungal invasive disease affecting approximately 1 in 10,000 patients in the United States. Globally, cryptococcal meningitis contributes to a substantial burden of disease, particularly in HIV positive individuals. The paucity of available antifungal treatments contributes to the morbidity and mortality of fungal infections. Classes of antifungals include azoles (e.g. fluconazole), echinocandins and amphotericin B. However, fluconazole is the only antifungal drug available in many parts of the world. Additionally, fungal diseases can be difficult to diagnose due to nonspecific patient symptoms, invasive tissue sampling, specific culture conditions, and identification requiring histopathologic, biochemical, or genetic means.

More recent studies have begun to explore how fungi are multi-faceted in their potential to lead to beneficial as well as pathogenic outcomes for the host. Commensalism in the context of human fungi is exemplified by colonization resistance against pathogens. An example of a beneficial effect is the dominant human skin associated Malassezia, which have adapted to their niche to utilize skin lipids as a nutrient, and then secrete antimicrobial products which deter bacterial pathogens (1). Another example of the importance of colonization resistance is Candida albicans commensalism in the gastrointestinal tract. In an evolutionary experiment, C. albicans strains acquired genetic mutations that enabled them to more stably colonize the mouse gastrointestinal tract. Interestingly, gut colonization with these evolved C. albicans strains provided protection against subsequent experimental challenges with different virulent fungi (fully virulent C. albicans, Aspergillus fumigatus) and bacteria (Staphylococcus aureus, Pseudomonas aeruginosa) (2). However, these C. albicans strains only evolved in antibiotic-treated mice and were unable to stably colonize mice with endogenous gut bacteria, highlighting the genetic tradeoffs of adaptation to the host and competition within mixed microbial communities. Complementary studies demonstrated that mutations in transcription factors that regulate morphology are key determinants of C. albicans’ gut commensal fitness (3). In addition to these direct host-microbial interactions, mouse gut colonization with C. albicans tuned host immunity with systemic increase in fungal-specific Th17 CD4+ T cells and IL-17-responsive circulating neutrophils, which protected against more invasive bacterial and fungal, but not viral, infections (4). Ex vivo experiments showed that C. albicans elicited robust IL-17A and IL-22 responses from peripheral C. albicans- and A. fumigatus-specific Th17 CD4+ T cells from healthy human donors, demonstrating C. albicans can distinctly modulate human immunity as well (5).

Given the complexity of host-microbial interactions, any alteration in the host or microbiota can result in infections, ranging from chronic chromoblastomycosis, dermatophyte nail infections, and acute vaginal yeast infections to devastating mucomycosis in diabetics, candidal sepsis, and disseminated aspergillosis. Deficiencies in human immunity and their association with fungal infections generate hypotheses about selective regulation of the host-microbiome interplay. For example, patients with advanced HIV infections suffer from specific opportunistic fungal infections such as cryptococcal meningitis, mucosal candidiasis, and Pneumocystis jirovecii pneumonia. In transplant patients, candidal bloodstream infections were preceded by blooms of intestinal Candida spp. with altered bacterial communities which could be used as biomarkers to identify and alter medical management in at-risk patients (6). Patients with genetically defined primary immunodeficiency syndromes, e.g. chronic granulomatous disease, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), or caspase recruitment domain-containing protein 9 (CARD9) deficiency, and the specific fungal infections they suffer reflect the specificity of fungal-host immune interactions. Other examples of host-specific fungal susceptibilities include mice without chemokine (C-X-C motif) receptor 1 (Cxcr1) which have impaired defective neutrophil killing of Candida with decreased survival and higher fungal burden, similar to disseminated candidiasis patients and healthy donors with the CXCR1-T276 allele who also demonstrate impaired neutrophil killing of Candida (7). Recurrent vulvovaginal candidiasis, a localized fungal susceptibility, is estimated to affect >100 million women worldwide annually and has been linked with a single nucleotide polymorphism (SNP) in the SIGLEC15 (sialic acid binding Immunoglobulin like lectin 15) gene (8). Expressed on immune cells, SIGLEC15 can bind C. albicans inducing IL-17A and IFNγ production, suggesting a role in anti-C. albicans immune responses. While many hosts have the potential to develop fungal infections in the instances of dermatophytes or penetrating wounds, host susceptibility contributes towards the development of more persistent or severe fungal infections. These and other similar reports of SNPs in immune-related genes that correlate with increased susceptibility to mucosal or life-threatening fungal infections hold promise for the future development of precision medicine approaches for risk stratification and prophylaxis of vulnerable patients at the highest risk for fungal disease.

Beyond infections, inflammatory and autoimmune diseases are increasingly being linked to alterations in fungal communities, particularly in genetically defined hosts. SNPs in CARD9 (caspase recruitment domain family member 9) and CLEC7A (C-type lectin domain containing 7A) that encodes Dectin-1, a C-type lectin receptor (CLR) that recognizes fungal cell wall β-glucan and signals, via CARD9, to induce inflammatory mediators and Th1/Th17 differentiation have been associated with inflammatory bowel diseases in humans. Indeed, mice deficient in Dectin-1 had more severe experimentally-induced colitis with increased C. tropicalis burden and a SNP in human CLEC7A is associated with more severe ulcerative colitis in patients (9). Similarly, Crohn’s disease patients have higher relative abundances of intestinal Malassezia compared with healthy controls, and oral gavage of Malassezia demonstrated worsening of experimental colitis in mice, again via activation of CARD9 signaling and downstream Th1/Th17 polarization (10). Genetic ablation of CX3CR1⁺ mononuclear phagocytes, which express antifungal CLRs, also exacerbated experimental colitis in mice; furthermore, a missense mutation in CX3CR1 in Crohn’s patients has been associated with reduced IgG responses to fungi (11). In another example of fungal-associated pathologic inflammation, while C. albicans may tune the human immune system by inducing anti-fungal Th17 cells, A. fumigatus-reactive Th17 cells, which were also cross-reactive to C. albicans, were increased in the peripheral blood of lung disease patients, particularly in acute allergic bronchopulmonary aspergillosis, suggesting an aberrant inflammatory Th17 response in sensitized patients (5). These and other studies have shown that inflammatory diseases may result from host-fungal imbalances.

Although bacteria and viruses have been implicated in cancer, fungi have not been typically linked with cancer pathogenesis. Following observations of higher levels of intratumoral fungi, specifically an enrichment of Malassezia, infiltrating human pancreatic ductal adenocarcinomas (PDA), mouse models of PDA demonstrated fungal translocation from the intestinal tract into the pancreas, increased burden of Malassezia accelerating PDA progression, and antifungal treatment slowing pancreatic cancer growth (12). The complement cascade integrates immune recognition and killing of fungi with tumor development by stimulating pro-inflammatory pathways. Expression of mannose binding lectin, which activates the complement cascade in blood, was associated with worse survival outcome in PDA patients. Similarly, PDA progression in mice depended on Malassezia stimulating mannose-binding lectin activation of the complement cascade, linking fungi, inflammation and tumorigenesis. This study has prompted researchers to reconsider the potential relationships between fungi and a broader range of human diseases.

Candida auris epitomizes the gravest concerns about an emerging fungal pathogen as it has evolved resistance to all classes of antifungal drugs, particularly azoles. A dozen countries have reported active outbreaks, with increasing cases of C. auris bloodstream infections. High levels of resistance render these infections difficult to treat with resultant high mortality. Over the last decade, four distinct strains of C. auris have emerged independently on different continents. The origin of C. auris as a human pathogen has remained a mystery since it was first identified in 2009. C. auris’ propensity to colonize human skin for long stretches of time, which is an atypical feature for non-auris Candida species, is of substantial concern as shedding from patients into the environment facilitates transmissions within healthcare facilities (13). The emergence of a new human fungal pathogen points to several urgent unmet medical needs: need for new antifungal drugs and environmental disinfectants as well as genomic datasets that include fungal sequences to map global fungal diversity and a coordinated global health response.

Investigations into the role of fungi in human health and disease are not without challenges. Despite a myriad of bacteria, fungi, and viruses existing together in and on humans, reductionist studies focusing on a few microbial species are more tractable. Fastidious culture conditions or inclusion of metabolically distinct forms of dimorphic fungi (3) should also be considered. In vivo systems can have limitations including conventional laboratory mice with a lower fungal burden and different immune profiles than wild or wildling mice (14). Additionally, adaptation to stress – cultivation conditions, passaging through mammalian hosts, antifungal pressures, high temperatures, acidic pH, etc. – can lead to alterations in morphology, fungal capsule, or cell wall components such as glucan, chitin, or mannan resulting in evasion or triggering of host immunity and radically altered genomes with only a minority of fungal cells retaining their original genomes. Genomic plasticity, including loss of heterozygosity, copy number variation, or aneuploidy, in fungi underscores the importance of incorporating metagenomic analyses into studying fungal community adaptation to different niches (15). The expansion of existing databases of fungal genomes is also needed to differentiate between strains and more deeply understand fungal virulence and pathogenesis.

Fungal research is an area of considerable potential. This includes understanding how fungi are beneficial to human health and mining the multiple compounds produced by fungi that may benefit clinical medicine. The emergence of multi-drug resistant C. auris has been postulated to be in part due to rising temperatures as well as the widespread use of azoles in agriculture and in the clinic. Since these pressures on host-microbial homeostasis are likely to persist and evolve, the development of newer antifungals and transmission barriers are critical to counteract outbreaks from existing and future pathogens. Novel scientific technologies, such as targeted CRISPR/Cas9-mediated gene deletion for fungal mutagenesis, can provide precise tools for testing genetic hypotheses and elucidating fungal pathophysiology. Genomics advances as well as newer experimental systems have spurred the growth of fungal studies in infections, inflammation, and other diseases and will continue to improve our understanding of how fungi affect human health and disease.

FIGURE. Fungi contribute to host health and disease.

Fungal-host interactions can be beneficial (pathogen resistance, tuning host immunity) or detrimental (infection, inflammation, cancer) to the host.