Evolution of the Human Pathogenic Lifestyle in Fungi

Abstract

Fungal pathogens cause more than a billion human infections every year, resulting in more than 1.6 million deaths annually. Understanding the natural history and evolutionary ecology of fungi is helping us understand how disease-relevant traits have repeatedly evolved. Different types and mechanisms of genetic variation have contributed to the evolution of fungal pathogenicity and specific genetic differences distinguish pathogens from non-pathogens. Insights into the traits, genetic elements, and genetic and ecological mechanisms that contribute to the evolution of fungal pathogenicity are crucial for developing strategies to both predict emergence of fungal pathogens and develop drugs to combat them.

Editor’s summary

Understanding the mechanisms and evolution of pathogenicity in fungi will bring us a step closer to reducing the annual toll of 1.6 million deaths from fungal disease.

Introduction

With ~150,000 species described, but with perhaps as many as a few million more species awaiting discovery^1,2, the kingdom of fungi is the lesser known of the “big” three eukaryotic kingdoms after animals and plants. And yet fungi are every bit as vital to our lives and the planet as animals and plants. Although fungi are intricately intertwined with human society, they remain largely unappreciated and understudied^3,4. Unlike bacterial and viral pathogens, fungi have received less attention in the context of human disease^5,6. However, recent data suggest that the global annual burden of fungal disease is enormous; superficial e.g., skin, hair, nail, and eye, infections are estimated to affect a billion people, mucosal e.g., oral, vaginal, infections ~135 million, allergic infections ~23.3 million, whereas chronic severe and acute invasive infections affect several additional millions of people and have extremely high mortality rates⁷. For example, mortality rates in certain groups of severely immunocompromised patients with invasive aspergillosis can be as high as 50%⁸. Fungal diseases are responsible for more than 1.6 million deaths annually, a rate on par with that of tuberculosis and more than three times higher than that of malaria⁷. These are staggering numbers, especially considering how little is known about the biology of fungal pathogens and the lack of recognition of the effects of fungal infections to human health^3,6.

One reason for the lack of attention to fungal pathogens lies in their opportunistic nature. In contrast to bacteria and viruses, fungi only emerged as important human pathogens in the past few decades, primarily owing to changes in the landscape of human disease⁹ (Figure 1); these changes include the dramatic increase of numbers of immunocompromised patients (owing to mutations that impair host immune function, cancer chemotherapy, or the effect of drugs that prevent transplant organ rejection), and the advent of new diseases that seriously compromise immune system function (e.g., AIDS). Unfortunately, but not surprisingly, fungal pathogens cause secondary infections in individuals with severe COVID-19 (Box 1). This opportunistic behavior also means that many of the traits and genetic elements that make fungal pathogens virulent are not unique or specific disease determinants but have likely evolved for survival in conditions independent of human infection. Therefore, understanding fungal virulence requires understanding the natural history, ecology, and adaptations of fungi that facilitate their success in their natural environments.

Figure 1.

Milestones in the study of fungal diseases.

Box 1. COVID-19 and fungal secondary infections.

Viral respiratory infections can predispose patients to secondary opportunistic infections or co-infections by fungi and bacteria¹⁰⁹. For example, it is well established that patients with severe influenza infections can sometimes acquire secondary Aspergillus infections¹¹⁰. More recently, secondary fungal infections have also been associated with COVID-19, the ongoing global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)¹¹¹. For example, a significant percentage of COVID-19 patients harbors secondary Aspergillus infections^112,113, and it is now widely recognized that CAPA (COVID-19-associated pulmonary aspergillosis) is an important complication among COVID-19 patients¹¹⁴, especially ones with severe lung damage, structural lung defects, or that received broad-spectrum antibiotics or corticosteroids¹¹⁵. More recently, an increase of secondary infections of COVID-19 patients with Mucor fungi has been noted, which too appears to be the result of opportunism, namely COVID-19 severe infections in patients with poorly controlled diabetes mellitus¹¹⁶. Fungal isolates from CAPA patients do not appear to differ in their genomic or phenotypic characteristics from those typically isolated from aspergillosis patients¹¹⁷, although only a few isolates have so far been examined.

This review discusses the where, why, and how of the evolution of human fungal pathogenicity. First, the repeated evolution of fungal pathogenicity and where it took place on the fungal tree of life is presented. Next, the opportunistic nature of human fungal pathogens is discussed, including how their ecological traits can help explain why some fungal species infect hundreds of thousands of patients annually while closely related fungal species are relatively harmless. Finally, this review tackles the question of how fungal pathogenicity evolved by discussing the types of genetic variation that give rise to variation in virulence. Fungal pathogenicity is the outcome of complex interactions between pathogens, human hosts, and their environments (Box 2). However, this review does not cover the role of the human immune system^10–12, nor the role of antifungal drug resistance for evolution of fungal pathogenicity^13,14, both of which are important topics and merit to be discussed in separate reviews.

Box 2. Pathogenicity and virulence.

The two terms are often used interchangeably but have somewhat different meanings. Pathogenicity refers to the ability of an organism to cause disease, whereas virulence refers to the degree to which an organism is pathogenic. However, both terms actually reflect the outcomes of complex interactions between microbes, their hosts, and their environments, so they are not absolute but relative¹¹⁸. For example, the virulence of a specific fungal strain is often measured as the minimum dose of fungal spores required to kill 50% of individuals from a given host model of fungal disease (e.g., mouse, fish, invertebrate). Furthermore, the virulence of a particular strain is typically compared to the virulence of a reference strain so that different strains can be readily classified as more or less virulent. Similarly, a fungal strain may be pathogenic, i.e., capable of causing disease, in one host genetic background or model of fungal disease but not in another¹¹⁹.

This relativism in the definitions of these two terms has led some to develop alternative approaches that aim to quantify the capacity for causing disease. For example, Casadevall recently developed the “pathogenic potential” measure, which takes into account the fraction of individuals that develop the disease, inoculum dose, host mortality, as well as variables such as communicability and time to disease¹¹⁸. Other efforts have instead aimed to use existing approaches, such as the survival curves drawn for assessing the virulence of different strains in a given model of fungal disease, in new ways. For example, Cramer and Kowalski recently argued that survival curve data can be used to identify and distinguish between “disease initiation” and “disease progression” virulence factors, which will enable study of not just disease establishment but also disease progression¹²⁰.

Fungal pathogenic traits have evolved repeatedly

The kingdom Fungi is extraordinarily diverse and contains more than two hundred orders and a dozen phyla^15,16, with new ones being described continuously¹⁷. However, the vast majority of infections and deaths caused by fungi result from a few hundred fungal species that belong to a few lineages (Table 1). These human fungal pathogens have repeatedly evolved from non-pathogens across major lineages of the fungal tree of life (Figure 2). Plotting the genera harboring the major human pathogens on the fungal tree of life reveals that human pathogenicity has evolved in more than a dozen different lineages. Interestingly, pathogenicity has also evolved repeatedly within some of these lineages, suggesting that they may harbor traits that preadapt them to human pathogenicity. For example, pathogenicity has evolved multiple times independently in Aspergillus fungi¹⁸. As such, the closest relatives of the two major pathogens causing aspergillosis, Aspergillus fumigatus (Figure 3) and Aspergillus flavus, are non-pathogenic^19–21. Human pathogenicity has also independently evolved within Onygenales, the order that contains dermatophytes and dimorphic fungi²², as well as within Mucorales which harbors the causative agents (Mucor, Rhizopus, and their relatives) of the devastating disease mucormycosis²³. Similarly, pathogenicity has independently evolved at least five times within budding yeasts^24,25, including in the causative agents of candidiasis, Candida (Nakaseomyces) glabrata and Candida albicans, and in the emerging pathogen Candida auris (Figure 2).

Table 1:

Human fungal diseases.

Disease	Major / notable pathogens	Region	Burden (all estimates from Ref.7 unless other reference is provided)	Genera involved	Phylum / Subphylum
Aspergillosis8	A. fumigatus, A. flavus	Worldwide	invasive: >300,000 annually chronic pulmonary: ~3 million global burden allergic bronchopulmonary: ~4.8 million global burden	Aspergillus	Ascomycota: Pezizomycotina
Blastomycosis128	B. dermatitidis	Regional (Central and Eastern US)	~3,000 global burden	Blastomyces	Ascomycota: Pezizomycotina: Onygenales
Candidiasis129	C. albicans, C. glabrata, C. auris	Worldwide	Invasive: ~750,000 annually Oral: ~2 million annually Oesophageal: ~1.3 million annually Vulvovaginal: ~134 million global burden	Many genera (Candida, Nakaseomyces, Clavispora, Pichia, Meyerozyma)	Ascomycota: Saccharomycotina
Coccidioidomycosis / Valley fever130	C. immitis, C. posadasii	Regional (Southwestern US, Central and South America)	~25,000 global burden	Coccidioides	Ascomycota: Pezizomycotina: Onygenales
Cryptococcosis131	C. neoformans, C. gattii	Worldwide (C. neoformans), Worldwide (C. gattii: expanding in California and Pacific Northwestern US)	~223,000 annually	Cryptococcus	Basidiomycota: Agaricomycotina
Emergomycosis132	E. pasteurianus	Regional (Southern Africa)	Tens / hundreds	Emergomyces	Ascomycota: Pezizomycotina: Onygenales
Fusariosis133	F. solani, F. oxysporum	Regional (South America)	Hundreds	Fusarium	Ascomycota: Pezizomycotina
Histoplasmosis134	H. capsulatum	Regional (Central and Eastern US, South America, Southern Africa, and Southeastern Asia)	Infections: ~500,000 annually / ~25,000 global burden Disseminated: ~100,000 annually	Histoplasma	Ascomycota: Pezizomycotina: Onygenales
Microsporidiosis46	Entercytozoon bieneusi, Encephalitozoon intestinalis	Worldwide	~10% prevalence135	Many genera (Encephalitozoon, Anncaliia, Enterocytozoon, Microsporidium)	Microsporidia
Mucormycosis136	Rhizopus arrhizus	Worldwide	>10,000 annually	Many genera (Mucor, Rhizopus, Lichtheimia, Apophysomyces, Rhizomucor, Cunninghamella)	Mucoromycota: Mucoromycotina
Paracoccidioidomycosis137	Paracoccidioides brasiliensis	Brazil, Central and South America	~4,000 global burden	Paracoccidioides	Ascomycota: Pezizomycotina: Onygenales
Pneumocystis pneumonia98	Pneumocystis jirovecii	Worldwide	~500,000 annually	Pneumocystis	Ascomycota: Taphrinomycotina
Sporotrichosis65	S. brasiliensis, S. schenckii, S. globosa	Worldwide, with increased prevalence in Central and South America	>40,000 annually	Sporothrix	Ascomycota: Pezizomycotina
Talaromycosis97	Talaromyces marneffei	South and Southeastern Asia	~8,000 annually	Talaromyces	Ascomycota: Pezizomycotina
Eye infections / fungal keratitis138	Aspergillus spp., Fusarium spp., Candida spp.	Worldwide	~1 million global burden	Multiple genera (Fusarium, Aspergillus, Candida)	Ascomycota
Skin, hair, nail infections64	Trichophyton rubrum, Trichophyton tonsurans, Microsporum canis, Malassezia globosa	Worldwide	~1 billion global burden	Multiple genera of dermatophytes (Trichophyton, Arthroderma, Microsporum) and Malassezia	Ascomycota: Pezizomycotina: Onygenales (for dermatophytes) and Basidiomycota: Ustilagomycotina (for Malassezia)

All estimates are from Ref. 7 unless another reference is provided.

Figure 2. Human pathogenicity has repeatedly evolved in fungi.

Genera and lineages harboring major and emerging fungal pathogens (see Table 1) are shown in red and non-pathogenic taxa are shown in black. The fungal tree of life based on a phylogenomic analysis of 1,644 species and 290 genes from Ref.¹⁶. Only species whose genomes have been sequenced are included. The tree with species names included is shown in Figure S1. Figure adapted with permission from ref. 16, Elsevier.

Figure 3. Repeated evolution of pathogenicity in Aspergillus section Fumigati lineage.

Species whose biosafety level (BSL) is 2 are considered pathogenic and shown in red. BSL1 organisms are shown in black. Estimated cases of invasive infection (K: in thousands) / year (y) are also shown. Phylogeny modified from^18,158–160; infection case estimates from^7,161. Figure adapted with permission from ref. 18, under a Creative Commons license CC BY 4.0.

In some instances, several species within a lineage are human pathogens. These closely related pathogenic fungi often exhibit significant differences in their pathogenicity. For example, although C. albicans and its closest known relative Candida dubliniensis are both human pathogens, C. albicans is much more virulent than C. dubliniensis²⁶. Similarly, the closely related pathogenic species in the genus Cryptococcus, which cause the potentially lethal fungal disease cryptococcosis, display substantial variation in their virulence and pathogenicity (Box 2)^27,28. For example, whereas Cryptococcus neoformans primarily infects immunocompromised individuals, Cryptococcus gattii infections primarily affect immunocompetent individuals^27,28. The dozen pathogenic species in the Aspergillus section Fumigati also display considerable variation in their virulence and antifungal drug resistance profiles²⁹.

Differences in the pathogenicity of closely related species can be observed in large lineages of major pathogenic species such as in Malassezia, a genus of basidiomycete yeasts that contains several species adapted to living on the human skin³⁰, as well as in the dermatophytes and dimorphic fungi in the order Onygenales. Onygenales harbors several different genera of so-called thermally dimorphic fungi, such as Blastomyces, Coccidioides, Histoplasma, and Paracoccidioides, which grow in mycelial form in typical environmental temperatures (e.g., 25°C) but switch to yeast growth at the human body temperature. These dimorphic fungi differ widely in their pathogenicity and disease profiles³¹. Another clade within Onygenales harbors multiple genera of dermatophyte fungi (e.g., Trichophyton, Epidermophyton, and Microsporum) that can cause skin infections, and which exhibit a wide variation in pathogenicity³².

Variation or heterogeneity in pathogenicity-associated genes and traits is not restricted between species and lineages but is also observed among strains within populations of fungal pathogens. This strain heterogeneity is both evolutionarily interesting (e.g., for revealing the genetic or epigenetic mechanisms that contribute to the evolution of pathogenicity) and clinically relevant (e.g., different strains often exhibit different virulence and antifungal drug resistance profiles). For example, strains of C. albicans^33,34 A. fumigatus^35–37, and of other Aspergillus pathogens³⁸ exhibit extensive genomic and phenotypic heterogeneity in their virulence and drug resistance profiles. Similarly, genetic diversity within the major pathogen C. neoformans is significantly associated with patient clinical outcome³⁹. Not much is known yet about the extent of this strain heterogeneity and how it may be influenced by differences in the sampling of strains between species, or how species boundaries are defined. However, studies in Aspergillus have shown that the amount of variation in virulence observed within a major pathogen is lower than that observed between the pathogen and its non-pathogenic closest relatives^20,36,37.

As mentioned above, the number of fungal species that are considered major pathogens is relatively small (Figure 2). However, it is worth noting that the spectrum of fungi capable of causing disease is likely much larger, and that nearly every fungus can be an opportunistic or accidental pathogen in a human host whose immune system is severely weakened (see also Box 2). Support for this hypothesis comes from clinical case reports of invasive infections by diverse, well-known species of fungi that are thought to be harmless to humans. These include, for example, the baker’s yeast Saccharomyces cerevisiae⁴⁰, the splitgill mushroom Schizophyllum commune⁴¹, the gray shag Coprinopsis cinerea⁴², and the basidiomycete pigmented yeasts in the genus Rhodotorula⁴³.

Human pathogenic fungi have originated independently multiple times across the fungal kingdom as well as within certain lineages, which highlights the remarkable versatility of these organisms and their ability to colonize new ecological niches, such as those provided by human hosts. It should be noted that most of the human pathogenic fungi, such as pathogenic species in the genera Aspergillus, Candida, Cryptococcus, Histoplasma, and Coccidioides, also infect many other animals, including other vertebrates and mammals⁴⁴. These animal infections, much like human infections, are caused via direct acquisition of fungal spores from the environment, but zoonotic outbreaks with direct transmission from animals to humans (e.g., cat to human transmission of Sporothrix brasiliensis⁴⁵) are also known⁴⁴. Thus, much like human pathogenicity (Figure 2), animal pathogenicity has also evolved multiple times independently across the fungal tree of life. Of course, the ability to cause disease in humans and the ability to cause disease in other warm-blooded animals are tightly linked since both rely on certain infection-relevant traits, such as thermotolerance (see next section). Are these infection-relevant traits shared by human pathogens across the fungal tree of life? Answering this major question requires understanding the lifecycle of a typical fungal infection and its opportunistic nature, which is discussed in the next section.

Ecological traits that aid opportunistic pathogenicity

Human pathogenic fungi differ greatly with respect to their degree of adaptation to their pathogenic lifestyles. At the one end of the spectrum, one finds obligate pathogens such as Microsporidia, a phylum of unicellular fungi that are intracellular parasites of a wide range of animal hosts⁴⁶. Passage through a host is a required part of microsporidian lifecycle and these pathogens have likely co-evolved with their hosts and possess adaptations for within-host survival. In the middle of the spectrum, one finds organisms that have a commensal relationship with their hosts. For example, the extracellular Pneumocystis yeasts cannot survive outside of a mammalian host (i.e., they are host-obligate), turning pathogenic in hosts with weakened immune systems⁴⁷. Budding yeasts that cause candidiasis are also commensal⁴⁸, although they are not host-obligate and recent studies have shown that these species are also present in the natural environment^25,49. It is likely that these commensals-turned-pathogens have also co-evolved, at least to some extent, with humans (in the case of budding yeasts) and mammals (in the case of Pneumocystis).

The great majority of the approximately 200 fungal pathogens that infect humans⁹, however, lie at the other end of the spectrum; they are typically not dependent on their hosts for survival and growth, and their pathogenicity is accidental or opportunistic^50,51. In nature, fungi are the primary decomposers of organic matter, growing on a variety of substrates and interacting with a wide range of organisms. Because most fungal pathogens are opportunistic, we can gain insights into their ability to infect humans by considering their natural environments. Soil, for example, is a common ecological niche where many opportunistic pathogenic fungi can be found. A single gram of soil harbors billions of microbial organisms from thousands of species belonging to dozens of taxonomic groups⁵². Survival in such a highly competitive environment requires many adaptations related to defense, feeding, and growth, and it has been argued that pathogenicity-associated traits are precisely those that also facilitate fungal survival in nature^53,54. A recent evolutionary ecological examination of more than 1,200 fungal species revealed a significant association between the ability to survive in multiple different types of extreme conditions (e.g., thermotolerance, osmotolerance, etc.) and opportunistic pathogenicity⁵⁵. For example, ascomycete fungi tend to be more widely distributed and thermotolerant than basidiomycete fungi^56,57, which may be part of the explanation as to why there are more lineages of opportunistic human pathogens in ascomycetes than in basidiomycetes (Figure 2, Table 1).

These observations suggest that understanding why fungi are such successful opportunistic pathogens will require detailed understanding of the natural fungal lifestyle and the ways in which the human host environment parallels their natural environment. One way to begin approaching this question is by examining the traits that distinguish pathogens from their non-pathogenic relatives. Differences in pathogenicity may stem from variation in a wide variety of ecological traits, such as in distribution and abundance^56,58, in the ability of fungi to grow at the human body temperature⁵⁷, in the ability to adapt to varying levels of oxygen⁵⁹, the preference for sexual versus asexual reproduction⁶⁰, and in the response to natural predators⁶¹.

If certain ecological traits are infection-relevant, it follows that we should expect human pathogens and their most closely related non-pathogenic relatives to exhibit significant differences in these traits. While most research so far has focused on just the pathogens, comparisons between pathogenic species and their non-pathogenic relatives provide support for this prediction. For example, the major pathogen A. fumigatus grows better at the human body temperature and is much more tolerant to oxidative stress or stress associated with nutrient and oxygen availability than its very close non-pathogenic relative Aspergillus fischeri²⁰. Differences in the ability to grow at the human body temperature are also observed between organisms in the pathogenic Cryptococcus species complex, which includes the major pathogens C. neoformans and C. gattii, and their closely related non-pathogenic relatives, such as Cryptococcus amylolentus^62,63.

Closely related pathogens also exhibit differences in ecological traits associated with human pathogenicity. For example, the pathogens C. neoformans and C. gattii differ substantially in their ecology (e.g., C. neoformans is more often associated with bird infections, whereas C. gattii with mammal infections), thermotolerance, and melanin production²⁷. For human skin commensals, such as Malassezia yeasts and dermatophytes, different species are typically associated with different body sites^30,64. Even in cases where little is known about the natural history of a lineage that harbors pathogenic species, the available evidence is suggestive of key differences in ecology. For example, the three most common causative agents of sporotrichosis, S. brasiliensis, S. schenckii, and S. globosa, show substantial differences in their geographic distributions and transmission routes⁶⁵. S. globosa, which is most prevalent in Asia, is commonly isolated from plant material, and wounds caused by such material are the main route of S. globosa human infections in this continent; infections by S. schenckii, which is most common in South Africa and Australia, also typically stem from an environmental transmission route⁴⁵. In contrast, the main route for human infections by S. brasiliensis, which is most prevalent in Brazil, is via infected domestic animals, such as cats and dogs⁴⁵.

Examination of some of these ecological traits has been key to our understanding of fungal pathogenicity and how it may have evolved. For example, many opportunistic fungal pathogens are saprophytic organisms that live in the soil where they are predated upon by diverse organisms, such as amoebae, whose functions in the natural environment can be perceived to parallel those of phagocytes in the human host environment⁶⁶. This hypothesis, which was first raised and tested in 2001 with C. neoformans, yielded two striking results: first, fungal interactions with amoebae were similar to interactions of the fungus with macrophages, and second, several traits, such as melanization, that contribute to fungal resistance against mammalian immune cells, also protect from amoeba predation⁶⁶. These discoveries have spearheaded a body of work examining how the coevolution of fungi with their natural predators may have accidentally favored or selected for the evolution of human pathogenicity and ability to withstand host defense strategies^61,67, not just in Cryptococcus^68,69 but also in other soil fungi, such as Aspergillus⁶¹ and Paracoccidioides⁷⁰. For example, a recent examination of the interactions between Paracoccidioides opportunistic fungal pathogens and their natural amoeba predators, showed that repeated exposure of Paracoccidioides to predatory amoebae increased the ability of these fungi to survive mammalian macrophages and to infect mice⁷⁰. Interestingly, studies on Cryptococcus have shown that prolonged growth in the presence of predatory amoebae selected for mutations that promote pseudohyphal (rather than yeast) growth, which increase resistance to macrophages but reduce virulence^68,69. Data are lacking on whether this variation is observed in the natural environment but raise the hypothesis that interactions of fungi with other organisms may generate substantial phenotypic diversity that is relevant for the capacity of individual strains to infect humans.

One prediction that follows from the repeated evolution of human pathogenic fungi is that several of their infection-relevant ecological traits may too have evolved repeatedly (convergent evolution). For example, thermotolerance is widely regarded as a key trait for fungal pathogens of humans and other warm-blooded animals and harbors this signature of convergent evolution⁵⁷. Another trait that has repeatedly evolved in human pathogenic fungi is osmotolerance⁵⁵. One particularly noteworthy example of convergent evolution is the developmental ability of certain human pathogenic fungi to switch between filamentous or mycelial growth and yeast growth, which has evolved multiple times independently across multiple fungal phyla⁷¹ and is observed in diverse pathogens, including C. albicans and C. neoformans. Some of the most notable examples of organisms that exhibit this morphogenetic switch are the thermally dimorphic fungi that have independently evolved in the orders Onygenales (e.g., Histoplasma, Blastomyces, Coccidioides, Paracoccidioides), and Ophiostomatales (which includes Sporothrix); the trait also evolved independently in Talaromyces marneffei (order Eurotiales)⁷². In these thermally dimorphic fungi, the switch from filamentous to yeast growth during infection confers protection against host defense responses⁷². Interestingly, whereas thermal dimorphism is widespread in the orders Onygenales and Ophiostomatales, only a single species from the order Eurotiales (T. marneffei) is known to be dimorphic⁷¹.

Once associated with human hosts, fungal survival and reproductive strategies may quickly diverge from strategies favored when they are in their natural environments. For example, comparisons of clinical and environmental strains of S. cerevisiae have revealed that clinical strains show higher levels of heterozygosity, a reduced ability for sexual reproduction, and an increased propensity for pseudohyphal development than environmental strains^60,73.

Finally, it is worth noting the potential limitation of this evolutionary approach, namely the assumption that diverse fungal pathogens share infection-relevant ecological traits. Although the examples discussed above suggest that this is indeed the case for traits such as thermotolerance and osmotolerance, the question remains whether there are other convergent traits shared by opportunistic human fungal pathogens. A non-mutually exclusive alterative is that understanding of fungal pathogenicity will require a detailed dissection of the interactions of each pathogen with the human host because each pathogen has its own unique suite of infection-relevant ecological traits. One notable example of this alternative hypothesis are secondary metabolites, which are small, bioactive molecules biosynthesized by certain fungi, and that play key roles to their ecology. Secondary metabolites produced by fungal pathogens, such as A. fumigatus, have been shown to influence host biology and pathogenicity⁷⁴. However, the ability of several other pathogens to biosynthesize secondary metabolites is either limited (e.g., C. albicans, C. neoformans) or is reduced relative to their non-pathogenic relatives (e.g., dimorphic fungi⁷⁵).

Bridging evolutionary analyses with targeted genetic studies can elucidate the genetic basis of several infection-relevant ecological traits in fungal pathogens and help refine our concept of how fungal pathogenicity evolves. The next section describes how genetic variation associated with these traits has contributed to the evolutionary origin and maintenance of fungal pathogenicity.

Fungal genomics and human pathogenicity

Fungal pathogenicity is the outcome of complex interactions between pathogens, human hosts (e.g., host immune system status), and their environments (e.g., spore availability) (Box 2). While host genetics, host immune system status, and environment certainly contribute to the manifestation of fungal disease, differences in genetic elements associated with infection-relevant ecological traits are also major contributors. Genetic variants that have contributed to the evolution of fungal pathogenicity can be broken down into two broad categories or types: larger-scale genomic changes that affect the entire genome or large parts of it, such as hybridization⁷⁶, introgression⁷⁷, transposon mobilization⁷⁸, loss of heterozygosity⁷⁹, and variation in ploidy⁷⁹, and smaller-scale changes that typically affect a single genomic region, such as copy number variation⁸⁰, gene duplication⁸¹, gene loss⁷⁵, horizontal gene transfer⁸², indels and single nucleotide polymorphisms³³ (Figure 4). It is also important to emphasize the remarkable plasticity of fungal genomes with respect to the range of mechanisms and processes that can give rise to this genetic variation, including their diversity of reproductive strategies⁸³.

Figure 4. Genetic variation and the evolution of infection-relevant traits.

Some of the types of genetic variation illustrated typically affect large genomic regions or entire genomes; these include (A) variation in ploidy, (B) loss of heterozygosity, (C) transposon mobilization, (D) introgression, and (E) hybridization. Note that the red branch leading to taxon D in the hybridization panel is meant to illustrate the origin of a new hybrid. Other types of variation typically affect a single locus; these include (F) copy number variation, (G) horizontal gene transfer, (H) single nucleotide polymorphisms, (I) cis-regulatory element variation, and (J) gene duplication and loss. Copy number variation could involve linear or circular DNA. Most examples of genetic variation concerning the evolution of human fungal pathogens identified focus on or concern variation in the protein-coding regions of the genome. However, variation of cis-regulatory elements (panel I), which can alter gene activity, can also have a major impact in fungal pathogen evolution. We currently lack understanding of the relative frequency with which these mechanisms operate in different fungal pathogens. It is also likely that these mechanisms differ in their prevalence in fungal genomes. Figure adapted with permission from: a,b, ref. 162, American Society for Microbiology; c, ref. 163, Springer Nature Ltd; d,e, ref. 164, Springer Nature Ltd; f, reprinted courtesy of the National Human Genome Research Institute, https://www.genome.gov; g, ref. 165, Springer Nature Ltd; h, ref. 166, Springer Nature Ltd; i, ref. 167, under a Creative Commons license CC BY 4.0; j, ref. 168, under a Creative Commons license CC BY 4.0.

Comparisons of the genomes of pathogenic fungi and their non-pathogenic relatives have identified numerous larger- and smaller-scale genomic differences associated with the origins of pathogenicity, implicating many genes with diverse functions⁸⁴. For example, one significant difference between thermally dimorphic fungal pathogens and their nonpathogenic relatives is that pathogens have lost secondary metabolic genes and genes associated with the degradation of plant material⁷⁵. Similarly, a recent comparative genomic examination of the host-obligate Pneumocystis species revealed extensive between-species variation in the msg superfamily, whose members are involved in pathogen-host interactions⁸¹. An examination of horizontal gene transfer in Malassezia identified more than two dozen genes that were likely acquired from bacteria, including a flavohemoglobin-encoding gene, which was shown to be involved in nitric oxide resistance and interaction with the human host⁸². Comparisons between Aspergillus pathogenic and closely related non-pathogenic species have revealed significant differences in the presence of biosynthetic gene clusters involved in secondary metabolite biosynthesis⁸⁵; several of these bioactive, small molecules are known to be important to Aspergillus ecology and to modulate human host biology⁷⁴.

While many of the known variants are from the protein-coding parts of the genome, differences in the regulation of genes that are conserved in both pathogens and non-pathogens can also contribute to differences in pathogenicity. As mentioned above, C. albicans and C. dubliniensis differ in their virulence but are very closely related and do not contain many differences in gene content⁸⁶. However, a systematic examination of differences in gene expression of orthologous genes between the two species revealed that all 15 genes involved in glycolysis were more highly expressed in C. albicans than in C. dubliniensis²⁶. Strikingly, genetic engineering of a C. dubliniensis strain that expressed all 15 glycolysis genes at higher than native expression levels led to an increase in virulence²⁶. Thus, much like is the case for other traits^87,88, changes in pathogenicity and infection-relevant traits may stem from genetic changes in both the protein-coding parts and the regulatory parts of the genome.

Genetic variants associated with pathogenicity are also found in examinations of within-species variation, an observation in line with the heterogeneity in infection-relevant traits observed between strains of individual fungal pathogenic species. Strains of the major pathogen A. fumigatus show variation in the structure of their biofilms, which influences the ability of strains to grow in low oxygen environments, such as that encountered inside human lungs. Interestingly, this variation stems from variation in the presence of the hrmA gene across A. fumigatus strains³⁵. Similarly, examination of genomic variation in strains of the major pathogen C. albicans identified numerous genetic changes, including single nucleotide polymorphisms, that contributed to strain variation in virulence and other infection-relevant traits³³. Comparison of S. cerevisiae clinical and environmental strains revealed higher levels of heterozygosity in clinical strains and identified significant associations between specific genetic variants and pathogenicity-associated phenotypes, such as increased copper resistance^60,73.

Looking into the past and reconstructing how pathogenicity evolved using comparative genomics is one approach toward understanding the observed differences between pathogens and non-pathogens. An independent approach is to ask how pathogenicity could evolve, which can be achieved through experimental evolution approaches⁸⁹. Such experiments typically select (over many generations) those individuals in a fungal population that show increased survival or growth in a particular environment (e.g., the oral cavity⁹⁰) or that exhibit a particular infection-relevant trait (e.g., thermotolerance⁹¹, and reduction⁹² or increase of virulence⁹³). For example, repeated passaging of environmentally derived isolates of C. neoformans through mice resulted in significant increases in virulence⁹³. This was, at least partly, due to the higher expression of the FRE3 gene, which encodes for an iron reductase. In the commensal C. albicans, experimental evolution for loss of virulence via repeated passaging through a mammalian host identified key genes and traits associated with the transition from commensalism to mutualism⁹². Additionally, repeated passaging of the same species through the mouse oral cavity⁹⁰ and gastrointestinal tract⁹⁴ led to the identification of a chromosome 6 trisomy that was shown to result in a commensal-like phenotype (in the oral cavity experiment⁹⁵) and of a chromosome 7 trisomy that increased fitness in the gastrointestinal tract⁹⁴.

Finally, it is worth noting that some of the examples above concern genetic mechanisms that contributed to the origin of human (and / or animal) pathogenicity in the first place (e.g., gene content variation stemming from differential gene duplication and loss between pathogens and their non-pathogenic close relatives). Other examples concern genetic mechanisms that shaped adaptation during evolution inside the human host or in response to interventions, such as treatment with antifungal drugs (e.g., variation in ploidy, heterozygosity, and gene copy number). We currently lack understanding of the relative frequency with which these mechanisms operate in different fungal pathogens and of their relative importance for the origin of pathogens vs. the maintenance of the human pathogenic lifestyle.

Outlook

In the last two decades, adoption of an ecological and evolutionary perspective, coupled with the huge advances in genomics, has revolutionized medical mycology^83,84, greatly illuminating the broad contours of the where, why, and how of the evolution of fungal pathogenicity. But several major gaps remain, including our understanding of the genetic and ecological factors that contribute to the emergence of new human fungal pathogens. In a planet with a rapidly changing climate that has witnessed the emergence of several new pathogens (Box 3), forecasting the emergence of new pathogens is becoming more urgent than ever^9,51. Understanding how pathogens evolve as well as figuring out the genetic determinants that contribute to the origin and maintenance of fungal pathogenicity could also aid in the identification of potential vulnerabilities that could be targeted for new therapeutics^13,14. Below I outline three grand challenges that, if tackled, promise to greatly advance our understanding of the origins of fungal pathogens of humans, potentially facilitating the development of models that predict the emergence of new ones and of therapeutics that better combat fungal infections.

Box 3. Climate change and the evolution of new fungal pathogens of humans.

Fungal populations can readily respond to selection for growth at higher temperatures in experimental evolution studies⁹¹. Given that most fungi typically grow on lower temperatures and that the high temperature of the human body likely acts as a preventive barrier for their growth, it has been hypothesized that increasing global temperatures will inadvertently select for thermotolerant fungi that are more likely to cause opportunistic human disease¹²¹. Evidence for this global warming emergence hypothesis has been increasing¹²².

Arguably one of the most fascinating (and frightening) examples concerns the emergence of the novel pathogen Candida auris. First discovered in 2009, this new pathogen is now known to have caused infections in more than 30 countries from six continents, including nosocomial outbreaks, and its clinical isolates exhibit resistance to all known antifungal drugs^123,124. Remarkably, infections on different continents occurred near simultaneously and originated from different lineages of the C. auris phylogeny¹²⁵. It therefore appears that there was not one emergence of C. auris, but multiple, simultaneous ones.

But how did C. auris emerge and where did it come from? The short answer is that we do not definitely know because little is known about the organism’s ecology and geographic distribution, but the current working hypothesis is that this might be the first fungal disease that has originated because of global warming¹²⁶. Recent support for this hypothesis came from the isolation of C. auris from the coastal wetlands of the tropical Andaman Islands, India¹²⁷, suggesting that the organism does have an environmental reservoir and that strains infecting patients could have been environmentally acquired.

Challenge #1: We have limited understanding of fungal biodiversity and ecology.

This is arguably the biggest knowledge gap, especially considering the opportunistic nature of most major fungal pathogens and the frequent emergence of new ones. We still lack fundamental understanding of the diversity of fungal species^1,2,96. Even for the small fraction of species that are known to science, including of most fungal pathogens, we do not typically know their natural distributions and ecological niches or how pathogenic and non-pathogenic fungi interact with other organisms in nature. While some of that knowledge is available for certain pathogens (e.g., T. marneffei natural reservoirs in wild rodents are well-defined, linking the organism’s ecology with disease epidemiology⁹⁷), it is lacking for many others, hindering efforts to understand pathogen biology and epidemiology. For example, we do not know the environmental reservoirs, if any, of Pneumocystis species⁹⁸, the zoonotic reservoirs for many microsporidian human pathogens⁴⁶, or the ecology of the Sporothrix emerging human pathogens⁶⁵.

Challenge #2: Lack of systematic characterization of pathogens and non-pathogens.

In most lineages harboring pathogens and their non-pathogenic relatives, we lack data on the phenotypic profiles of the non-pathogens and their growth characteristics in infection-relevant conditions; for example, for many non-pathogenic relatives of major pathogens (e.g., C. glabrata⁹⁹, A. fumigatus¹⁸) data are lacking on their ability to grow at 37°C or their tolerance to various infection-relevant stresses. This lack of systematic data collection with respect to both challenge #1 and challenge #2 makes it difficult to begin to understand and make predictions of where new human pathogens are more likely to emerge from. From lineages that are thermotolerant or extremotolerant? From lineages that harbor fungal pathogens of other mammals? From lineages that live in extremely competitive environments or are widely geographically distributed? For example, temperature growth assays have shown that species in the pathogenic C. neoformans / C. gattii clade have the capacity to grow at the human body temperature, whereas closely related non-pathogenic relatives do not, suggesting that the ability to grow at the human body temperature evolved in the ancestor of the pathogen clade. Thus, evolution of thermotolerance is tightly coupled to the evolution of pathogenicity in Cryptococcus, but whether this pattern is observed in other clades containing fungal pathogens remains unknown. While several hypotheses have been proposed and associations have been drawn, systematic testing of these hypotheses is lacking, largely because of the historical unavailability of large, synthetic data sets that contain the high-quality data necessary to address these questions. Large-scale data sets of fungal genomic¹⁰⁰, evolutionary^16,101,102, taxonomic¹⁰³, and ecological¹⁰⁴ diversity are going to be invaluable in this synthesis.

Challenge #3: Lack of understanding of the relationship between genotype and phenotype for pathogenicity- and virulence-related genes and traits.

The amount of genomic and phenotypic variation or heterogeneity in pathogenicity across human fungal pathogens remains largely unknown. Research in S. cerevisiae, where clinical strains show a reduced ability for sexual reproduction and an increased propensity for pseudohyphal development, raises the hypothesis that fungal survival and reproductive strategies favored inside human hosts will be distinct from those in their natural environments^60,73, but we still lack understanding of the degree and speed with which fungi can alter these key traits to adapt to the human host environment. We also lack understanding of how much of the observed phenotypic variation has a genetic basis, and the heritability of most infection-relevant traits in most pathogens remains unknown. This makes it challenging to infer what cellular pathways should be targeted for the development of fungal vaccines and antifungal drugs. For example, a recent study showed that genetically identical asexual spores of different fungal species, including those of the pathogens A. fumigatus and T. marneffei, exhibit substantial phenotypic diversity¹⁰⁵. However, diverse approaches are now available to study the proportion of phenotypic variation that stems from genetic variation, including genome-wide association studies (GWAS)^106,107, reverse ecology⁸⁴, as well as a range of phylogenetic methods¹⁰⁸. These analyses can link the genotypic and phenotypic variation observed not only within but also between fungal species.

Conclusion

Now that fungal genomes can be sequenced in the hundreds and thousands, and tools that enable the genetic and molecular dissection of infection-relevant traits are developed, the limiting factor in tackling pathogenic fungi is a better understanding of fungal ecology and natural history. Which lineages and ecological lifestyles will human fungal pathogens emerge from? What are the key genetic and ecological differences that distinguish pathogenic fungi from their non-pathogenic relatives? Answering these vital questions will require the synthesis of genomic, evolutionary and ecological features of fungal lineages.