Short-term evolution strategies for host adaptation and drug escape in human fungal pathogens.

Research on human fungal pathogens has historically taken a backseat to other infectious diseases, perhaps due to a common misperception that fungi largely cause superficial infections [1]. In reality, fungi can be life-threatening to those who become immunocompromised during medical procedures or through conditions such as HIV and diabetes. Invasive fungal infections are estimated to kill over 1 million people every year, with mortality rates reaching 50% [2]. Significant challenges to the treatment of fungal infections include the limited availability of antifungals and the innate ability of fungi to rapidly evolve and adapt to fluctuating conditions. This adaptive ability is partially driven by extensive genomic plasticity, with many species acquiring diverse ploidy states, chromosomal rearrangements, and point mutations during host colonization [3–8]. Genetic plasticity enables rapid increases in virulence and antifungal drug resistance, which often translate to poor disease outcomes. Short-term evolution (microevolution) strategies in fungal pathogens are therefore essential for environmental adaptation in the mammalian host, and their study can inform adaptive mechanisms in other eukaryotes.


Loss of heterozygosity as a driver of phenotypic variation in diploid species
Another strategy that has emerged as an important mechanism for adaptation is loss of heterozygosity (LOH) or the loss of genetic information from one chromosome homolog. LOH can arise via chromosomal double-strand breaks (followed by break-induced replication or other repair mechanisms) as well as via recombination between homologous chromosomal regions, which can occur both mitotically and meiotically. LOH events can vary dramatically in size, depending on the type of event, affecting single polymorphisms (i.e., gene conversions) to whole chromosomes (i.e., non-disjunctions). Large LOH events can single-handedly impact multiple genes through the homozygosis of hundreds to thousands of positions [19]. While diploid genomes minimize the impact of de novo mutation by maintaining an ancestral copy of an allele, LOH can unmask recessive variants under conditions where they endow a fitness benefit. This is particularly relevant for heterozygous diploid Candida species but also for C. neoformans VNIII (AD) hybrids [20]. LOH events across short regions via gene conversion or via segmental or whole chromosome loss are common in C. albicans clinical isolates [8,17] and strains passaged in mice [3,4,10,21]. Studies of LOH distribution found the majority of LOH breakpoints within or adjacent to repeat sequences, suggesting repetitive DNA may promote genetic recombination between or within homologs [17,19]. Such recombination events can lead to subsequent de novo mutations via error-prone DNA repair mechanisms, further adding to the ability of LOH to accelerate fungal evolution. Evidence of this phenomenon can be seen in the significant enrichment of heterozygous single nucleotide polymorphisms (SNPs) adjacent to LOH breakpoints in C. albicans [19]. LOH can also maximize the impact of de novo point mutations. Analysis of serial isolates from 11 candidiasis patients detected large LOH events in isolates from all patients, resulting in the homozygosis of 130 de novo SNPs [8]. Several C. albicans studies found that LOH on one chromosome significantly increases the likelihood of LOH at other loci, thereby amplifying the impact of LOH in this species and suggesting that LOH may be a concerted process [4,21,22].
A couple of recent examples highlight how LOH across recessive alleles can alter both virulence and commensalism in C. albicans. In one study, passage through the mouse gastrointestinal (GI) tract selected for LOH events, which inactivated the hyphal-regulator FLO8 via homozygosis of nonsense and frameshift mutations [10]. Loss of Flo8 led to a commensal phenotype, with strains losing their virulence and providing immune-priming against secondary fungal or bacterial infections [10]. Loss of function of another central regulator of filamentation, Efg1, was similarly associated with increased fitness in the mouse GI tract [23]. Interestingly, heterozygous null mutations in EFG1 are common across C. albicans clinical isolates, and frequent LOH at this locus led to EFG1 inactivation during GI tract passage [24]. These findings demonstrate how LOH events can significantly impact the evolutionary trajectories of C. albicans during host colonization. Furthermore, LOH of hemizygous alleles of key regulators may represent a common mechanism by which C. albicans can increase its fitness in the host.

Small-scale genetic variation with genome-wide consequences
Large-scale genomic changes such as aneuploidy and long-tract LOH events can affect hundreds to thousands of genes in a single cell cycle but also carry a significant deleterious risk. It is therefore not surprising that the majority of mutational events observed during fungal microevolution are represented by small-scale mutations, including SNPs, insertions and deletions (indels), and short-tract LOH [7,8,19]. These small variants can also have extensive downstream consequences when they affect critical genes. One example are mutations in central transcription factors, in which a single mutation can alter the expression of hundreds of genes. In C. neoformans VNI, the commonly used laboratory lineage H99 contains a deletion in the transcriptional regulator Sgf29 encoding a histone acetylase [25]. Several clinical isolates independently evolved loss of function mutations in the SGF29 gene, the inactivation of which impacted acetylation at more than 700 loci and led to a hypervirulent phenotype [25].
Mutations within DNA repair genes can also impact trajectories of evolution. Mutations in the mismatch repair gene MSH2 have been reported in Candida glabrata [26] and C. neoformans VNI [27], where they produce a hypermutator phenotype. In C. neoformans VNI, MSH2 loss of function mutations produced strains with high mutation rates and extensive phenotypic variability [27]. In C. neoformans VNI and VNIV, an amino acid substitution in the DNA polymerase gene POL3 also caused rapid microevolution without an apparent fitness cost [28]. In both species, the disruption of mismatch repair mechanisms enabled development of drug resistance, demonstrating how accelerated mutation rates can enable rapid adaptation [27] [28].

Escape from antifungal treatment
The ability of fungal pathogens to evolve resistance within the time frame of a single infection can be life-threatening. Development of antifungal resistance is common during human infections by the major pathogens C. albicans [8], A. fumigatus [29], and Cryptococcus species [5]. Experimental evolution using different in vitro antifungal treatments have further demonstrated the ability of fungal pathogens to rapidly acquire drug resistance [12,21,30]. Some studies indicate that acquisition of drug resistance may carry a fitness cost in vitro, as resistance is often lost during subsequent passage in the absence of drug [5,31]. However, the impact of drug resistance on fitness within the host is more complex as resistant isolates can arise in patients even without antifungal treatment via mutations that provide protection against chemically-similar compounds [32]. This is seen in Candida lusitaniae lung isolates for which mutations in the MRR1 transcription factor gene confer azole resistance and also contribute to resistance against the host antimicrobial peptide histatin 5 and bacterial phenazines [32].
The types of genetic mutations underlying antifungal resistance are diverse across species and drugs. As discussed above, both Candida [9,30] and Cryptococcus [5,12] utilize aneuploidy and associated increases in gene expression of antifungal targets and efflux pumps to escape drug treatment. In Candida species, single base pair mutations in key cellular efflux regulators MRR1 [32], TAC1 [33], or PDR1 [34] have been linked to multidrug resistance. LOH can further contribute to this process by enabling homozygosis of the mutated alleles, as observed for both MRR1 [32] and TAC1 [33] genes. Point mutations within drug target genes themselves can also drive resistance in Candida species, as in the case of β-glucan synthase genes FKS1 and FKS2 (targets of echinocandins) [35,36] and ergosterol pathway gene ERG11 (target of azoles) [37]. In Aspergillus, similar mutations in CYP51 (the ERG11 homolog) reduce the affinity between azole and drug target, thereby leading to drug resistance [38]. In addition to providing a strong selective pressure, exposure to antifungals may themselves promote genetic variation. This is seen in C. albicans, in which antifungal treatment results in increased genome instability and higher frequencies of LOH and point mutations [22,39]. Antifungals therefore drive fungal microevolution by both increasing the rates of genetic variation and by selecting for tolerant and/or resistant isolates.

Microevolution in complex host environments
In contrast to the defined environments in which fungi are passaged in the laboratory, selective pressures encountered in the host are complex and multifaceted. Here, commensal and pathogenic fungi must contend with constant host immune surveillance, fluctuating pH and nutrient availability, as well as compete with resident microbes. This array of selective pressures results in a wider range of genetic variability than typically seen following in vitro passage. Indeed, for C. albicans, higher numbers of mutations and rates of recombination were estimated to occur during passage in the host (bloodstream infection and GI tract colonization) relative to growth in rich laboratory media [4,19]. While it is extremely challenging to evaluate the relative contribution of individual selective forces, mutations accumulated within the host can provide important clues. In C. lusitaniae, mutations in MRR1, which endow azole resistance, also protect against histastin-5, an antimicrobial peptide of the innate immune system [32]. Alterations in cell wall-related genes were also common in serial clinical isolates of this species obtained from blood and lung samples [6]. Similarly, C. albicans passage through bloodstream and GI mouse infection models resulted in frequent mutations in cell wall adhesin genes relative to other coding regions [19], perhaps due to the large number of tandem repeats that these genes carry. Passage of a yeast-locked C. albicans strain within murine macrophages led to restored filamentation and virulence via a single nucleotide change in the Mediator component SSN3, thereby illustrating that complex regulatory networks can be easily rewired through microevolution [40]. Together, these studies indicate that the host immune system is a key driver of microevolution and the cell wall surface is likely an important site of genetic variation.
In addition to host immunity, host-resident microbes also exert substantial pressure on fungal pathogens. In fact, GI passage of C. albicans within immunodeficient and antibiotic treated neonatal mice showed that adaptation to this niche was dependent upon inhibition of the microbiota, suggesting that host microbes limit the adaptation of this commensal species to the mouse GI [10]. Metabolic pressures within the host may also drive fungal microevolution. In C. neoformans VNI, serial passage through the mouse brain resulted in a mutant with increased expression of an iron reductase [41]. Host-mediated nutritional immunity of micronutrients such as iron and zinc (reviewed in [42]) is likely an important selective pressure in diverse host environments. C. albicans strains passaged in the mouse GI repeatedly evolved trisomies for chromosome 7, which resulted in increased fitness for this niche [19]. This aneuploidy was later implicated in susceptibility to medium chain fatty acids [43]. Oxidative metabolism may also be under strong selection within the host. In one example, passage of C. albicans through a systemic infection model yielded a respirationdeficient strain, which was resistant to phagocytosis and proliferated in the host with decreased morbidity [44]. In A. fumigatus, fitness in low oxygen was associated with higher virulence in a murine lung infection model [45]. Further investigation revealed that adaptation to low oxygen in this species resulted in altered growth morphology, cell wall architecture, and hyphal adhesion, which in turn increased virulence via immunopathological inflammation [46]. These studies illustrate the complexity of fungal microevolution in the host environment, demonstrating how seemingly unrelated processes such as metabolic adaptation and immune evasion can be linked.

Balancing novel genetic variation, genome integrity, and fitness trade-offs
Human fungal pathogens utilize a broad array of strategies to generate genetic diversity and adapt to fluctuating environments. This genomic flexibility enables rapid adaptation to antifungal drugs or selective pressures within the host. While the ability of fungi to tolerate significant genomic plasticity provides increased adaptability, this raises the question of how this ability is balanced against the need to maintain genome integrity. This requirement may be relaxed in host niches with limited timescales due to rapid host death or those that do not facilitate pathogen transmission (e.g., deep-seated infections). However, genome integrity is crucial for fungal viability in host niches with sustained colonization such as skin, lungs, genitourinary, and GI tracts.
The need to maintain genome integrity becomes even more pressing when considering that aneuploidy and LOH are associated with a global increase in genome instability [4,21,47]. Aneuploidy is often unstable in the absence of selection [12,31] and is associated with delayed cell division and increased proteotoxic, oxidative, and hypo-osmotic stress [48,49], therefore additional fitness costs may be incurred. To mitigate these fitness costs, fungi may employ chromosomal rearrangements only as a temporary solution to sudden stress, thus "buying time" to acquire less costly mutations such as SNPs and indels [50] (Fig 1). However, how these species balance genomic plasticity with genomic integrity remains a large gap in our understanding of fungal pathogens.
The need to better understand fungal microevolution is emphasized by our inability to effectively treat invasive fungal infections, a major challenge given the limited repertoire of antifungals available. A better understanding of the relationship between commensalism and virulence is also fundamental. Given the diversity of host niches that fungi can occupy, microevolution can rapidly tilt the balance between commensalism and pathogenesis. This is demonstrated in C. albicans in which strains adapted to the mouse GI [10,24] or oropharyngeal tracts [11] lost the ability to cause lethal systemic infection. Conversely, isolates recovered from kidneys during serial systemic infection display accelerated host killing [51]. Understanding the selective pressures within specific host niches and how fungal genomes respond to them during microevolution is central to defining the mechanisms by which fungi adapt and evolve, particularly in the context of mammalian host infection.