Genetic and environmental control of parasexual reproduction in the pathogenic Candida species

SUMMARY

Candida species are major yeast pathogens that cause both mucosal candidiasis and life-threatening invasive infections. Most Candida species, including Candida albicans, have long been thought to be “imperfect” due to the lack of a complete sexual reproduction cycle. Since the discovery of the mating type-like locus in C. albicans in 1999, the regulation of (para)sexual reproduction has been intensively investigated in this organism as well as in several phylogenetically closely related species. The (para)sexual cycle is not only critical for the generation of genetic and phenotypic diversity but is also involved in the regulation of other biological processes, such as morphological transitions, biofilm development, and virulence in pathogenic fungi. In this review article, we focus on the unique characteristics and genetic and environmental regulatory mechanisms of parasexual reproduction in the pathogenic Candida species. We discuss the relationship between the white-opaque switching and mating in the Candida species, particularly in C. albicans. We describe recent findings on environmental factors, genetic regulators, and key signaling pathways involved in sexual mating in C. albicans and related species. Finally, we discuss the mating potential and associated regulatory machinery in several Candida species, where parasexual reproduction has not been observed and bring to light some open-ended questions regarding the unique features of parasexual reproduction that should be addressed in future studies in the field.

INTRODUCTION

Fungi are eukaryotes capable of both asexual and sexual reproductions (1). In the classical sexual cycle, cells of opposite types fuse to form a zygote, which then undergoes meiosis to produce genetically diverse progeny (2). This hallmark of eukaryotic life is broadly conserved across fungi, from the budding yeast Saccharomyces cerevisiae to mushrooms (2, 3). By producing genetically varied offspring, sexual reproduction acts as an evolutionary driver and supports fungal adaptation and survival under adverse conditions (4–7). However, most pathogenic Candida spp. lack a conventional sexual cycle and instead rely on an alternative parasexual pathway to produce genetic diversity.

Pathogenic Candida species cause not only mucosal candidiasis but also systemic and bloodstream infections, especially in immunocompromised individuals (8). Six major fungal pathogens among Candida species, including Candida albicans, Candida auris (Candidozyma auris), Candida glabrata (Nakaseomyces glabratus), Candida tropicalis, Candida parapsilosis, and Candida krusei (Pichia kudriavzeveii), were included in the priority list of fungal pathogens published by the World Health Organization in 2022 (9). Among them, C. albicans, C. auris, C. tropicalis, and C. parapsilosis belong to the CTG clade, where the CTG codon is translated as serine rather than the canonical leucine (10, 11). C. glabrata is a member of the Saccharomyces clade and is closely related to the model yeast S. cerevisiae, whereas C. krusei belongs to the genus Pichia in the family Pichiaceae (12, 13). Owing to recent genus-level taxonomic revisions, these species are now classified into three distinct phylogenetic groups (14). The close evolutionary relationships provide insights into the similarities and differences of sexual reproduction among the important pathogenic Candida species (Fig. 1).

FIG 1.

Phylogenetic tree of the pathogenic Candida species and Saccharomyces cerevisiae. Maximum-likelihood phylogenetic tree of the pathogenic Candida species and S. cerevisiae was constructed based on 1,570 core genes and 1,000 bootstrap replicates. Cartoons on the right of the figure illustrate the features of virulence, ploidy, and mating and meiosis competence. CTG, the CTG clade (species of this clade translate the CTG codon as serine instead of leucine); Deb, Debaryomyces; Met, Metschnikowia; WGD, the WGD lineage (arose from a whole-genome duplication). Homotypic synonyms: Candida auris, Candidozyma auris; Candida haemulonii, Candidozyma haemuli; Candida pseudohaemulonii, Candidozyma pseudohaemuli; Candida duobushaemulonii, Candidozyma duobushaemulonii; Candida lusitaniae, Clavispora lusitaniae; Candida krusei, Pichia kudriavzevii; Candida glabrata, Nakaseomyces glabratus. FPPL, WHO Fungal Priority Pathogens List.

Unlike S. cerevisiae (5–7), most Candida species have traditionally been recognized as “imperfect” yeast species due to their lack of a defined sexual cycle (15, 16). Parasexual reproduction, an alternative mechanism for generating genetic recombination, has been extensively investigated in several Candida species, including C. albicans and C. tropicalis (16, 17). Mating products of parasexual reproduction undergo concerted chromosome loss (CCL) rather than meiosis to generate cells of various ploidy states (2, 18, 19). Parasexual reproduction can generate genetic and phenotypic diversity and eliminate deleterious alleles, ultimately benefiting long-term adaptation in stressful environments, such as exposure to antifungal drugs and other in vitro and in vivo hostile factors (17, 20, 21). Given the wide distribution of sex-related genes, many pathogenic Candida species likely maintain the ability for sexual or parasexual reproduction, although their strategies and processes could be cryptic.

There are multiple species-specific strategies known for sexual and parasexual reproductions in the human fungal pathogens (2). Perhaps as a result of their distinct regulatory machineries for mating, natural ecological habitats, and diverse genetic backgrounds, these species-specific regulatory features are highly diverse, providing additional layers of regulation for sexual reproduction and a balance of trade-offs between sexual and asexual lifestyles. In this review, we focus on the characteristics and mechanisms for the environmental and genetic regulation of parasexual reproduction in C. albicans and its closely related species, such as C. tropicalis and Candida dubliniensis, which are considered “obligate” diploid organisms (22, 23). Mating between diploid cells generates unstable tetraploids (18, 19). Recent discovery of the haploid form in C. albicans and C. tropicalis complicates their life cycles (24–26). We also discuss the mating potential of more frequently isolated pathogenic Candida species, such as C. auris, C. glabrata, and C. parapsilosis.

GENETIC BASIS FOR PARASEXUAL REPRODUCTION IN THE CANDIDA SPECIES

Mating-type loci and mating-type switching in S. cerevisiae

The budding yeast S. cerevisiae is not only a genetically tractable eukaryotic model organism for the study of the sexual cycle but also an excellent model system for studying the biology of the Candida species. S. cerevisiae possesses three mating-type (MAT) loci, which control mating and meiosis (27). One active MAT locus determines cell identity, while HML and HMR are two silent MAT loci located on the same chromosome (28, 29). The MAT locus carries either the MATα allele, which contains MATα1 and MATα2, or the MATa allele, which contains MATa1 (Fig. 2A) (29). MAT genes (α1, α2, and a1) encode three homeodomain-containing transcription factors. A haploid a or α cell has the MATa or MATα allele at the active locus, respectively. In haploid α cells, together with the Mcm1 protein, the α1 transcriptional regulator promotes the expression of α-specific genes, while the α2 protein represses a-specific gene expression. a cells constitutively express a-specific genes due to the lack of the α2-Mcm1 repressor. In diploid a/α cells, a-specific and α-specific or haploid-specific genes are suppressed by the a1-α2 complex (28, 30, 31). Cell identity is determined by the expression of a or α cell type-specific genes.

FIG 2.

Organization of the mating-type loci in S. cerevisiae and the pathogenic Candida species. (A) S. cerevisiae. Three MAT loci (MATa or MATα, HMRa, and HMLα) are shown. (B) C. glabrata. (C) Mating-type like (MTL) loci of C. albicans, C. tropicalis, C. dubliniensis, C. auris, C. lusitaniae, C. parapsilosis, Candida orthopsilosis, Candida metapsilosis, Lodderomyces elongisporus, and C. haemulonii. PIK, PAP, and OBP are non-sexual genes located at the MTL locus of C. albicans and its close relatives. Crosses indicate gene losses or pseudogenes. Dotted lines represent the HO endonuclease sites in S. cerevisiae and C. glabrata. The same color was used to indicate orthologs in different species. Arrows indicate transcriptional orientation. OBP, oxysterol-binding protein; PAP, poly(A) polymerase; PIK, phosphatidyl inositol kinase.

The MAT active allele can be replaced with the silent HMLα or HMRa locus through homologous recombination (HR) (32, 33). This mating-type switch is dependent on the HO endonuclease in S. cerevisiae (28, 34). The HO enzyme creates a site-specific double-strand break (DSB), which is then repaired by HR. Only haploid cells undergo mating-type switching, and the HO gene is tightly regulated to ensure the existence of both mating types in a lineage to promote mating (34, 35). Similar to S. cerevisiae, one active and two silent mating-type loci were found in the genome of C. glabrata, and mating-type switching was observed in C. glabrata (Fig. 2B) (36).

Mating-type loci in the Candida species

In 1999, a mating-type locus was identified on chromosome 5 of C. albicans, similar to the MAT locus in S. cerevisiae, and was denoted the mating type-like (MTL) locus (37). Key genes encoding the sexual cycle regulators MTLa1, a2, α1, and α2 were identified in this report and in a later study by the same group (38). The C. albicans MTL genes are arranged on homologous chromosomes in a similar manner to that of the S. cerevisiae MAT alleles. However, the MTL locus of C. albicans is much larger than the MAT locus of S. cerevisiae and additionally contains a set of “non-sexual” genes (Fig. 2C). These genes encode phosphatidylinositol kinases (PIKs), oxysterol binding protein-like proteins (OBPs), and poly(A) polymerases (PAPs) (37). In a/α cells, PIK, PAP, and OBP play roles in fungal growth, biofilm formation, and fungal virulence (39); however, the roles of these genes in the sexual cycle have yet to be identified. The DNA sequences of these genes located on the two MTL homologous chromosomes exhibit significant differences, suggesting that they may play distinct yet-to-be-determined roles in the regulation of sexual reproduction in C. albicans.

Unlike in S. cerevisiae, C. albicans has a single MTL locus in its genome. In diploid C. albicans, the MTLa and MTLα alleles can form three mating-type configurations, namely, MTLa/a, α/α, and a/α. The majority of C. albicans isolates are MTLa/α strains (40, 41). In addition to the three homeodomain-containing transcriptional factors—a1, α1, and α2—which are found in S. cerevisiae, there is a fourth regulator, a2, encoded by the MTLa allele in C. albicans (38). The presence of the a2 in C. albicans makes the regulation of mating type-specific genes in C. albicans different from those in S. cerevisiae (38). In C. albicans α cells, the α1 protein acts as an activator of α-specific gene expression, while the α2 protein does not repress a-specific gene expression. In a cell, a2 functions as an activator to promote a-specific gene expression (38, 42). Thus, a2 and α1 serve as key regulators of cell identity, while the a1-α2 complex specifies the a/α cell type and underlies the mating-incompetent state of MTL-heterozygous C. albicans (38, 42). Despite the differences in regulatory logic, the overall outputs of the cell type-specific genes are similar between C. albicans and S. cerevisiae. We note that there are no homologs of the HO endonuclease-encoding genes in C. albicans (37, 43); thus, the processes of homothallic mating in C. albicans and S. cerevisiae are distinct.

Pheromone-sensing mitogen-activated protein kinase signaling pathway in C. albicans

In S. cerevisiae, MATa cells secrete a-pheromone and express the α-pheromone receptor Ste2 to sense the α-pheromone secreted by α cells. Similarly, MATα cells are able to sense a-pheromone via the a-pheromone receptor Ste3 (44). Genes homologous to the S. cerevisiae genes encoding pheromone precursors and corresponding receptors have been identified in C. albicans and related CTG clade species (22, 45–47). In addition, the conserved serine proteinase Kex2, which is required for the generation of mature α-pheromone, has also been found in several Candida species (48–50). The presence of pheromone and corresponding receptor-encoding genes indicates a similar pheromone response pathway in C. albicans and S. cerevisiae.

The heterotrimeric G-protein complex (Gαβγ) and conserved mitogen-activated protein kinase (MAPK) pathway function downstream of the pheromone and Ste2/3 receptors (51, 52). In S. cerevisiae, the pheromone signal activates the heterotrimeric G-protein complex via dissociation of the Gβ (Ste4) and the Gγ (Ste18) subunits from the Gα subunit (Gpa1). The Gβγ complex activates Ste20, leading to the activation of the Ste11-Ste7-Fus3 MAPK cascade (Fig. 3A). The Fus3 kinase phosphorylates the transcription factor Ste12 to activate the pheromone-responsive genes and promote the formation of mating shmoos or projections. The central components of the pheromone-sensing signaling pathway are known to be conserved and required for mating in C. albicans (Fig. 3B) (53). The homologs of Ste20, Ste5, Ste7, Fus3, and Ste12 in S. cerevisiae have been named Cst20, Cst5, Hst7, Cek1/Cek2, and Cph1 in C. albicans, respectively. The Cek1 and Cek2 kinases appear to play redundant roles in mating in C. albicans. In addition to its roles in pheromone sensing and mating regulation, this MAPK pathway in C. albicans is also important for filamentous growth (54). The multiple roles of the MAPK pathway in C. albicans are important for its host adaptation and virulence.

FIG 3.

Comparison of the pheromone signaling pathways in S. cerevisiae and C. albicans. (A) The pheromone signaling pathway in S. cerevisiae. (B) The pheromone signaling pathway in C. albicans. Mating pheromone binds to its receptor and activates the Ste20-Ste11-Ste7-Fus3 kinase cascade in S. cerevisiae and the Cst20-Ste11-Hst7-Cek1/Cek2 kinase cascade in C. albicans. The Ste12 (Cph1 in C. albicans) transcription factor, downstream of the MAPK pathway, is then phosphorylated and activates the expression of mating-associated genes.

MATING AND PHENOTYPIC SWITCHING IN C. ALBICANS

Discovery of mating in C. albicans

The discovery of an intact mating-type locus in C. albicans suggested its capacity for mating (37). In 2000, two independent studies demonstrated that engineered diploid C. albicans cells were able to mate and generate tetraploid progeny under both in vitro and within an animal host (55, 56). These findings challenged the long-standing view of C. albicans as an imperfect asexual fungus. To assess the abilities of C. albicans to mate, the two research groups used different strategies to convert MTL-heterozygous strains to MTL-homozygous strains (55, 56). The standard laboratory strain of C. albicans, SC5314, and its derivatives are heterozygous at the MTL locus. Hull et al. (56) constructed “a” and “α” strains by deleting one MTL allele in strain CAI4, a uridine auxotroph derived from SC5314 (56). The authors mixed the derivatives of opposite mating types and injected the mixture into mice via the tail vein. C. albicans cells indeed underwent mating and generated tetraploid mating products after 24 hours of infection. Magee and Magee (55) obtained similar results under in vitro culture conditions using a culture method called sorbose utilization (55). C. albicans grows poorly in media with sorbose as the sole carbon source, and colonies formed on plates containing sorbose often lose one copy of chromosome 5, where the MTL locus is located. Taking advantage of the relationship between sorbose utilization and loss of chromosome 5, the authors generated a set of C. albicans strains containing only MTLa or MTLα loci from strain CAI4. They then cross-streaked the MTLa and MTLα strains on several rich media types, incubated the plates for 36 hours, and replated the strains onto minimal medium for selectable growth of the mating progeny. The authors observed that C. albicans cells under these conditions could mate and form tetraploid progeny (55). Both studies observed that overall mating efficiencies were extremely low in C. albicans. However, by extending the incubation time of the mixed mating strains prior to their growth on selectable medium, more efficient mating resulted. This observation suggests that nutritional conditions are involved in the regulation of mating. Taken together, these findings indicate that C. albicans has maintained its mating machinery and is capable of mating under specific environmental conditions.

Relationship between white-opaque switching and mating

It has long been observed that C. albicans has multiple morphological types and can switch among different phenotypes, which are critical for pathogenesis (57, 58). White-opaque transitions represent a typical heritable phenotypic switching system in C. albicans, which was first observed in the clinical strain WO-1 (59, 60). White cells are relatively round and form white, dome-shaped colonies, while opaque cells are larger, more elongated, and form darker, flatter colonies on solid agar media. The two types of cells can be differentially stained using the dye phloxine B. Colonies formed by opaque cells can be stained red, while colonies of white cells appear white and shine in the presence of phloxine B (61). Both white and opaque cell states are heritable across many generations and can switch reversibly and spontaneously at very low frequencies. In the WO-1 strain, the frequency of white to opaque switching occurs around 10⁻⁴ under ambient conditions (60). Other than the differences in cellular and colony morphologies, white and opaque cells exhibit distinct gene expression profiles (62–65), environmental responses (66, 67), metabolic preferences (62), virulence properties (68, 69), and mating abilities (Fig. 4) (70).

FIG 4.

Colony and cellular morphologies of white, gray, and opaque cells of C. albicans strain BJ1097 on yeast extract peptone dextrose agar with phloxine B. G-Sec, gray sector. Scale bar, 10 μm. This figure is adapted from reference 71.

Although C. albicans has intact mating machinery, mating frequencies are extremely low under both in vitro and in vivo conditions (55, 56), suggesting that additional tiers of regulation are likely involved in mating. In 2002, Miller and Johnson found that the switch from the white to opaque cell type is controlled by the mating-type locus and is required for efficient mating in C. albicans (41, 70). Opaque cells mate one million times more frequently than white cells. The authors presumed that only opaque cells are mating competent, and the extremely low efficiency of mating detected in white cells was likely due to spontaneous white-to-opaque switching of the mating partners (70). Mating between opaque a and α cells results in the formation of a/α heterozygous tetraploid progeny, which returned to the white state. Another interesting finding of this study was that only engineered MTLa/Δ or MTLΔ/α strains could switch to the opaque phase, while MTLa/α heterozygous strains were unable to form opaque cells (70). A later study further confirmed that only MTL homozygotes of clinical isolates (MTLa/a or α/α) are able to switch to the opaque phenotype, whereas the majority of isolates with the heterozygous mating type (MTLa/α) maintain the white phenotype (41). These studies imply that, to mate, MTLa/α strains must first undergo homozygosity at the MTL locus and then switch to the opaque cell type.

The MTL locus controls both white-opaque switching and mating in C. albicans, and the opaque phenotype is a prerequisite for mating (70). The links between white-opaque switching competence, mating, and MTL configuration indicate that the a1 and α2 transcription factors are key regulators of phenotypic transitions and mating (70). The a1-α2 heterozygous complex represses the expression of mating-associated genes and white-opaque switching-associated regulators, including the master regulator WOR1 in C. albicans. We note that the WO-1 isolate is homozygous at the mating-type locus and contains only the MTLα allele and thus cannot form the a1-α2 complex to repress white-to-opaque switching (70). Besides the a1-α2 complex, many environmental and genetic factors are involved in the regulation of white-opaque switching in C. albicans.

Environmental and genetic regulation of white-opaque switching in C. albicans

The discovery that the opaque state is a prerequisite for mating in C. albicans sparked significant interest and research in the field (59, 70). Given that the opaque phenotype is important for mating, one would expect that environmental and genetic factors governing phenotypic switching would affect the efficiency of mating and/or parasexual reproduction in this fungus. The underlying mechanisms driving white-to-opaque switching have been extensively investigated (see the review by Soll [59]). Despite the heritable nature of this phenotypic switch, epigenetic rather than genetic alterations are involved in its regulation (59, 64, 65, 72, 73). It has been demonstrated that a plethora of environmental factors including temperature (74), ultraviolet irradiation (75), CO₂ levels (76), carbon source (e.g., N-acetylglucosamine [GlcNAc]) (77), pH (78), and genotoxic or oxidative stresses (79), regulate white-to-opaque or opaque-to-white transitions. Multiple conserved signaling pathways, including the Ras-cAMP/protein kinase A (PKA) (77, 80), Hog1-MAPK (81), pH sensing (78), and Vps21 (82) pathways, are involved in the regulation of phenotypic switching in C. albicans in response to environmental cues (Fig. 5).

FIG 5.

White-opaque switching in C. albicans is regulated by multiple environmental factors and signal transduction pathways. The cAMP/PKA, MAPK, pH sensing, Hog1, and Vps21 signaling pathways converge on the regulation of the WOR1 master regulator of white-opaque switching. In MTLa/α strains, transcriptional regulators Efg1, Rfg1, Brg1, Sfl2, and a1-α2 repress the expression of WOR1. Arrows indicate activating effects, and lines with bars indicate inhibiting effects.

Moreover, together with a number of transcription factors, the master transcriptional regulators Wor1 and Efg1 form a transcriptional circuit and function as major controllers of white-opaque transitions (65). Wor1 is a fungal-specific transcription factor containing a conserved WOPR domain and a potential PKA phosphorylation site (83). Deletion of WOR1 locks C. albicans cells in the white phenotype, while overexpression of WOR1 promotes the opaque phenotype (83–85). Several binding sites of the a1-α2 co-repressor have been identified in the promoter region of WOR1, suggesting that the a1-α2 complex regulates white-opaque switching by controlling WOR1 expression (85). Several transcription factors have been identified to regulate the expression of WOR1 and EFG1, and a genetic network between the core transcriptional regulators has been identified (63, 65, 86, 87). These regulators include the transcription factors Czf1, Ahr1, Wor2, Wor3, Wor4, and Ssn6 (Fig. 5) (63, 88–92).

A number of host-associated environmental factors regulate white-opaque switching in C. albicans (59, 66). It is intriguing that the mating-competent opaque phenotype is unstable at the host physiological temperature of 37°C (60, 74) and that low environmental temperatures (≤10°C) promote opaque-to-white transitions (60, 74) under common laboratory culture conditions. The underlying mechanisms of temperature-regulated phenotypic switching remain unclear. Metabolites of commensal bacteria, CO₂, and GlcNAc are abundant in the gut of mammalian hosts, which represent natural niches for C. albicans colonization (76, 77). It has been demonstrated that high levels of CO₂ (≥5%) and the addition of GlcNAc to the media not only promote white-to-opaque switching but also stabilize the opaque state at 37°C (76, 77). The combination of high levels of CO₂ and GlcNAc has a synergistic effect on the induction of the opaque phenotype (40, 59, 71, 87). It has been demonstrated that these two environmental cues can induce filamentous growth of C. albicans through the conserved Ras-cAMP/PKA signaling pathway (58). This pathway consists of the adenylyl cyclase Cyr1, PKA regulatory subunit Bcy1, and PKA catalytic subunits Tpk1 and Tpk2 (93). The small GTPase Ras1 is the upstream activator of Cyr1, which transfers outside signals to Cyr1 and stimulates cAMP production (94). The second messenger molecule cAMP binds to Bcy1 and activates PKA catalytic subunits to phosphorylate downstream transcription factors (58, 95, 96). By activating the downstream master regulator Wor1, the Ras-cAMP/PKA pathway plays a similar role in CO₂-induced and GlcNAc-induced white-to-opaque switching (58, 97). Multiple environmental signals are transduced through pathways that converge on the master regulator Wor1 to control white-opaque switching.

Although the a1-α2 complex represses white-to-opaque switching in MTLa/α strains under routine laboratory culture conditions, the combined use of GlcNAc and high levels of CO₂ can induce the formation of opaque cells in clinical C. albicans strains with the MTLa/α configuration (40, 59, 71, 87). Opaque cells of MTL heterozygous strains exhibit similar characteristics to MTLa/α opaque cells in terms of cellular sizes, shapes, and morphologies (40). However, MTLa/α opaque cells are unable to undergo mating. Screening of a library of transcription factor deletion mutant strains in an MTLa/α background identified that transcription factors Rfg1, Brg1, and Efg1 may be involved in this regulation (40). Another study demonstrated that inactivation of the transcription factor Sfl2 promoted opaque cell formation in MTLa/α strains (98). Several studies on the regulation of white-opaque switching in MTLa/α cells have also been reported (99–101). Together, these findings imply that white-opaque transitions are not restricted by MTL configurations and could be a general feature of natural isolates of C. albicans under certain culture conditions and/or in certain host environments. The switch from white to opaque in MTLa/α strains in C. albicans would have a direct impact on mating, such that MTLa/α opaque cells undergoing MTL homozygosis could become mating competent immediately.

Other signal transduction pathways, including the pheromone-sensing MAPK, Hog1-mediated osmotic sensing, pH signaling, and Vps21 signaling pathways, have also been shown to play roles in the regulation of white-opaque switching in C. albicans (Fig. 5) (78, 81, 82, 102) (reviewed by Huang [58] and Soll [59]). Taken together, these studies indicate that a variety of environmental factors and multiple signaling pathways control white-opaque transitions in C. albicans. Environmental and genetic perturbations of these pathways could have direct or indirect impacts on mating in this fungal pathogen.

Mating competency of the gray cell type of C. albicans

Other than the white and opaque phenotypes, a subset of clinical strains of C. albicans, C. dubliniensis, and C. tropicalis can form another heritable cell type, namely, the gray phenotype (71, 103, 104). Although the gray cell state is less frequently observed than the white and opaque cell states in C. albicans, together, the three cell types form a tristable phenotypic switching system under laboratory culture conditions. Both MTL heterozygous and homozygous strains can form gray cells, suggesting that this switch is independent of MTL configurations (71). C. albicans gray cells are elongated but much smaller than opaque cells. Gray cells form smooth colonies on solid culture media and appear light pink in the presence of phloxine B (71). The mating competencies of MTL-homozygous gray cells are intermediate between those of white and opaque cells, suggesting that the opaque cell type is not the sole mating-competent cell type in C. albicans.

The stable and unique features of the gray cell type indicate that it is a distinct phenotype of C. albicans, C. dubliniensis, and C. tropicalis and is not simply a transitional phenotype between the white and opaque phenotypes (71, 103, 104). The key regulators of the white-opaque transition, Wor1 and Efg1, which are essential for maintaining the opaque and white cell phenotypes, respectively, are not required for gray cell formation in C. albicans. In contrast, inactivation of both Wor1 and Efg1 locks C. albicans cells in the gray phenotype, suggesting that these two transcriptional regulators could function coordinately in the regulation of white-gray-opaque transitions (71). Liang et al. (105) demonstrated that C. albicans cells heterozygous for EFG1 can undergo phenotypic switching (105). Notably, the majority of gray-competent clinical isolates exhibit hemizygosity at the EFG1 locus. The C. albicans BJ1097 strain, where the gray phenotype was first observed (71), is an EFG1 heterozygote containing a nonsense mutation in EFG1 in its white cell form. However, the derived gray cells of BJ1097 exhibit complete EFG1 inactivation (105). EFG1 null mutant strains, which frequently arise through genetic alterations or loss of the functional allele from EFG1-hemizygous strains, are also capable of forming gray cells (105). Multiple studies have confirmed that EFG1 null mutant strains can form gray cells (87, 106, 107). Collectively, these findings suggest that diverse genetic alterations contribute to regulating phenotypic switching in C. albicans.

GENETIC AND ENVIRONMENTAL REGULATION OF MATING IN C. ALBICANS

As in other fungal species, environmental perturbations influence key processes of sexual reproduction in C. albicans (108–113). A variety of environmental cues can modulate mating efficiency through the regulation of MTL homozygosity and opaque cell formation (59, 114). These findings suggest that additional layers of mating regulation in C. albicans are finely tuned by both genetic and environmental factors to balance asexual and parasexual reproduction.

Sorbose-induced loss of the MTL locus in C. albicans

Sorbose induces the loss of chromosome 5, which harbors the MTL locus in C. albicans (114). The L-sorbose dehydrogenase-encoding gene SOU1 is essential for sorbose utilization. However, the expression of SOU1 is transcriptionally repressed by regulators on chromosome 5 (114). When grown in sorbose-containing media, one copy of chromosome 5 is often lost, and monosomic strains, carrying only a single MTL locus (a or α) and now capable of assimilating sorbose, are formed. This monosomic chromosome could undergo duplication to obtain two MTL-homozygous chromosomes (a/a or α/α) (114, 115). Mitotic recombination of chromosome 5 is the major mechanism of MTL homozygosis, and loss of one homolog following duplication is frequently observed to achieve MTL homozygotes in C. albicans (116). The majority of C. albicans clinical isolates are heterozygous at the MTL locus (a/α), a state that may confer heterozygosity of chromosome 5, which has been associated with fungal virulence (117).

Antifungal-induced MTL homozygosis in C. albicans

Many genes associated with antifungal resistance, including ERG11 encoding a lanosterol 14-α-demethylase required for ergosterol biosynthesis, are located on the same chromosome as the MTL locus in C. albicans (118–120). It has been demonstrated that exposure to fluconazole, a first-line azole drug, leads to gene conversion, mitotic recombination, and the generation of aneuploidy on chromosome 5 (121–123). Thus, when treated with fluconazole, MTL heterozygous strains of C. albicans become resistant and homozygous at the mating-type locus due to genome rearrangements or replacement of one allele with a duplicate of the other (20). Consistently, there is an obvious correlation between MTL homozygosis and azole resistance in clinical or evolved isolates. Only ~3.2% of natural strains are MTL homozygous, whereas 48.6% of fluconazole-treated strains are MTL homozygous (124, 125). These findings suggest that azole treatment promotes drug resistance concomitant with the loss of heterozygosity at the MTL locus in C. albicans. Since this loss of MTL heterozygosity results in white-opaque switching and mating competency, MTLa and MTLα cells generated in the same population could possibly undergo mating and form unstable tetraploid mating products. Additionally, genomic alterations of mating products could further promote the development of drug resistance in C. albicans.

Hbr1 and Ofr1 regulate MTL gene expression in C. albicans

The cell identity of C. albicans is not only determined by the genomic presence of MTL alleles but also dependent on the expression of MTL genes. Transcriptional repression of MTL genes could lead to similar effects as those caused by loss of heterozygosity at the MTL locus, thereby permitting white-opaque switching and mating in MTL heterozygotes of C. albicans (126, 127). Hemoglobin regulates the expression of MTLa1 and MTLα2 via the hemoglobin response protein Hbr1. Disruption of a single allele of HBR1 in C. albicans MTLa/α cells confers these cells with the ability to mate with MTLα cells (126). The reduced dosage of Hbr1 in the HBR1/hbr1 heterozygous strain with an MTLa/α configuration results in decreased expression of MTLα1 and MTLα2 and enhanced expression of MTLa1 (126). This change in MTLα2 expression levels in the HBR1/hbr1 strain could be sufficient to activate the expression of mating-related genes (126). Conversely, exposure to hemoglobin or overexpression of HBR1 in MTL heterozygous cells could increase the expression of MTLα (126).

Sun et al. (127) found that deletion of OFR1, which encodes a Yci1-domain-containing protein, enables opaque cell formation and promotes mating in MTLa/α strains (127). Intriguingly, the ofr1/ofr1 MTLa/α cells can mate with MTLa, MTLα, and even homothallically with ofr1/ofr1 MTLa/α cells (127). Transcriptional profiling analysis revealed that genes encoding both a- and α-mating pheromone precursors (MFA1 and MFα) and corresponding pheromone receptors (STE3 and STE2) are expressed in the ofr1/ofr1 MTLa/α cells. Since they can produce and sense pheromones, the ofr1/ofr1 MTLa/α cells are capable of mating with both MTLa and MTLα partners (127). These findings imply that MTL heterozygous cells of C. albicans can mate under specific conditions. For example, certain environmental factors could repress MTL gene expression and confer mating competency of C. albicans cells via the loss of original cell identity.

Nutrient conditions regulate mating in C. albicans

Replacement of glucose with GlcNAc as the sole carbon source and high levels of CO₂ induces white-to-opaque switching in C. albicans (76, 77). These inducing effects would indirectly increase mating frequencies due to the formation of mating-competent opaque cells. GlcNAc and CO₂ function primarily through the activation of the Ras1-cAMP/PKA pathway (58, 76, 77). Although the activation of this pathway promotes the opaque phenotype, inactivation of key components of the pathway, such as the PKA subunits Tpk1 and Tpk2, increases mating efficiency in opaque cells of C. albicans (80). These findings indicate that the Ras1-cAMP/PKA pathway positively regulates white-to-opaque switching and negatively regulates mating in C. albicans (Fig. 6A).

FIG 6.

Environmental regulation of heterothallic and homothallic mating in C. albicans opaque cells. (A) Nutrients impact mating efficiency through the regulation of mating-associated genes by the cAMP/PKA pathway during heterothallic mating in C. albicans. Acidic pH inhibits the expression of MFA1 and STE2 and mating in opaque a cells. (B) Glucose depletion, oxidative stress, and atmospheric humidity regulate homothallic mating in C. albicans.

Other than GlcNAc, replacement of glucose with other carbon sources and alteration of nutritional conditions can also have remarkable impacts on mating in C. albicans (53, 128). Chen et al. (53) observed that the mating efficiency of C. albicans was highly dependent on the culture media and incubation time (53). C. albicans underwent more effective mating on Lee’s mannitol medium than other nutrient media types, such as yeast extract + peptone (YP) + glucose, YP + succinic acid, and Lee’s + glucose. Interestingly, they found that mating was completely blocked on synthetic complete + glucose medium. Extended incubation time on mating media increases mating efficiency in C. albicans. The deprivation of nutrients that results from extended incubation time could promote mating in C. albicans in a manner similar to that of S. cerevisiae (53). Consistently, Bennett and Johnson (128) demonstrated that the degree of pheromone responses in C. albicans is nutrient dependent (128). External nutritional cues regulate this response through Gpa2-mediated signaling. They found that deletion of GPA2, which encodes a G-protein subunit in C. albicans, resulted in an enhanced pheromone response and efficient cell cycle arrest (128). Taken together, these findings suggest that C. albicans integrates signals of nutrient sensing and the pheromone-MAPK pathway to regulate mating.

Effect of pH on mating in C. albicans

CO₂ and pH are interconnected environmental signals since CO₂ dissolves in water to form carbonic acid. Consistently, acidic pH conditions favor the formation of opaque cells, whereas basic pH conditions repress white-to-opaque switching in C. albicans (78). Sun et al. (78) reported that pH plays opposing roles in the regulation of C. albicans mating (78). Acidic pH conditions decrease mating efficiency due to the repression of the pheromone response (Fig. 6A) (78). Other than the Rim101-mediated pH sensing pathway, a recent study demonstrated that the endosomal GTPase Vps21-mediated signaling pathway also regulates white-to-opaque switching and mating in C. albicans (82). This conserved signaling pathway regulates vacuolar biogenesis, trafficking, and morphological transitions in fungi (129, 130). Inactivation of the endosomal GTPase Vps21 and its major downstream components Vps9, Vps3, Vac1, or Pep12 promotes opaque cell formation in the presence of GlcNc but decreases the expression of pheromone and mating-associated genes and results in reduced mating efficiencies (82).

HOMOTHALLIC MATING IN C. ALBICANS

Discovery of homothallic mating in C. albicans

Candida spp. typically reproduce clonally and do not produce sexual spores in nature. Since a and α cells rarely coexist within the same environmental niche, heterothallic mating between C. albicans a and α cells is expected to occur at very low frequencies in nature. Homothallic mating, a sexual reproduction strategy that does not require a partner of the opposite mating type, represents a predominant mode of sexual reproduction in S. cerevisiae and Cryptococcus neoformans (28, 34, 131). In contrast, C. albicans possesses only a single mating-type locus and is unable to undergo mating-type switching to generate cells of the opposite mating type (37, 43). Consequently, the mechanism of homothallic mating in C. albicans differs from that observed in S. cerevisiae and C. neoformans (Fig. 6B and 7).

FIG 7.

Comparison of homothallic mating in S. cerevisiae, C. albicans, and C. neoformans. (A) In S. cerevisiae, mating-type switching allows for the formation of cells of the opposite mating type in MATa or α cells. Cells of the opposite mating types then undergo mating. (B) In C. albicans, environmental stresses overwhelm the heat shock protein 90 (Hsp90)-mediated stress-response pathway and activate the master regulator of a-type mating, MTLa2, and the pheromone precursor-encoding gene, MFα. The activation of both a- and α-pheromone-response pathways in a cells confers the “bimater” feature to the cells and promotes homothallic mating in C. albicans. (C) In C. neoformans, the majority of natural isolates are MATα. MATα cells can mate and produce mating products in the absence of a partner of the opposite mating type.

Alby et al. reported that C. albicans undergoes homothallic mating either through autocrine pheromone signaling or in the absence of Bar1 (132), an aspartyl protease that acts as an extracellular barrier that antagonizes the function of α-pheromone in both S. cerevisiae and C. albicans (132–134). C. albicans opaque cells of the bar1/bar1 mutant strain exhibit wrinkled colonies and form elongated cells confirmed to be mating projections with increased expression of the mating-specific genes FIG1 and FUS1 (132). This finding suggests that opaque cells of the bar1/bar1 mutant strain can mate in the absence of mating partners of the opposite mating type. Phenotypic and genetic analyses indicate that a-pheromone or α-pheromone precursors and receptor-encoding genes are required for this autocrine pheromone sensing and homothallic mating. C. albicans opaque a cells of the bar1/bar1 mutant strain express both a-pheromone and α-pheromone. This study detected no mating products from crosses between two wild-type a cells (<0.00002%), while the mating efficiency increased to 0.037% from crosses between two bar1/bar1 a strains (132). C. albicans bar1/bar1 a cells are able to mate with either a or α cells, implying that Bar1 acts as a repressor of self-fusion events within unisexual populations.

The mating-like responses observed in bar1/bar1 a cells, similarly observed in opaque a cells in the presence of synthetic α-pheromone, indicate the critical role of sufficient α-pheromone in the homothallic mating of a cells in C. albicans. Indeed, co-culture with opaque α cells promotes a-a mating at the frequency of 0.22%, possibly due to the fact that sufficient α-pheromone secreted by α cells can overcome Bar1 activity in culture (132). This so-called “ménage à trois mating” system was also observed in α-α mating in the presence of opaque a cells as pheromone donors (132). In addition, cells of the opposite mating type in both white and opaque states can serve as pheromone donors to drive homothallic mating in this co-culture system (135). Whether natural niches or environmental factors that suppress Bar1 activity exist remains to be investigated. Notwithstanding, the downregulation of Bar1 may be a prerequisite for homothallic mating in C. albicans.

Glucose depletion promotes homothallic mating in C. albicans

For most microbes in natural environments, carbon sources are the most limiting nutrients. In natural niches, such as environmental and host mucosal surfaces (e.g., the mammalian lower gut), C. albicans often suffers from glucose starvation (110). Rich YPD medium (1% yeast extract, 2% peptone, and 2% glucose [wt/vol]) is used for the routine growth of C. albicans in the lab. Guan et al. (110) found that when grown on YP-K medium (a modified rich medium containing 1% yeast extract and 2% peptone, without glucose and with K₂HPO₄ for pH buffering), opaque a cells developed mating projections and formed wrinkled colonies (110). The increased expression of mating-associated genes, including MFA1, MFα, STE2, BAR1, FIG1, and FUS1, confirmed that the elongated morphologies of the opaque cells were indeed mating projections (110). Interestingly, the proportion of cells forming projections exhibited a dose-dependent relationship with decreasing glucose concentrations. Activation of pheromone sensing and the mating MAPK signaling pathway promoted homothallic mating (1.3 × 10⁻⁶ vs <2.6 × 10⁻⁹) in C. albicans under conditions of glucose depletion. Further investigation indicated that the generation of intracellular reactive oxygen species (ROS), the transcription factor Hsf1, and the heat shock protein 90 (Hsp90) (both required for stress responses) was involved in the regulation of homothallic mating induced by glucose depletion (Fig. 6B). Via its downstream effectors Cta4 and Cwt1, the Hsf1-Hsp90 pathway activates the pheromone response and mating in C. albicans by activating the expression of MTLa2 and MFα. Given that carbon source limitation is a commonly encountered environmental stress, the occurrence of homothallic mating could be more frequent in nature than previously thought. Indeed, opaque cells undergo the development of mating projections as well as homothallic mating under specific conditions mimicking natural niches, such as on nutrient-deprived agar, agar containing mouse feces, and agar containing C. albicans debris (110). Such homothallic mating bypasses the need for an opposite mating partner as well as for Bar1 downregulation, and thus may represent an important mode of sexual reproduction for C. albicans in nature. These observations also provide examples of how homothallic mating can be induced by environmental stresses in fungi.

Effect of atmospheric humidity on homothallic mating in C. albicans

Atmospheric humidity is an important environmental factor impacting fungal pathogenesis and sexual reproduction (136–139). In a recent study, Li et al. (108) found that atmospheric conditions could be a major contributor to mating behaviors and that low atmospheric humidity conditions could favor homothallic mating (1.1 × 10⁻⁶ vs <2.9 × 10⁻⁸) in C. albicans, especially under conditions of glucose depletion (108). The authors found that low atmospheric humidity increased the expression of mating-associated genes and promoted the formation of mating projections (108). Under conditions of high atmospheric humidity, homothallic mating was nearly completely blocked (108). In addition, low atmospheric humidity promoted ROS production and osmotic stress.

Deletion of the cytoplasmic trehalose-encoding gene NTH1 increases intracellular trehalose levels and promotes opaque cell mating, which resembles the effect of addition of exogenous trehalose to the culture medium (108). The conserved aquaporin Aqy1 and G protein-coupled receptor Gpr1, which are required for humidity sensing in fungi (140, 141), play negative roles in homothallic mating in C. albicans. Deletion of GPR1 leads to a decrease in expression of NTH1 and AQY1 and reduces Hog1 phosphorylation, indicating that the interaction between trehalose and the osmotic stress response regulates homothallic mating in C. albicans. Genes involved in trehalose synthesis, however, regulate mating of C. albicans through a different pathway. Since trehalose has a protective effect on fungal cells under oxidative stress conditions (142), it is reasonable that the decrease of trehalose synthesis results in the accumulation of intracellular ROS and the activation of the Hsf1-Hsp90 signaling pathway (108). Finally, the Hog1 osmotic and Hsf1-Hsp90 signaling pathways function coordinately to control homothallic mating in C. albicans (Fig. 6B). These findings suggest that atmospheric humidity regulates sexual reproduction via conserved signaling pathways in C. albicans.

ROLES OF WHITE CELLS IN MATING OF C. ALBICANS

White cells facilitate opaque cell mating

C. albicans opaque cells are more vulnerable to environmental and host-associated stresses (143). Although white cells are the default cell state and are mating incompetent under standard laboratory culture conditions, white cells can respond to pheromone by increasing the expression of mating-associated genes (110, 135, 144). When exposed to mating pheromone or opaque cells of the opposite mating type, white cells can form “sexual biofilms,” which are distinct from conventional biofilms that are produced by MTL-heterozygous cells (Fig. 8A) (144–146). Conventional biofilms involve processes such as cell adherence and invasion, hyphal formation, and extracellular matrix deposition, while sexual biofilms are initiated in response to mating pheromone (144). A detailed comparison of sexual and conventional biofilms is reviewed by Perry et al. (146). Interestingly, biofilm thickness is doubled with the addition of a minority of opaque cells to a majority of white cells during sexual biofilm development, suggesting that the presence of rare opaque cells can stimulate biofilm formation by white cells. Opaque cells release gradients of pheromone, directing the elongation of mating projections of opposite mating types, a process referred to as “chemotropism.” The formation of a three-dimensional cell matrix by white cells during sexual biofilm formation could stabilize these pheromone gradients over long distances, which could facilitate opaque cell mating in biofilms via enhanced chemotropism (144, 145).

FIG 8.

Role of white cells in mating. (A) White cell responses to pheromone. The MAPK pathway mediates pheromone-induced biofilm development and mating in white cells when treated with synthetic pheromone or in the presence of opaque cells of the opposite mating type. (B) Glucose depletion induces white cell mating. The Cph1 and Tec1 transcription factors play positive and negative roles, respectively, in the regulation of mating.

While white and opaque cells share the same pheromone response pathway, they target different downstream transcription factors to regulate sexual biofilm formation or mating, respectively (54, 147). The minority opaque cells can induce sexual biofilm formation by white cells, and deletion of STE2 or MFα in white a cells can lead to reduction in biofilm formation, suggesting that pheromone signaling in white a cells is involved in sexual biofilm formation. Pheromones, either produced by cells of opposite mating types or by opaque cells of the same mating type, as well as pheromone receptors, are essential for the formation of sexual biofilms.

In addition, white cells can be induced to secrete mating pheromones in the presence of pheromones of the opposite mating type (Fig. 8A) (135). In a mating experiment where white a and opaque α cells were mixed together, a high proportion of opaque α cells were observed to form mating projections. To exclude the possibility of spontaneous white-to-opaque switching of white a cells inducing the production of mating projections of α cells, wor1/wor1 a cells were mixed with opaque α cells. In the latter experiment, a similar outcome was observed, where mating projections of opaque α cells formed, indicating that white cells can produce pheromone in the presence of opaque cells. Pheromones produced by white cells, thus, can facilitate both heterothallic (six- to ninefold increase in mating efficiency) and homothallic mating (1.1 × 10⁻⁶ vs <5.1 × 10⁻¹⁰) of opaque cells in C. albicans likely by maintaining a proper level of pheromone in the mixture. These results suggest that the coordination between white and opaque cells through formation of sexual biofilms or by maintaining pheromone gradients could increase the mating efficiency of C. albicans in certain natural niches.

Glucose depletion promotes white cell mating

In response to pheromone of the opposite mating type, white cells are induced to secrete pheromone and increasingly express a set of mating-associated genes (135, 144). This white cell pheromone response phenomenon is highly similar to that of the opaque cell pheromone response, implying that white cells, under certain conditions, could become mating competent. Although many components of the pheromone-MAPK pathway are induced in white cells (54, 148, 149), the low expression levels of key mating-associated genes, such as STE4, CST5, CEK1, and CEK2, inhibit mating of white cells (149). Ectopic expression of these genes, either individually or in combination, promotes mating projection formation and mating in white cells at comparable levels to those of opaque cells (149). This finding suggests that white cells could become mating competent under culture conditions that promote the expression of mating-associated genes.

A recent study demonstrated that mating between white cells could be induced by environmental conditions, such as glucose depletion and oxidative agents, stresses that C. albicans cells routinely encounter in natural settings (Fig. 8B) (109). Guan et al. (109) found that when grown on YP-K medium, a set of genes encoding the major components of the pheromone MAPK pathway and mating-associated genes, including MFA1, MFα, STE2, STE3, CEK1, CEK2, FIG1, and FUS1, had increased expression levels in the white “locked” wor1/wor1 strain under conditions of glucose depletion (109). The frequency of mating between white a and α cells was 1.4 × 10⁻⁶ on YP-K medium, while no mating between white cells was observed on YPD-K medium (<3.0 × 10⁻⁹). Activation of the pheromone-MAPK pathway promotes mating projection formation and confers mating competency in white cells. As expected, inactivation of the pheromone-MAPK pathway through deletion of STE2 blocked white cell mating, while disruption of either CEK1 or CEK2 decreased mating efficiency. However, overexpression of CEK2 (but not CEK1) increased mating efficiency of the wor1/wor1 mutant strain, suggesting that the Cek2 kinase plays a major role in white cell mating. The authors further showed that the downstream transcription factors Cph1 and Tec1 play positive and negative roles, respectively, in the regulation of white cell mating; however, there is some controversy over whether white cells respond to pheromone via signaling mediated by Cph1 or Tec1 (54, 148, 149). Cph1 is known to be essential for opaque cell mating, and Tec1 is thought to be required for pheromone-induced sexual biofilm formation of white cells (53, 54, 150). Moreover, the Dig1 transcription factor, which inhibits the expression of Cph1 during filamentous growth in C. albicans (151), negatively regulates white cell mating. Even in the presence of glucose, the dig1/dig1 wor1/wor1 double-mutant strain can undergo mating. Consistently, ectopic expression of DIG1 blocked white cell mating under conditions of glucose depletion. Taken together, the regulators Cph1, Tec1, and Dig1 function downstream of the MAPK pathway to regulate white cell mating. Further investigation has shown that white cells can mate even more efficiently under extreme nutrient-limiting conditions (e.g., on medium containing only agar), providing additional evidence that white cells could become mating competent under specific inducing conditions. Given that white-opaque switching is a newly evolved species-specific characteristic that is only found in C. albicans and its closely related species, white cell mating could represent a more ancient mode of sexual reproduction in nature than opaque cell mating.

FATE OF C. ALBICANS MATING PRODUCTS

Most natural C. albicans strains are diploid (19), and mating between diploid a and α cells produces tetraploid progeny (55, 56). Although the genome of C. albicans contains most, if not all, meiosis-associated gene homologs, no meiotic events have been observed in C. albicans to date (152). Tetraploid mating products are unstable, and chromosome loss can be triggered by stress (153, 154). A subset of tetraploid cells formed through nuclear fusion of two diploid protoplasts has been shown to be induced to return to diploid or near diploid states after brief heat treatment at 50°C for 1–2 min (153) or by selection for drug resistance (154). To explore conditions that induce chromosome reduction, a marked tetraploid strain formed between diploid a and α cells, carrying heterozygous URA3 and GAL1 genes and four MTL configurations, was grown under different test conditions. This strain contains one copy of URA3 and two copies of GAL1 and cannot grow on media containing either 5-fluoroorotic acid (5-FOA) or 2-deoxygalactose (2-DOG). When cultured on pre-sporulation (pre-spo) medium—a medium used for meiosis in S. cerevisiae—at 37°C, tetraploid cells produced 10-fold more colonies on the 5-FOA or 2-DOG selective media than under any of the other conditions tested (19). The progeny displayed a range of ploidy, with ~33% returning to diploid or near-diploid states. Analysis of MTL alleles indicated that chromosome loss, rather than gene conversion, accounted for the allele loss (19). The genetic markers URA3 and GAL1 and the MTL locus are located on chromosomes 1, 3, and 5, respectively, and all three chromosomes showed similar loss rates. Loss of one or more chromosomes often occurred in concert with others, a process termed CCL. MTL-homozygous diploid progeny generated by CCL are mating competent, and mating between a and α progeny forms tetraploids that undergo CCL to return to the diploid state and complete the parasexual cycle in C. albicans (18, 19).

While the pre-spo condition efficiently induces chromosome loss in tetraploids, diploid cells remain largely stable (19). Diploid strains with wild-type GAL1 and URA3 genes showed low growth efficiency on 5-FOA or 2-DOG media, and no MTL changes were observed, confirming that the diploid state is more stable than the tetraploid state under these conditions. L-Sorbose was implicated in the loss of one homolog of chromosome 5 in diploids (114), promotes extensive chromosome reduction in tetraploids, including chromosome 5 and at least two others, generating more diploid progeny with only one copy of chromosome 5 than the pre-spo medium (19). These observations indicate that sorbose is a general inducer of chromosomal instability. Similar CCL processes have been observed in S. cerevisiae, and both species converge toward diploidy despite haploid, diploid, and tetraploid viability, highlighting the role of environmental selection in maintaining diploidy (155).

The mechanisms underlying CCL remain unclear. Endogenous metabolic processes producing high ROS promote chromosome loss in tetraploids of C. albicans (156). High glucose in the pre-spo medium drives hyperactive metabolism, increasing ROS, activating Cap1 (a ROS-responsive transcription factor), and inducing DNA DSBs (156). Analysis of diploid progeny revealed that true diploids are not immediately generated, and multiple homologous recombination events complicate the CCL process (18). Recombination efficiency during CCL is lower than meiosis but is 1,000-fold higher than normal mitosis. The meiosis-specific protein Spo11, required for DSB formation during meiosis (157), reduces chromosome loss and mediates genetic recombination in C. albicans; Spo11 deletion decreases recombination in tetraploids and also functions in mitotic recombination in C. albicans (18, 158). Rec8, a meiosis-specific kleisin, also affects chromosome stability and genetic recombination during CCL (158). Collectively, these findings support the notion that the parasexual cycle is an alternative mechanism to conventional meiosis for generating genetic diversity in C. albicans.

SEXUAL REPRODUCTION IN NON-ALBICANS CANDIDA SPECIES

The processes and regulation of parasexual cycles in different fungal pathogens often have unique species characteristics (16). For example, the epigenetic white-opaque phenotypic switch in C. albicans and closely related species is involved in the regulation of mating (41, 70, 103, 159), while mating and meiosis are coupled and regulated by the pheromone MAPK signaling pathway in Candida lusitaniae (15, 160–162). Homothallic mating and coupled homothallic and heterothallic mating have been identified in C. tropicalis (163, 164). The similarities and distinctions among Candida species are shown in Table 1. Although genes homologous to mating and meiosis regulators in S. cerevisiae are present in the genomes of some Candida species, neither mating nor meiosis has been observed in these fungi. These genes may have evolved functions unrelated to sexual reproduction, and whether certain Candida species retain the capacity for mating remains to be determined.

TABLE 1.

Similarities and differences of genetic and biological features among key Candida species^a

Species	Clade	Major niches	Predominant ploidy state	Phenotypic switch-regulated mating	Ability to mate	Ability to undergo meiosis	Sexual cycle
C. albicans	CTG	Humans and warm-blooded animals	Diploid	Yes	Yes	NA	Parasexual reproduction
C. tropicalis	CTG	Humans and warm-blooded animals, natural environment	Diploid	Yes	Yes	NA	Parasexual reproduction
C. dubliniensis	CTG	Humans and warm-blooded animals, natural environment	Diploid	Yes	Yes	NA	NA
C. lusitaniae	CTG	Humans and warm-blooded animals, natural environment	Haploid	No	Yes	Yes	Sexual reproduction
C. parapsilosis	CTG	Humans and warm-blooded animals, natural environment	Diploid	NA	NA	NA	NA
Lodderomyces elongisporus	CTG	Humans and warm-blooded animals, natural environment	Diploid	NA	NA	NA	NA
C. auris	CTG	Humans and warm-blooded animals, natural environment	Haploid	NA	NA	NA	NA
C. glabrata	WGD	Humans and warm-blooded animals, natural environment	Haploid	NA	NA	NA	NA
S. cerevisiae	WGD	Natural environment	Diploid	No	Yes	Yes	Sexual reproduction

^aNatural environmental niches include soil, plants, fruits, seawater, etc. CTG, the CTG clade; NA, not applicable or not yet found; WGD, the whole-genome duplication clade.

Candida dubliniensis

First identified in 1995, C. dubliniensis is the closest known relative of C. albicans (165). Although the two species share genomic similarities and close phylogenetic relatedness, they differ in several important aspects, including epidemiology, virulence, and antifungal resistance (166). C. dubliniensis is frequently associated with oral candidiasis in patients infected with human immunodeficiency virus. However, in invasive infection models, C. dubliniensis exhibits substantially reduced virulence compared to C. albicans (167). Comparative genomic studies indicate that the expansion of the SAP, ALS, and IFF gene families facilitated fungal adaptation, whereas amplification of the TLO and IFA gene families contributed to the increased pathogenic potential of C. albicans (167). In contrast, C. dubliniensis shows extensive gene loss and limited genomic innovation, likely reflecting its pronounced genetic instability and high rates of mitotic recombination (167).

There are three MTL configurations (MTLa/a, α/α, and a/α) that have been observed in clinical isolates of C. dubliniensis, where the proportion of MTL homozygotes is ~33%, 10-fold higher than that of C. albicans (Fig. 2C) (168). Similar to C. albicans, MTL-homozygous strains of C. dubliniensis can also undergo white-opaque bistable switching, white-gray-opaque tristable switching, and mating (103, 165, 166). White, gray, and opaque cells of C. dubliniensis exhibit mating abilities similar to their counterparts in C. albicans (103). Mating has been documented not only between C. dubliniensis cells of opposite mating types but also between C. dubliniensis and C. albicans cells of opposite mating types (168). Remarkably, the efficiency of interspecies mating between C. dubliniensis and C. albicans was even higher than that of intraspecies mating within C. dubliniensis (168). This capacity for interspecies mating underscores their close genetic relationship. Although nuclear fusion (karyogamy) occurs during interspecies crosses, no evidence of genetic recombination between the genomes of C. albicans and C. dubliniensis has been detected in nature (168). Nonetheless, fusion of cells and nuclei from different species may influence epigenetic states through cytoplasmic mixing, potentially enabling rare recombination events under stressful conditions. Moreover, such interspecies mating provides a valuable system for investigating cytoplasmic incompatibility in eukaryotes.

Candida tropicalis

C. tropicalis, another diploid species of the CTG clade (10, 11), is a common cause of infection in patients with neutropenia and hematological malignancies and represents the second or third etiological agent of life-threatening Candida infections (169–172). The MTL locus of C. tropicalis is similar in structure to that of C. albicans and can also undergo white-opaque and white-gray-opaque switching under specific inducing conditions (Fig. 2C) (104, 159, 164, 173). Although GlcNAc has opposing effects on filamentous growth in C. tropicalis and C. albicans, it promotes the gray and opaque phenotypes in C. tropicalis (104, 159). Of the C. tropicalis cell types, opaque cells are the most efficient at mating, while white and gray cells exhibit moderate mating competencies even under standard culture conditions (104). Therefore, unlike in C. albicans, mating in C. tropicalis is not dependent on phenotypic switching. Seervai et al. (164) demonstrated that C. tropicalis diploid cells of opposite mating types can mate and form tetraploids, which are unstable and return to the diploid state through chromosome loss when grown on sorbose medium or when incubated for an extended period of time on rich medium (164). Although diploid and tetraploid C. tropicalis cells display similar doubling times, diploids exhibit greater fitness than tetraploids under in vitro culture conditions.

C. tropicalis is capable of homothallic mating when exposed to cells of the opposite mating type or to the pheromone of the opposite mating type (163). This homothallic mating between diploid cells produces MTL-homozygous tetraploid progeny (163). Interestingly, these MTL-homozygous tetraploids are mating competent and able to further mate with diploid cells. The combined process of heterothallic and homothallic mating generates unstable hexaploidy in C. tropicalis, which leads to rapid chromosome loss and the generation of genetically and phenotypically diverse offspring (163). Consistently, a recent study found that some natural strains of C. tropicalis exhibited high levels of intragenomic heterozygosity, indicating that mating or hybridization occurred between different parental strains (174). Overall, these findings demonstrate that a unique parasexual cycle exists in C. tropicalis, which drives ploidy and genetic diversity. The differences in mating regulation between C. tropicalis and C. albicans may reflect their distinct ecological adaptation strategies.

Candida lusitaniae

C. lusitaniae is a clinically rare non-albicans pathogenic Candida species that is notorious for developing resistance to antifungal agents, especially the azoles (175). C. lusitaniae is considered to be a haploid organism and possesses a defined sexual cycle where it undergoes meiosis and sporulation (162). Haploid MTLa and MTLα C. lusitaniae cells can mate and produce diploid MTLa/α heterozygotes. Notably, the absence of the MTLα2 gene in C. lusitaniae (Fig. 2C) (162) suggests that its mating regulatory mechanisms may have been rewired. Moreover, despite the lack of multiple key meiosis-associated genes, C. lusitaniae is still capable of undergoing meiosis (162). Unlike other yeast species with conventional sexual cycles, C. lusitaniae sporulation generates unusually high proportions of aneuploid and diploid progeny, perhaps due to the loss of key meiotic components and the increased frequency of meiotic errors (162). This ploidy variation may enhance its adaptability as a human pathogen. Interestingly, a subset of genes, including those involved in the pheromone MAPK signaling pathway, is expressed during both mating and meiosis in C. lusitaniae, potentially supporting its predominantly haploid lifestyle (22). Comparative genomic analyses further revealed that, like other CTG Candida species, C. lusitaniae lacks several conserved meiosis-associated genes. The presence of a complete mating-meiosis cycle highlights the evolutionary plasticity of meiotic machinery and suggests the existence of alternative regulatory strategies in this species (162).

Candida parapsilosis

C. parapsilosis is the second or third most frequently isolated Candida species from blood cultures, accounting for approximately 15% of Candida infections in clinical settings (176). This species is particularly associated with infections in neonatal and ICU patients (177). C. parapsilosis spreads readily in hospital settings and is associated with treatment failure, largely because of its capacity to form robust biofilms and its decreased susceptibility to antifungal drugs. In C. parapsilosis, only the MTLa idiomorph has been identified, and the MTLa1 gene has degenerated into a pseudogene, containing four stop codons within its open reading frame (Fig. 2C) (178, 179). Candida orthopsilosis and Candida metapsilosis are members of the C. parapsilosis complex and are closely related species to C. parapsilosis (179, 180). All three idiomorphs (MTLa, MTLα, and MTLa/α) have been found in C. orthopsilosis and C. metapsilosis (179, 181). Although C. parapsilosis, C. orthopsilosis, and C. metapsilosis possess functional pheromone-encoding genes and their synthesized α-pheromones can trigger mating responses in C. albicans, mating has not been observed in these species. Interestingly, a hybrid between two C. orthopsilosis subspecies was identified, suggesting that C. orthopsilosis may be capable of mating under specific inducing conditions (182, 183). Why have only the MTLa strains of C. parapsilosis been isolated in clinical settings? It remains to be investigated whether the mating-type locus plays a critical role in host and environmental adaptation and whether the a mating type confers an advantage for survival over the α mating type.

Lodderomyces elongisporus

L. elongisporus is a diploid ascomycete yeast that belongs to the CTG clade and is phylogenetically closely related to C. parapsilosis (184). Infections caused by L. elongisporus are becoming more common in clinical settings (185, 186). Since it highly resembles C. parapsilosis in cellular morphology, filamentation, and biofilm formation (187), L. elongisporus was initially considered to be a teleomorph of C. parapsilosis and was often misidentified as C. parapsilosis in clinical settings (188, 189). A striking feature of the MTL configuration of L. elongisporus is the lack of all four MTL transcriptional regulators (Mtla1, a2, α1, and α2) (22). A recent genomic analysis revealed that certain clinical isolates of L. elongisporus contain partial sequences homologous to C. parapsilosis MTLa1 and C. albicans MTLα2 (Fig. 2) (186); however, these sequences do not appear to encode functional MTL proteins. Although mating has not been detected in L. elongisporus, some strains have been observed to form ascospores (186, 188). It has been proposed that L. elongisporus may be capable of mating via an MTL-independent mechanism (186).

Candida auris

The emerging fungal pathogen C. auris was first described in Japan in 2009 (190). It is a haploid CTG species closely related to Candida haemulonii and C. lusitaniae (Fig. 1) (191). C. auris has garnered considerable attention due to its rapid transmission and notable antifungal-resistant properties (191). As of December 2023, C. auris-associated infections have been reported in at least 60 countries across 6 continents (192). Based on whole-genome analysis and the geographic origins of initial isolates, six distinct genetic clades of C. auris have been identified to date: clades I (South Asian), II (East Asian), III (South African), IV (South American), V (Iran), and VI (Singapore and Bangladesh).

Two MTL idiomorphs (a and α) have been discovered in C. auris (Fig. 2C). All isolates of clades I, IV, V, and VI carry an MTLa mating type, while all isolates of clades II and III harbor an MTLα locus (191, 193). Multiple cell types were observed in C. auris populations, and white-brown switching occurs reversibly and frequently (194). Despite the presence of most genes associated with mating and meiosis in its genome (161), sexual reproduction has not been observed in C. auris. Population genomic analyses indicate that recombination and ongoing genetic exchange have occurred in natural populations of C. auris, although at low frequencies, particularly following the divergence of its genetic clades (15, 195).

It has been proposed that two potential barriers may prevent mating in C. auris (161). The first is a geographic barrier: each genetic clade, identified from distinct geographic regions, consists exclusively of either MTLa or MTLα isolates. This distribution would limit heterothallic mating in C. auris. The second is a chromosomal barrier: significant karyotype differences between mating partners of different C. auris clades may hinder the likelihood of successful genetic exchange. In addition, recent findings have revealed that many conserved genes required for mating and meiosis in C. auris carry mutations (J. Bing and G. Huang, unpublished data, and reference 15). Notable mating-associated genes in C. auris that are missing or that contain nonsense mutations include AXL1, STE2, STE6, STE50, and FUS1. These genes are involved in the regulation of mating pheromone production (AXL1 and STE6), signal transduction (STE2 and STE50), and cell fusion (FUS1). Notable meiosis-associated genes in C. auris that are missing or that carry mutations include CHL1, RAD57, IME2, UME6, and RAD9. The widespread presence of mutations in genes associated with mating and meiosis suggests that sexual reproduction in C. auris may have undergone degeneration.

Candida glabrata

C. glabrata (also known as Nakaseomyces glabratus) is considered an asexual, haploid fungal pathogen of the Nakaseomyces genus and represents the second most commonly isolated Candida species in clinics (12). C. glabrata is phylogenetically more closely related to S. cerevisiae than to other Candida species of the CTG clade, such as C. albicans and C. tropicalis (Fig. 1) (196). Although C. glabrata is incapable of forming true hyphae, it can form pseudohyphae and chlamydospores, and it undergoes phenotypic switching under specific culture conditions, such as on medium containing CuSO₄ (197–199).

In C. glabrata, three mating-type loci (MTL1–MTL3) are located on two separate chromosomes (Fig. 2B). C. glabrata also contains a gene that encodes an HO endonuclease (200, 201). The majority of clinical isolates have the MTLa idiomorph (a mating type at the active MTL1 locus). Ectopic expression of the HO gene in C. glabrata can trigger mating-type switching (36). This artificial induction led to high cell mortality and even triggered the conversion of the “inactive” HML cassette (MTL3) (36). DSBs at the mating-type loci may underlie cell death induced by HO expression, as the enzyme can remain bound to DNA ends after cleavage, thereby blocking homologous recombination (202).

Unlike in S. cerevisiae, where the expression of MTLa and MTLα genes is mating type specific, C. glabrata MTLa1 is expressed in both a and α strains, possibly due to a deficiency in effective gene silencing (203). However, MATα1 is expressed only in C. glabrata α strains (204). In addition, the expression of a-pheromone and α-pheromone receptor genes (STE3 and STE2, respectively) is also independent of mating type in C. glabrata. No mating pheromone response phenomena have ever been observed in C. glabrata when exposed to synthetic pheromone (204). Nonetheless, C. glabrata possesses a number of mating and meiosis-associated genes comparable to its closest phylogenetic relative, Nakaseomyces delphensis, which is mating competent (201, 205–207). Although genomic and population studies suggest that C. glabrata could harbor a cryptic sexual cycle (208–210), it remains unclear whether this fungus can undergo sexual or parasexual reproduction under specific environmental conditions that have yet to be identified.

ASEXUAL PLOIDY SHIFTS IN THE CANDIDA SPECIES

Ploidy plasticity is a common trait among pathogenic fungi (211–213). Ploidy shifts or changes in entire chromosome sets that are not the result of sexual reproduction have been reported in several pathogenic Candida species (211). For example, the so-called obligate diploid fungal pathogens, C. albicans and C. tropicalis, have been documented to form haploid or hyperdiploid cells after exposure to azoles (24, 26, 214). The haploid yeasts C. auris and C. glabrata can spontaneously transition to a diploid state under in vitro culture conditions or during infections in animal models (210, 215). Given the predominant clonal growth patterns of the Candida species, the generation of genetic diversity through sexual or parasexual reproduction seems unlikely to occur in nature (15, 16, 216). Thus, asexual ploidy variations are likely to play key roles in generating phenotypic diversity and in enabling rapid adaptation to environmental perturbations and may represent an important alternative strategy to sexual reproduction in fungal pathogens.

Azole-induced ploidy shifts in C. albicans and C. tropicalis

With the exception of a handful of azole-resistant clinical isolates that exhibit aneuploidy, C. albicans and C. tropicalis are generally considered to be diploid organisms (22, 23). Exposure to azole drugs has been shown to induce the formation of aneuploid, hyperploid, and haploid cells in C. albicans and C. tropicalis (24, 26, 214). Harrison et al. (214) observed that C. albicans grew abnormally and formed a three-lobed multinucleated morphology termed a “trimera” when exposed to fluconazole (214). This trimera then underwent abnormal mitotic division and produced aneuploids with varying numbers of chromosomes (214). These aneuploids may have selective advantages under stressful conditions and/or during host infection. Interestingly, short-term fluconazole exposure can promote CCL and trigger the formation of haploid cells in C. albicans (24, 25, 214). This CCL-driven mechanism resulting in the formation of haploids may be similar to the process of parasexual tetraploid formation in C. albicans (214). Similar to their diploid parental strains, haploid strains of C. albicans are also capable of phenotypic switching and mating (24). Mating between haploid opaque cells of opposite mating types can produce diploid progeny with enhanced fitness. Moreover, haploid cells can undergo spontaneous autodiploidization, resulting in the formation of homozygous diploids (24).

In contrast to C. albicans, C. tropicalis is broadly distributed across diverse natural environments and is frequently exposed to agricultural azoles, such as fungicides (26, 172). Recently, Hu et al. (26) demonstrated that treatment with the agricultural azole tebuconazole resulted in significant genomic instability in C. tropicalis, leading to the formation of aneuploid cells with distinct chromosomal alterations (26). Some strains resulting from the tebuconazole treatment even possessed a haploid or near-haploid genome (26). The genomic diversity in C. tropicalis induced by tebuconazole may reflect a similar phenomenon to the ploidy shifts observed in C. albicans following fluconazole treatment. As expected, the resulting C. tropicalis aneuploid/haploid strains were resistant to azoles. Given the diverse ecological niches of C. tropicalis, agricultural azoles may act as an evolutionary driver to promote genetic and phenotypic diversity. In summary, the discovery of the haploid form has not only improved our understanding of the biology of C. albicans and C. tropicalis but also offers a powerful tool for advancing future molecular and genetic research in the field.

Spontaneous ploidy shifts in C. auris and C. glabrata

The recently emerged fungal pathogen C. auris is generally considered to be a haploid yeast (217). When grown under stressful conditions, clinical isolates of C. auris display considerable karyotypic variability (217). Spontaneous diploidization has been observed in the haploid state of the model yeast S. cerevisiae, potentially aiding the survival of the haploid population under stressful environmental conditions (218). Fan et al. (215) discovered that C. auris can spontaneously switch to a diploid state under laboratory culture conditions (215). C. auris diploid cells grow faster than haploid cells and exhibit survival advantages during systemic infections. Both diploid and haploid cells of C. auris express a set of cell type-specific genes involved in regulating key important biological processes, including metabolism and cell wall maintenance. This cell type-specific gene expression profile could contribute to the fitness of both C. auris diploid and haploid cells in adapting to different ecological niches.

Despite its close phylogenetic relationship to S. cerevisiae, sexual reproduction has not been observed in C. glabrata (208–210). Zheng et al. (210) reported that clinical isolates of C. glabrata exhibited high levels of ploidy variation and were able to undergo spontaneous switching between haploid and diploid states under in vitro culture conditions or during systemic infections (210). More interestingly, the authors identified diploid strains of C. glabrata from clinical samples, indicating that ploidy changes could occur during host colonization or infection (210). Analysis of 500 C. glabrata clinical isolates indicated that approximately 3% of the strains were stable diploids (210). Cells of both haploid and diploid isolates demonstrated distinct features of antifungal susceptibility, morphology on CuSO₄-containing medium, and virulence (210). Considering the potential absence of a sexual cycle in both C. auris and C. glabrata, ploidy changes may serve as an alternative means of generating genetic and phenotypic diversity in these species in order for them to rapidly adapt to environmental changes. Further research is needed to reveal the mechanisms governing ploidy shifts in these species.

CONCLUDING REMARKS AND OPEN QUESTIONS

Sexual reproduction is widely prevalent in fungal species and serves as an engine for the generation of genetic and phenotypic variability, which is important for the evolution of new traits and adaptation to changing environments. The major components of the conserved machinery for mating and meiosis are maintained in the pathogenic Candida species (22). Several species, including C. albicans, C. tropicalis, C. dubliniensis, and C. lusitaniae, are able to undergo sexual or parasexual reproduction under specific inducing conditions (55, 56, 159, 162, 173, 219). However, the mechanisms of sexual and parasexual reproduction vary between species and often exhibit species-specific features.

A notable characteristic of C. albicans and its close relatives, C. tropicalis and C. dubliniensis, is the integration of the white-opaque epigenetic switching system into their mating processes (70, 103, 159), which adds an extra layer of regulation to sexual reproduction in these species. Many host- or niche-associated environmental factors that regulate the efficiency of white-opaque switching could therefore indirectly affect mating in C. albicans, C. tropicalis, and/or C. dubliniensis. For the Clavispora clade species C. lusitaniae, its sexual cycle exhibits a high similarity to that of the model yeast Schizosaccharomyces pombe, where mating and meiosis regulation are tightly intertwined (162). The pheromone MAPK signaling pathway is involved in the regulation of both mating and meiosis in C. lusitaniae, suggesting that substantial rewiring has taken place to accommodate its unique lifestyle. While sexual and parasexual reproduction processes are generally conserved among the pathogenic Candida species, species-specific genetic and environmental regulators could likely reflect and be associated with adaptations to their natural ecological niches or host environments.

Sexual and parasexual reproduction have many advantages as well as costs compared to asexual reproduction. Given that sexual and parasexual reproduction rarely occur in the pathogenic Candida species in nature (220), their genetic regulators and associated signaling pathways are likely also involved in the regulation of other important biological processes, such as antifungal resistance, biofilm development, morphological transitions, and virulence. As a result, environmental factors that affect mating in the pathogenic Candida species are often critical regulators of phenotypic transitions, virulence, and/or other biological processes. These common regulatory mechanisms thus moderate the transitions between sexual and asexual reproductive cycles.

Despite the significant advances made over the past two decades, many questions about the sexual and parasexual cycles in the Candida species remain unanswered. For example, is mating via the white-opaque switching common in nature for C. albicans and its close relatives? Given that white cells are mating competent under specific environmental conditions, why do these species need to incorporate this additional phenotypic switching system into the regulation of mating? Are there similar phenotypic transition systems involved in the regulation of sexual reproduction in other fungal species? Given the propensity for clonal reproduction in the Candida species, does homothallic mating occur more frequently than heterothallic mating in nature? Are the non-sexual genes located at the MTL loci important for sexual reproduction, virulence, or some other important biological processes? Do ploidy shifts triggered by spontaneous or environmental factors represent an alternative process to sexual reproduction to generate genetic diversity? Homologs of meiosis-specific protein family members in the model yeast S. cerevisiae, such as Spo11 and Rec8, positively regulate CCL-mediated genetic recombination in C. albicans. What is the underlying mechanism regulating chromosome loss and recombination in tetraploid cells of the Candida species? Further research is essential to address these questions and to deepen our understanding of the life cycle and biology of the pathogenic Candida species.

Biographies

Chengjun Cao is a researcher of College of Pharmaceutical Sciences at Southwest University. She received her Ph.D. degree in Microbiology under the guidance of Dr. Guanghua Huang at the Institute of Microbiology, Chinese Academy of Sciences (IMCAS) in 2017. Following her Ph.D., she joined Dr. Chaoyang Xue’s laboratory as a postdoctoral fellow at Rutgers University, where she investigated ubiquitin E3 ligase-mediated pathogenicity in Cryptococcus neoformans. In 2023, she started her independent research group at Southwest University (Chongqing, China). Her research interest is the regulatory mechanisms of pathogenicity and antifungal drug resistance of human fungal pathogens, including Cryptococcus and Candida species.

Li Tao is an associate professor of Microbiology at the School of Life Sciences at Fudan University. She obtained her M.S. and Ph.D. in Microbiology from China Agricultural University. During her Ph.D. studies, she conducted two years of joint research at University of Wisconsin-Madison, focusing on fungal morphogenesis and pathogenesis mechanisms. After returning to China, she was appointed to the Institute of Microbiology, Chinese Academy of Sciences (IMCAS), where she discovered the C. albicans gray phenotype and demonstrated a new role for white cells in C. albicans mating (2014). Since 2018, she has worked at Fudan University, where her research is focused on morphogenesis, mating and pathogenesis in Candida species.

Tianren Hu is a post-doctoral researcher at Shanghai Pulmonary Hospital, Tongji University School of Medicine. He received his Bachelor of Science from China Agricultural University in 2017. He obtained his Ph.D. in 2024 from Fudan University under the mentorship of Prof. Guanghua Huang. His research now centers on unraveling the evolutionary dynamics and antifungal mechanisms of Candida species through the lens of the ‘One Health’ approach. His recent work discovered that ploidy plasticity drove azole resistance in agriculture and clinics.

Haiqing Chu is a professor, chief physician, and director of the Department of Respiratory and Critical Care Medicine, Shanghai Pulmonary Hospital, Tongji University. Her laboratory studies the epidemiology, mechanisms of pathogenesis and drug resistance of non-Tuberculous mycobacteria and pathogenic Candida species. She has published over 50 SCI-indexed papers in high-impact journals such as Nature Communications and PLOS Biology.

Austin M. Perry received his B.S. in Biology with an emphasis in Microbiology and Immunology in 2017 from the University of California, Merced. He joined the Quantitative and Systems Biology (QSB) graduate program at the University of California in 2017, where he worked under Prof. Clarissa Nobile to study sexual biofilm formation and the mating type-like (MTL) locus in the Candida species of human fungal pathogens. He obtained his Ph.D. in 2024 from the University of California, Merced.

Clarissa J. Nobile is the Kamangar Family Chair in Biological Sciences and Professor of Molecular and Cell Biology at the University of California, Merced. She is also co-founder and Chief Scientific Officer of BioSynesis, Inc., a Bay Area startup company whose mission is to establish biofilm-specific diagnostics and therapeutics to diagnose and treat recalcitrant hospital infections. Prior to joining the faculty at the University of California, Merced in 2014, Dr. Nobile undertook her postdoctoral studies at the University of California, San Francisco in Prof. Sandy Johnson’s lab, and earned her doctoral degree in Microbiology with distinction from Columbia University in Prof. Aaron Mitchell’s lab. Prof. Nobile’s research is directed towards understanding the molecular and mechanistic bases of microbial communities. Prof. Nobile has published extensively and has received numerous extramural grants for her research. She is also a standing member of the NIH study section Genetic Variation and Evolution (GVE). She is the recipient of several scientific awards, including awards from the American Society for Microbiology and the Genetics Society of America. In 2015, she was selected as a Pew Biomedical Scholar, a recognition given annually by The Pew Charitable Trusts for her seminal research contributions to human health. In 2019 and again in 2024, she was the recipient of the distinguished Kamangar Family endowed chair, which provides additional support for her work in the biomedical sciences. In 2022, she was selected as a Pew Innovation Fund Investigator for her innovative work on understanding how microbial communities interact with their hosts. Website: http://faculty.ucmerced.edu/nobilelab Twitter handle: @cnobile1

Guanghua Huang is a professor in the School of Life Sciences at Fudan University. He is a graduate of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and received his doctorate in Biochemistry and molecular biology in 2006. He did his post-doctoral training in Prof. David R. Soll’s lab at the University of Iowa from 2007 to 2011. The Huang laboratory focuses on studying the molecular mechanisms of morphogenesis, pathogenesis, and sexual reproduction in pathogenic Candida species. Recent discoveries in his laboratory have focused on understanding the development of drug resistance and pathogenic features in the emerging fungal pathogen Candida auris.