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The emerging fungal pathogen Cryptococcus gattii: Epidemiology, pathogenesis, immunomodulatory attributes, and drug susceptibility [1]

['Chengjun Cao', 'College Of Pharmaceutical Sciences', 'Southwest University', 'Chongqing']

Date: 2025-08 

Abstract The emerging fungal pathogen, Cryptococcus gattii, causes infections in both immunocompromised and immunocompetent individuals, often resulting in high mortality rates. While Cryptococcus neoformans is predominantly associated with cryptococcosis in immunocompromised patients, the significance of C. gattii infections has garnered attention due to its prevalence among seemingly healthy individuals. Notably, C. gattii exhibits distinct epidemiological patterns, geographic distribution, genotypes, and phenotypes compared to C. neoformans. However, the comprehension of C. gattii’s virulence characteristics, regulatory mechanisms, and therapeutic avenues has lagged behind those of C. neoformans. The less robust clinical and epidemiological data, coupled with the limitations of effective treatment options, underscore the urgency in addressing C. gattii as a serious public health threat. In this review, I discuss the epidemiology, virulence factors, regulatory mechanisms, immunomodulatory attributes, and drug susceptibility of C. gattii. This comprehensive discussion aims to enhance our understanding of this emerging fungal pathogen and potentially contribute to the development of more effective prevention and management strategies.

Citation: Cao C (2025) The emerging fungal pathogen Cryptococcus gattii: Epidemiology, pathogenesis, immunomodulatory attributes, and drug susceptibility. PLoS Negl Trop Dis 19(7): e0013245. https://doi.org/10.1371/journal.pntd.0013245 Editor: Angel Gonzalez, Universidad de Antioquia, COLOMBIA Published: July 3, 2025 Copyright: © 2025 Chengjun Cao. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Natural Science Foundation of China (grants 82472305) and the research foundation of Southwest University (No. SWU-KR24012/5330501123) to C.C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: APCs, antigen-presenting cells; cAMP, cyclic AMP; CGSC, C. gattii species complex; DCs, dendritic cells; GM-CSF, granulocyte-macrophage colony-stimulating factor; HOG, high osmolarity glycerol response; MAPK, mitogen-activated protein kinase; PBMCs, peripheral blood mononuclear cells; PKA, protein kinase A

Introduction Cryptococcus species, notably Cryptococcus neoformans and Cryptococcus gattii, are major causes of life-threatening fungal meningitis [1]. These two species complexes exhibit distinct epidemiological patterns: C. neoformans predominantly affects immunosuppressed individuals, whereas C. gattii has been observed to cause cryptococcosis in both immunocompromised patients and those without apparent immune deficiency [2,3]. C. gattii has been recognized as an emerging fungal pathogen with an expanding environmental niche [4]. Within the C. gattii species, isolates can be further subdivided into two serotypes (B and C) and six genotypes (VGI, VGII, VGIII, VGIV, VGV, and VGVI), facilitated by advanced molecular and phenotypic analyses [2]. These geographical distinctions among the VG lineages reflect the intricate microevolutionary patterns within the C. gattii species complex (CGSC) over time. These ongoing processes underscore the continuous evolution of the CGSC, ensuring its adaptation to diverse environmental niches. Infectious propagules of C. gattii were inhaled into the lungs to establish fungal infection. Several virulence factors and signaling pathways that contribute to the pathogenicity of C. gattii have been identified. Alarmingly, the absence of dedicated antifungal drug trials to guide treatment of C. gattii infections underscores the urgency for effective therapeutic strategies. This environmental fungus has garnered global attention due to its increasing prevalence and associated high morbidity and mortality rates. Thus, a comprehensive understanding of its pathogenic characteristics and the development of targeted therapeutic interventions are vital in addressing the challenge posed by C. gattii infections.

Methods To prepare a review on the emerging fungal pathogen Cryptococcus gattii, the keywords “Cryptococcus gattii” and “fungal virulence” were used to identify relevant articles published from 2010 to 2024 using the PubMed and ScienceDirect databases. The search was limited to English-language articles. Relevance was assessed through title and abstract, and selected full-text articles were reviewed based on the information regarding the epidemiological patterns, virulence factors, molecular regulation pathways, the immune response, and drug susceptibility of C. gattii. Additional references were identified through citation tracking of selected articles.

Epidemiology Recent estimates indicate that cryptococcal meningitis affects more than 190,000 individuals, resulting in approximately 150,000 fatalities worldwide annually [1]. Prior to the advent of acquired immunodeficiency syndrome (AIDS), the incidence of cryptococcosis was relatively low. However, the HIV pandemic has dramatically reshaped the landscape of Cryptococcus infections, with cryptococcosis accounting for approximately 19% of HIV/AIDS-related deaths [5]. C. neoformans is the leading cause of cryptococcosis in immunocompromised individuals, particularly those afflicted by HIV. While the vast majority of C. gattii-infected patients are immunocompetent and apparently healthy individuals without HIV infection [2,6]. The VGI genotype prevails globally in both environmental and clinical settings, serving as the most common isolate [7]. Outbreaks on Vancouver Island and the Pacific Northwest (PNW) have been traced primarily to the VGII genotype. When considering the high global incidence rates of C. gattii infections, genotypes VGI and VGII are the primary C. gattii pathogens among non-AIDS patients [8]. Lineages VGIII and VGIV are dominant C. gattii types associated with AIDS patients [9]. C. gattii has the capacity to infect humans as well as domestic, terrestrial, and marine animals [10]. Cryptococcosis is not contagious. Both humans and animals can be infected by inhalation of desiccated yeast cells or basidiospores from the environment [10]. Increased environmental exposure is a significant reason for C. gattii infection [11]. The ability of healthy individuals’ host defense systems to eliminate fungal pathogens ensures that only a fraction of the exposed population ultimately develops C. gattii infection. However, predisposing risk factors significantly contribute to this outcome. These include the presence of autoantibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF), preexisting use of oral steroid use, a history of cancer or chronic lung disease, and the virulence of the C. gattii genotype [9].

Virulence factors The classic cryptococcal virulence factors, such as the polysaccharide capsule, melanin, and the ability to grow at physiological temperature, are involved in the pathogenicity of C. neoformans and have been reviewed in detail [2]. Although these virulence strategies are shared by pathogenic C. gattii, there are some crucial differences between the two species complexes (Fig 1). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Virulence factors identified in C. gattii. (A) Host conditions fitness, capsule and melanin production of C. gattii (VGI and VGII genotypes) and C. neoformans (VNI genotype). Growth assays at 30° in the air, at 37° in 5% CO 2 . India ink staining under light microscopy reveals the capsule. Melanin production at 30° on Niger seed medium. Bar, 5 μm. (B) Virulence factors associated with fungal pathogenesis and function in C. gattii. These virulence factors include polysaccharide capsule, melanin, cell wall components such as chitin and chitosan, titan cells, extracellular vesicles (EVs), and extracellular enzymes. https://doi.org/10.1371/journal.pntd.0013245.g001 Genotypes Different lineages of CGSC have been isolated from patients, VGIII and VGIV infections occur mainly in HIV/AIDS patients, whereas VGI and VGII were isolated in immunocompetent hosts [12–14]. Survival data using a Drosophila infection model found that VGIII was the most virulent molecular type [15]. Studies on the Vancouver Island outbreak strains showed the virulence difference within VGII that the subtype VGIIa is highly virulent compared to VGIIb based on a murine infection model [16]. These findings point to the influence of molecular subtypes on fungal virulence of C. gattii. However, an intriguing complexity arises when considering the virulence spectrum across different isolates within each major molecular type. Galleria mellonella infection studies demonstrate that C. gattii strains, regardless of their molecular types, encompass a broad range of virulence capabilities [14,17]. The coexistence of both low and highly virulent strains within all major molecular types suggests that virulence is not solely determined by molecular type but influenced by additional virulence factors. Capsule The polysaccharide capsule of Cryptococcus is composed of a major glucuronoxylomannan (GXM, formed by mannose backbone with xylose and glucuronic acid side chains) and two minor galactoxylomannan (GXMGal, galactan backbone with galactomannan side chains that are further substituted with variable numbers of xylose residues) and mannoproteins [18]. The GXM structures of C. gattii and C. neoformans differ in the degree of mannose backbone substitution, which can divide cryptococcal strains into different serotypes (A, B, C, D, and AD) [18]. The function and structure of the capsule have been well studied and documented in C. neoformans. The polysaccharide capsule is critical for host infection of C. neoformans [2,18]. During lung invasion, the capsule undergoes dramatic expansion and density modulation, a phenomenon intricately influenced by host factors like serum, high CO 2 concentration, low iron, and nutritional deprivation. The capsule provides a physical barrier that protects the fungus from desiccation and phagocytic predators, such as amoeba [18]. The enlarged capsule confers resistance to oxidative stresses to protect yeast cells against host phagocytic cells [18]. However, the sole reliance on capsule size as a virulence biomarker is tenuous, given the conflicting findings across various studies. Therefore, the relationship between capsule size and fungal virulence remains a subject of ongoing investigation. The capsule function of C. gattii is different from C. neoformans, which was discovered when comparing the virulence difference between an acapsular cryptococcal mutant strain coated with capsular extract from C. gattii and from C. neoformans [19]. Although the fungal biology of the capsule is likely to be conserved in C. gattii, the impact of capsule composition on C. gattii virulence remains unclear. Acapsular yeast cells of C. gattii were virulence attenuated in a mouse infection model, indicating that the capsule of C. gattii is required for pathogenicity and evasion from the host immune system [20,21]. The relationship between capsule size and fungal virulence is further complicated by observations that exposure of C. gattii to agrochemical benomyl and antifungals can diminish capsule size without compromising fungal virulence [22]. Thus, capsule size and structure play important and complex roles in C. gattii. Melanin The production of melanin pigment serves as a pivotal virulence factor of cryptococci, playing a crucial role in the pathogenicity of these fungi [23]. The dark macromolecular melanin maintains cell wall integrity and protects fungal cells against environmental stressors, including ultraviolet and ionizing radiation, as well as oxidative stress [23]. Laccase catalyzes the biosynthesis of melanin in the presence of phenolic compounds, such as L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine, epinephrine, norepinephrine and caffeic acid [24]. Notably, neurotransmitters can also serve as substrates to produce melanin during host infection in Cryptococcus species [24], indicating the association of melanin production with the central nervous system (CNS) infection. Moreover, the expression of laccase promotes intracellular proliferation in macrophages, while laccase-deficient mutant displays attenuated pulmonary dissemination [12,25], highlighting the enzyme’s significance in virulence. It has been reported that the ability of melanin production was directly associated with fungal virulence in C. gattii in a G. mellonella infection model [14]. Melanin production protects against reactive oxygen species and evades the host immune system, possibly correlating with fungal virulence and subsequent morbidity and mortality. Chitin and chitosan The fungal cell wall is an essential structure that provides the primary barrier against host defenses. Chitin and chitosan are important components in the cell wall that significantly modulate the interplay between host and pathogen. Chitosan-deficient strains of C. neoformans induce robust host immune responses during infection while exhibiting attenuated virulence, indicating the important role of chitin and chitosan in Cryptococcus virulence [26]. Whole-genome transcriptome analyses of C. gattii and C. neoformans have shown notable differences in expression levels of genes involved in chitin and chitosan biosynthesis, suggesting the transcriptional regulation divergence of chitin and chitosan biosynthesis between the two species [27,28]. Notably, C. gattii R265 cells have been found to produce a substantially larger amount of chitosan in their cell wall during host infection compared to C. neoformans KN99 [28]. Intriguingly, despite no discernible impact on melanin production, a chitin deacetylase mutant cda3Δ exhibits a notable virulence defect in the murine intranasal infection model [28], further emphasizing the crucial role of cell wall chitin and chitosan in facilitating C. gattii infection. Biofilms Biofilms, highly structured microbial communities enclosed in an extracellular polymeric matrix, are ubiquitous among microorganisms in nature and provide various adaptive attributes for microorganisms, including increasing the concentration of nutrients, enhancing cell-to-cell interactions, and stress resistance [29]. When biofilms form on implanted biomedical devices, they pose significant challenges, leading to device dysfunction, antimicrobial resistance, and host defense. Some strains of C. gattii have a great ability to form highly organized and complex biofilm on abiotic surfaces like polyvinyl chloride and silicone catheter [29]. Yeast cells adhere to the substratum in a monolayer in the early stage of biofilm formation. Then, C. gattii cells produce extracellular fibrils to connect yeast cells and the abiotic surface until the mature biofilm formation [29]. Transcriptomic profiling of C. gattii biofilm formation on polystyrene surfaces, as compared to free-floating planktonic cells, has unveiled differential gene expression patterns. These changes encompass metabolic pathways, information processing mechanisms, stress response systems, and cell-to-cell adhesion factors [29]. The comparative proteome of C. gattii grown under planktonic and biofilm conditions found that up-regulated proteins are related to oxidative stress, mitochondrial electron transport, metabolic process-related proteins, and transcription, aligning with prior transcriptomic findings [30]. The biofilm’s adaptation to utilize alternative carbon sources enhances fungus survival fitness during infection. Additionally, the biological processes enriched in the biofilm proteome exhibit similarities to those observed in the transcriptome of C. gattii R265 recovered from bronchoalveolar lavage of infected mice [31], suggesting shared pathways underpinning both biofilm development and lung infection by C. gattii. The ability of forming biofilms has been associated with fungal virulence, as evidenced by the heightened virulence displayed by the biofilm-forming C. gattii cells compared to their planktonic counterparts in the G. mellonella infection model. Furthermore, this biofilm formation is correlated with significant histopathological damage to pulmonary tissues observed in animal infection models, underscoring its pivotal role in disease pathogenesis [32,33]. Cell gigantism In addition to capsule structure and size change that occur during lung infections of Cryptococcus species, cellular size heterogeneity has been recognized as an important virulence factor during infection [34–36]. Enlarged cryptococcal cells, also known as giant or titan cells, were observed in clinical specimens and have been largely studied in Cryptococcus [37]. The defining attributes and regulatory mechanisms of titan cells have been predominantly elucidated in C. neoformans [37]. The important phenotypical feature of titan cells is their huge size, far exceeding the size of typical cells, which are 5–8 µm in diameter. Typical cells are 5–8 µm in diameter, and the cell body sizes of titan cells range from 25 to 30 µm in diameter, with some reaching extraordinary dimensions of up to 100 µm in diameter in the lungs of infected mice [37]. Titan cells isolated from infected lungs exhibit more phenotypical characteristics, including a single nucleus with an augmented genome copy number, a thicker cell wall, and a large vacuole. The enlarged titan cells confer protection of the entire population of cryptococcal cells from phagocytosis and play a critical role in establishing pulmonary infection [37]. Examination of phenotypic variation of cryptococcal isolates from HIV/AIDS patients in Botswana indicated that C. gattii seems to have a greater propensity to form titan cells than C. neoformans in response to a host-relevant environment [38]. During G. mellonella infection, the cell size of all C. gattii strains studied underwent significant enlargement, while cells grown under nutrient conditions exhibited normal cell body size, indicating the involvement of cell size enlargement during infection [14]. To understand the biology and mechanism underlying titanization, in vitro titan induction systems have been performed in C. gattii [39,40]. The capacity to form titan cells in C. gattii strains was significantly higher than in C. neoformans and all C. gattii isolates (VGI–VGIV) tested were able to form titan cells in the in vitro conditions. Cell enlargement is asynchronous with DNA replication. The polyploid titan cells are unbudded in C. gattii R265, which differs from C. neoformans in that titan cells undergo cell division to produce normal-sized daughter cells [40]. This difference may explain the lower ability of cell dissemination outside the lungs in C. gattii compared to C. neoformans. The ability to grow at physiological temperature and CO 2 level Upon host infection, Cryptococcus species must adapt to various host-specific stress conditions, such as higher physiological temperature, higher carbon dioxide (CO 2 ) concentration, and nutrient limitation. High-temperature growth is one of the canonical virulence factors of C. gattii and C. neoformans. Although the ability to grow at a physiological temperature or higher alone is insufficient to cause disease in mammals, it is essential for invasive infection [41]. Gene deletions with growth defects under high temperatures also exhibit virulence attenuation [42]. The calcineurin pathway is critical for cryptococcal growth at 37 °C, and mutant strains disrupting the calcineurin genes are avirulent in C. neoformans and C. gattii [43,44]. The strain R265 lacking calcineurin function is thermotolerant but shows virulence defect [44], indicating that high-temperature tolerance is not sufficient but necessary for fungal virulence. The CO 2 concentration is very low (approximately 0.04%) in the environment. In contrast, the CO 2 levels in mammalian host tissues range from 4.5% to 30%. Environmental isolates of C. neoformans have reduced fungal virulence compared to clinical isolates, even though strains have similar ability to form canonical virulence factors (physiological temperature growth, melanization, and capsule production). The ability to grow in higher levels of CO 2 is different between environmental and clinical isolates and can also be used to distinguish fungal virulence during host infection [45]. Additionally, CO 2 tolerance is important for antifungal drug susceptibility and dissemination of C. neoformans from the lung to the brain [46,47]. The ability to grow in the host levels of CO 2 has been recognized as a virulence trait for C. neoformans. High CO 2 levels promote titan cells and polysaccharide capsule formation in C. gattii [39,40], indicating the role of CO 2 tolerance in the virulence of C. gattii, although it has not been directly investigated. Extracellular vesicles Extracellular vesicles (EVs) are bilayered lipid membranous structures that export molecules from cells to the extracellular milieu [48]. Components characterized in cryptococcal EVs contain ribosomal proteins, proteins associated with virulence and stress response, capsular GXM, laccase, urease, and RNAs. Thus, they have been termed as ‘virulence bags’ [49]. As facultative intracellular pathogens, Cryptococcus species utilize EV-boosted phagocytosis to offer survival advantages in the host. C. gattii EVs with significantly increased GXM concentration were detected in the mutant with increased capsule size [50]. Analysis of the small molecule (molecular mass <900 Daltons) composition of EVs obtained from solid cultures of C. gattii revealed lots of previously unknown components. One of the vesicular peptides protects G. mellonella from Cryptococcus infection, indicating the potential of extracellular peptides produced by C. gattii as a fungal vaccine [51]. Although the role of C. neoformans-derived EVs could potentially be applied to C. gattii, distinctive features of EVs, including EV size, protein preference, and function in the two species complexes, have been identified [19,49]. C. gattii EVs isolated from virulent strains accumulate in the phagosomes and trigger intracellular growth of hypovirulent strains [52]. This discovery of C. gattii EV-based pathogen-to-pathogen communication and coordination has not been reported in C. neoformans. C. gattii-specific EV features broaden our knowledge of cryptococcal EVs and indicate the diversity of pathogenic functions.

Signaling pathways involved in fungal virulence of C. gattii The conserved signaling pathways, such as the cyclic AMP (cAMP)/protein kinase A (PKA), high osmolarity glycerol response (HOG), calcineurin, and cell wall integrity pathways, play critical roles in the stress responses and virulence factors production of C. gattii (Fig 2). PPT PowerPoint slide

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TIFF original image Download: Fig 2. Signaling pathways involved in fungal virulence in C. gattii. The conserved signaling pathways, such as the cyclic AMP (cAMP)/protein kinase A (PKA), high osmolarity glycerol response (HOG), MAPK, and calcineurin pathways, play critical roles in the stress responses and virulence factors production of C. gattii. https://doi.org/10.1371/journal.pntd.0013245.g002 cAMP functions as the second messenger-mediated PKA signaling pathway that is critical in the pathogenicity and morphological differentiation of fungal pathogens. The cAMP/PKA pathway responds to a variety of environmental cues, such as higher CO 2 levels, glucose starvation, low nitrogen, and methionine, to regulate melanin and capsule biosynthesis, titan cell production, environmental stress response, and mating process in C. neoformans [37,53,54]. PKA is also involved in the formation of virulence factors in C. gattii. Identification of the role of Pka1 and Pka2 catalytic subunits in melanin and capsule production found that both Pkas share redundant roles in regulating capsule production, and only Pka2 is responsible for melanin formation [55]. The high-osmolarity glycerol mitogen-activated protein kinase (HOG-MAPK) pathway has a notable effect on environmental stress adaptation and is essential for virulence regulation and sexual differentiation of C. neoformans [56,57]. The regulatory mechanism of Hog1, a key component of the HOG1-MAPK pathway, seems differ between C. neoformans and other fungi in the phosphorylation state under stress conditions. Hog1 of most C. neoformans isolates is highly phosphorylated under normal conditions and dephosphorylated in response to various deleterious stimuli such as osmotic stress, oxidative response, high ion concentration, and high temperature [57–59], which in contrast to other fungi (such as Saccharomyces cerevisiae) that Hog1 undergoes phosphorylation under stress conditions [60]. While the phosphorylation state of Hog1 has been studied in C. neoformans, similar investigations in C. gattii remain unclear. It has been characterized that Hog1 is involved in the stress response and fungal virulence in C. gattii [59]. Similar as C. neoformans, Hog 1 positively regulates stresses responses in C. gattii. Deletion of HOG1 decreases capsule production, melanin synthesis, and fungal virulence in C. gattii [59]. However, Hog1-mediated control of capsule and melanin production is distinct in different serotypes of C. neoformans [61]. These observations suggest that HOG1 has developed distinctive virulence regulatory mechanisms in the two Cryptococcus species and provides an understanding of the pathogenesis of C. gattii. The Ca2+/calcineurin pathway responds to environmental signals, including thermotolerance, high CO 2 , and alkaline pH [60,62]. External cues elevate intracellular Ca2+ levels by extracellular Ca2+ transportation and internal pooled Ca2+ release. Increased Ca2+ binds to calmodulin and activates serine/threonine protein phosphatase, calcineurin. The roles of C. gattii calcineurin have been characterized in different molecular types, and studies found that VGIIa isolates were more tolerant to 37 °C in the presence of calcineurin inhibitors compared to isolates of other molecular types. However, the cna1Δ mutant of VGIIa isolate exhibited a clear growth defect at 37 °C, indicating that calcineurin is critical for thermotolerance and fungal virulence of C. gattii [44]. In addition, C. gattii calcineurin positively regulates cell membrane and cell wall integrity, as well as in ER stress responses may also contribute to its pathogenicity. Additional genes linked to the virulence of C. gattii have been identified and reviewed in detail [12,25,63]. Deleting the signal transduction pathway molecule Mpk1 causes defect of melanin production, cell wall integrity, and fungal virulence in C. gattii. Superoxide dismutase Sod1 and Sod2 are required to produce virulence factors and fungal virulence during mouse inhalation or intravenous inoculation. Tps1 and Tps2 are critical for thermotolerance, capsule and melanin production, and pathogenicity in C. gattii. Transcription factor Ste12α regulates melanin, mating, virulence, and ecological fitness of C. gattii. The GATA transcription factor Gat2 plays an important role in the virulence in C. gattii but not in C. neoformans during infection in the mouse intrapharyngeal instillation model. Other genes involved in C. gattii virulence include D-amino acid oxidase genes (DAO2) [64], β-carbonic anhydrase (CAN2) [65], CAP59 and CAP60 [66], zinc finger proteins (ZAP1, ZAP2, and ZIP3) [67–69], Deubiquitinase Ubp5 [42], chitin deacetylases (CDA3) [28], small heat shock protein Hsp12.1, and candidate genes identified by microarray and transcriptional analysis [31,70].

Immunomodulatory attributes of C. gattii The ability of C. gattii to infect apparently immunocompetent individuals suggests a distinct immune response of the host to this fungal pathogen compared to C. neoformans. The innate immune system constitutes the first line of defense against cryptococcal infection, and an adaptive immune response is critical for the control of disease progression. Lung-resident macrophages are the first host immune cells that interact with inhaled fungi [71]. GXM of cryptococcal polysaccharides can activate the toll-like receptor (TLR)-mediated innate immune response [72,73]. GXM samples from C. gattii strains induced nitric oxide (NO) production by RAW264.7 macrophages, while C. neoformans GXM did not [73]. Dectin-3, a member of the C-type lectin receptors (CLRs) family, has been identified as a direct receptor for the capsular GXM from C. gattii serotype B, but not C. gattii serotype C. Comparative studies between bone marrow-derived macrophages (BMDMs) from wild-type (WT, Clec4d+/+) and dectin-3-deficient (Clec−/−) mice found that the nuclear translocation of NF-κB p65 subunit, phosphorylation of IκBα, degradation and phosphorylation of extracellular signal-regulated protein kinase (ERK), and the secretion of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) production were different when exposed to GXM. These observations indicate that GXM of C. gattii serotype B are recognized by dectin-3 and mediated the activation of NF-κB and ERK pathways [74]. Caspase recruitment domain family member 9 (CARD9) is an adaptor protein that functions downstream of CLRs, facilitating the activation of NF-κB and ERK pathways [75]. Dectin-3- or CARD9-deficient mice infected with C. gattii had decreased survival and increased fungal burden in the lung due to impairment of alveolar macrophage activation [74]. Notably, variations in genotype among Cryptococcus strains impact their immunological properties, necessitating further inquiry into whether dectin-3 plays a role in recognizing diverse genotypes of C. gattii. During the activation of adaptive immune responses to pathogens, dendritic cells (DCs) are the most efficient antigen-presenting cells (APCs) that present antigens to T cells. C. gattii is capable of evading host cell-mediated immune defenses initiated by DCs. C. gattii is killed by DCs, but failed to induce DCs maturation, leading to defective T cell responses [76]. The capsule of C. gattii masks an essential ligand that associates and activates DC surface receptor and is involved in the process of DC maturation, which is sufficient to recover T cell responses against C. gattii [77]. Additionally, C. gattii can escape innate immune responses by altering capsule structure since purified GXM from C. gattii produced lower levels of inflammatory cytokines from DCs, and an acapsular cap60Δ mutant was easily phagocytosed and killed by DCs [20,21]. Further study found that sustained filamentous actin (F-actin) formed a cage-like structure to conceal the phagosomes from recognition and block their maturation to phagolysosomes. Phagosomal maturation is essential for intracellular fungal killing by DCs and subsequent antigen processing and presentation [78]. These observations reveal a unique mechanism of DC immune evasion that permits C. gattii infection in immunocompetent individuals. C. gattii causes different cytokine responses in the host when compared to C. neoformans. The cytokine profile of human peripheral blood mononuclear cells (PBMCs) of healthy individuals after being stimulated with heat-killed isolates of two Cryptococcus species was analyzed. Clinical C. gattii isolates induce higher levels of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 and the T-cell cytokines IL-17 and IL-22 than that of C. neoformans [79]. The examination of peripheral blood transcriptional changes in response to infection with C. gattii and C. neoformans in a mouse infection model found that components of the classical complement activation pathway and genes involved in M1 macrophage activation are up-regulated in C. gattii, indicating the alternative Th2-activated M2 macrophage polarization [80,81]. The cytokine types depend on the recognition of microbial components presented in the cells of the innate immune system. TLR4 and TLR9, but not TLR2, are pattern recognition receptors (PRRs) involved in the host’s cytokine response to C. gattii, suggesting the different pathogen-associated molecules on the cell surface of C. gattii and C. neoformans since TLR2 is known to recognize chitin in C. neoformans [82,83]. C. gattii and C. neoformans have different preferences for organ colonization that C. gattii is more likely to cause pulmonary infection, and the central nervous system is the primary target organ of C. neoformans [2]. The C. gattii strains suppress the protective immune responses by inhibiting the migration of neutrophils to the lungs of infected mice compared to C. neoformans infection [84,85]. In addition, the study of C. gattii strains showed lower levels of neutrophil infiltration and reduced cytokine production in the lungs of C. gattii-infected mice compared to those of mice infected with C. neoformans [85]. The study addresses the impact of C. gattii infection on the development of adaptive T helper cell immune response and found that fewer pulmonary Th1 and Th17 cells could be detected in mice infected with C. gattii strains when compared to C. neoformans infection. The expression levels of Th1-attracting chemokines were significantly reduced in the lungs of virulent C. gattii-infected mice. DCs in C. gattii-infected mice failed to induce effective Th1 and Th17 differentiation, suggesting that C. gattii infection diminished the DC-mediated protective Th1/Th17 immune responses [86]. The screening of the plasma samples of “apparently immunocompetent” patients with meningoencephalitis found that granulocyte-macrophage colony-stimulating factor (GM-CSF) neutralizing antibodies may be a risk factor for infection of C. gattii in HIV-negative and apparently healthy individuals. GM-CSF is a family of glycoprotein cytokines essential for the differentiation of monocytes to macrophages and modulating the immune response [11]. High titers of neutralizing anti-GM-CSF Abs were identified in 15 patients with cryptococcosis (15/39, 38.5%). Over 90% of GM-CSF autoantibodies-positive cryptococcosis clinical cases had central nervous system infection. The majority of these patients were confirmed to be infected with C. gattii [87]. GM-CSF autoantibodies are more strongly associated with C. gattii infection than with C. neoformans infection, which explains the difference in host immune responses between the two Cryptococcus species.

Antifungal susceptibility A limited number of antifungal drugs are available for the treatment of cryptococcosis. The most important treatment options for cryptococcosis are fluconazole, amphotericin B, and flucytosine [88]. Both C. neoformans and C. gattii are resistant to echinocandin class of drugs [88]. Clinical outcomes of fungal disease are influenced by multiple factors, including host immune status and treatment compliance, fungal virulence, drug characteristics, and antifungal susceptibility [2,6]. The in vitro antifungal susceptibility is related to the outcome of cryptococcal infection, especially in patients failing to respond to antifungal therapy [88]. These susceptibility testing methods provide microbiologic techniques and laboratory standardization to predict clinical outcomes based on in vitro data. Different antifungal susceptibility profiles have been reported among cryptococcal species [89–91]. In general, several studies have reported that C. gattii isolates show comparatively low MICs of standard antifungals compared with those of C. neoformans [25,92]. Azoles are the most commonly used drugs to treat cryptococcal infections. Antifungal susceptibility assays showed that C. gattii isolates have the same MIC value for itraconazole as C. neoformans isolates, but were less susceptible to fluconazole and voriconazole than C. neoformans isolates [92]. An antifungal susceptibility study including both clinical and environmental isolates showed that C. gattii strains were less susceptible than C. neoformans to azoles but not amphotericin B and 5FC [93]. In vitro antifungal susceptibility of Cryptococcus isolates from Brazil showed higher MIC values for fluconazole, voriconazole, amphotericin B, and 5FC to C. gattii than for C. neoformans [94]. The different patterns of antifungal susceptibility within isolates of C. gattii have also been documented [95–98]. C. gattii from Taiwan showed comparable MICs of antifungal agents (itraconazole, fluconazole, voriconazole, posaconazole, flucytosine, and amphotericin B) against isolates from environmental and clinical samples. Significant differences in antifungal susceptibility among C. gattii strains of different genotypes were detected, and strains of VGI were more susceptible to azoles and flucytosine compared with strains of VGII [95]. Antifungal susceptibility of clinical C. gattii isolates from Colombia varies among molecular types VGI, VGII, and VGIII. VGI and VGII were less susceptible to 5FC and azoles, respectively than other molecular types [96]. However, the lack of data on clinical outcomes relative to MICs has prevented the establishment of clinical interpretive breakpoints (CBPs) for Cryptococcus species. Thus, in vitro susceptibility testing based on MICs is not currently recommended for the treatment of cryptococcosis. C. gattii frequently resists limited antifungal drugs, especially azoles, because of long-term azole therapies for fungal disease treatment. Studies have examined the underlying mechanisms of antifungal drug resistance [92,99,100]. Azoles interact with the lanosterol 14-α-demethylase, encoded by the ERG11 gene, resulting in cell membrane integrity defect of fungal pathogens [101]. The role of the ERG11 gene in azole resistance of clinical isolates of C. gattii with higher MICs from the PNW has been investigated, and the study found that neither ERG11 overexpression nor mutations in ERG11 coding sequences contribute to the high azole MICs observed [102]. The antifungal susceptibility profile of Colombian clinical isolates of C. neoformans and C. gattii to fluconazole, voriconazole, and itraconazole showed that C. gattii isolates were less susceptible to azoles than C. neoformans isolates, which indicates that differences in the amino acid composition and structure of ERG11 of the two species correlated with the MIC difference. Based on the analysis of the ERG11 gene sequences of clinical isolates of both C. neoformans and C. gattii, a G973T mutation resulting in the substitution R258L was identified in the substrate recognition site of ERG11 in a C. gattii isolate with high MICs for fluconazole and voriconazole [92]. The molecular mechanisms of differences in azole susceptibility in different subtypes of C. gattii have been investigated using clinical C. gattii strains comprising 5 VGI subtypes and 3 VGII subtypes. Similar to previous reports, VGI showed lower MICs compared to VGII [95,97,102]. Furthermore, analysis of transcriptional profiles of VGI and VGII strains found that genes related to transporter activities were differentially expressed, and ABC transporter genes were significantly higher expressed in VGII strains than in VGI strains [100], indicating the involvement of ABC transporter in azole sensitivity in C. gattii. Deletion of FCY2 in C. gattii confers resistance to 5FC [103]. It has been reported that DNA mismatch repair confers an elevated mutation rate also enables rapid resistance to 5FC in C. gattii [104]. Through analysis of whole genomic sequence from 16 independent isolates, mutations associated with 5FC resistance were identified. These mutations were found in the known resistance genes, such as FUR1 and FCY2, and UXS1, gene associated with alterations in capsule production and nucleotide metabolism [104]. Furthermore, multiple 5FC-resistant strains lacked mutations in any genes already known to cause 5FC resistance, indicating unknown mechanisms are responsible for resistance to 5FC in C. gattii.

Conclusions Initially considered as a variety of C. neoformans, C. gattii has since been elevated to a separate species, and has been grouped into several major molecular types based on genetic diversity. Given the close relationship between C. gattii and C. neoformans, the two species exhibit differences in patient predisposition, preferred body sites of infection, and environmental niche. C. gattii is an emerging fungal pathogen that causes lung and CNS infection in individuals both with and without apparently immune defects worldwide. It is valuable to understand the epidemiologic and pathogenic features of C. gattii. In this context, I have compiled a summary of the reported virulence factors in C. gattii. While several virulence factors, including capsule, melanin, and biofilm formation, have been widely studied in C. neoformans, with their regulatory mechanisms well-established to enhance fungal virulence, the precise contribution of these features to C. gattii infections remains enigmatic. Further investigation into how these pathogenic traits interplay in C. gattii infections is crucial for advancing our understanding of this emerging pathogen. The clinical outcome of cryptococcosis caused by C. gattii is associated with early and accuracy of diagnosis, the host’s immune response, and the efficacy of the adopted treatment strategies. However, the prolonged latent period and the absence of specific symptoms associated with C. gattii infection pose challenges to achieving early and accurate diagnoses. Notably, C. gattii has the capability to infect even healthy individuals, suggesting a distinct host immune response pattern compared to C. neoformans infections. One of the host-dependent risk factors implicated in C. gattii infection is the GM-CSF neutralizing antibodies. This underscores the importance of investigating the immunomodulatory properties of C. gattii infections. To gain insights into these aspects, understanding patient status and intensifying research efforts are needed to elucidate the immunological interplay between C. gattii and its hosts. Such endeavors will be instrumental in refining diagnostic approaches, optimizing treatment strategies, and ultimately improving patient outcomes. The development of antifungal drug treatment trials specific to C. gattii infections are important for the clinical management of this pathogen. Given the emergence of C. gattii as a public threat, it is imperative to establish large-scale, international collaborative networks that integrate expertise across various disciplines, including ecology, epidemiology, molecular biology, and clinical practices. These collaborative efforts will facilitate the understanding of biology, transmission dynamics, and host-C. gattii interactions, thereby enabling the design of more precise and effective therapeutic interventions. Epidemiology of C. gattii The majority of C. gattii-infected patients are immunocompetent and apparently healthy individuals. GM-CSF autoantibodies in “apparently immunocompetent” patients significantly contribute to C. gattii infection. Fungal pathogenicity Cryptococcus gattii shares conserved virulence determinants, including capsule, melanin, and thermotolerance, biofilm production, titan cell formation, and CO 2 tolerance, with C. neoformans. However, there are some distinct divergences between the two species. The conserved signaling pathways, such as the cyclic AMP/protein kinase A (cAMP/PKA), high osmolarity glycerol response (HOG), calcineurin, and cell wall integrity (CWI) pathways. Immunomodulatory strategies of C. gattii C. gattii exhibits distinct immunomodulatory strategies compared to C. neoformans. C. gattii preferentially induces pro-inflammatory cytokine responses (IL-1β, TNF-α, IL-6) and Th17/IL-22 polarization while impairing neutrophil recruitment. Additionally, the prevalence of GM-CSF-neutralizing autoantibodies in immunocompetent hosts constitutes a critical susceptibility factor for C. gattii infection. Antifungal resistance pattern in C. gattii The most important treatment options for cryptococcosis are fluconazole, amphotericin B, and flucytosine. In general, C. gattii shows less susceptible to azole drugs, particularly to fluconazole and voriconazole, compared to C. neoformans. Erg11 and ABC transporter contribute to the azole drug resistance observed in C. gattii. Five key papers in the field Stott KE, Loyse A, Jarvis JN, Alufandika M, Harrison TS, Mwandumba HC, et al. Cryptococcal meningoencephalitis: time for action. Lancet Infect Dis. 2021;21(9):e259–e71. Epub 2021/04/20. https://doi.org/10.1016/S1473-3099(20)30771-4. PubMed PMID: 33872594. Galanis E, MacDougall L, Rose C, Chen SC, Oltean HN, Cieslak PR, et al. Predictors of Cryptococcus gattii clinical presentation and outcome: An international study. Clin Infect Dis. 2025. Epub 2025/01/08. https://doi.org/10.1093/cid/ciae640. PubMed PMID: 39774783. Huang Y, Zang X, Yang C, Deng H, Ma X, Xie M, et al. Gene, virulence and related regulatory mechanisms in Cryptococcus gattii. Acta Biochim Biophys Sin (Shanghai). 2022;54(5):593–603. Epub 2022/05/21. https://doi.org/10.3724/abbs.2022029. PubMed PMID: 35593469; PubMed Central PMCID: PMCPMC9828318. Saidykhan L, Onyishi CU, May RC. The Cryptococcus gattii species complex: Unique pathogenic yeasts with understudied virulence mechanisms. PLoS Negl Trop Dis. 2022;16(12):e0010916. Epub 2022/12/16. https://doi.org/10.1371/journal.pntd.0010916. PubMed PMID: 36520688; PubMed Central PMCID: PMCPMC9754292. Chen SC, Meyer W, Sorrell TC. Cryptococcus gattii infections. Clin Microbiol Rev. 2014;27(4):980–1024. Epub 2014/10/04. https://doi.org/10.1128/CMR.00126-13. PubMed PMID: 25278580; PubMed Central PMCID: PMCPMC4187630. Key learning points

Acknowledgments I thank Dr. Chaoyang Xue and Dr. Guanghua Huang for valuable discussions and comments on this review.

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