Turning on virulence: Mechanisms that underpin the morphologic transition and pathogenicity of Blastomyces

ABSTRACT This review article focuses on the mechanisms underlying temperature adaptation and virulence of the etiologic agents of blastomycosis, Blastomyces dermatitidis, Blastomyces gilchristii, and Blastomyces percursus. In response to temperature, Blastomyces undergoes a reversible morphologic switch between hyphae and yeast known as the phase transition. The conversion to yeast for Blastomyces and related thermally dimorphic fungi is essential for virulence. In the yeast phase, Blastomyces upregulates the essential virulence factor, BAD1, which promotes attachment to host cells, impairs activation of immune cells, and blunts cytokine release. Blastomyces yeast also secrete dipeptidyl-peptidase IVA (DPPIVA), a serine protease that blunts the action of cytokines released from host immune cells. In vivo transcriptional profiling of Blastomyces yeast has uncovered genes such as PRA1 and ZRT1 involved in zinc scavenging that contribute to virulence during murine pulmonary infection. The discovery and characterization of genes important for virulence has led to advances at the bedside regarding novel diagnostics, vaccine development, and new targets for drug discovery.


Introduction
Blastomyces dermatitidis, Blastomyces gilchristii, and Blastomyces percursus are the causal agents of blastomycosis [1,2]. Blastomyces species belong to a subset of pathogenic fungi within the Ascomycota phylum that include Histoplasma capsulatum, Coccidioides immitis, Coccidioides posadasii, Paracoccidoides brasiliensis, Paracoccidioides lutzii, Sporothrix schenckii, Talaromyces marneffei (formerly Penicillium marneffei), and Emmonsia species. These fungi are thermally dimorphic, which means they grow as filamentous mold in the environment and budding yeast in human tissue. The ability for the thermally dimorphic fungi to convert between hyphal and yeast forms is a fundamental part of their biology and is essential for pathogenesis. These fungi are capable of infecting humans and other mammals such as dogs and cats [1]. The majority of the dimorphic fungi can infect persons with both normal and impaired immune defenses [1]. This review of blastomycosis will highlight the link between morphologic switch and virulence as well as illustrate the innate and adaptive immune responses during infection.

Mycology
Blastomyces species are haploid ascomycetes that undergo a reversible, temperature-dependent morphologic switch between hyphae at 22-25 C (ambient temperature) and yeast at 37 C (core human body temperature). Blastomyces yeast are multi-nucleate, have a doubly refractile cell wall when visualized by light microscopy, and divide by budding with mother and daughter cells separated by a broad-based bud [3]. The presence of broad-based budding yeast in a specimen (e.g., sputum, tissue) directly obtained from a patient is highly specific for Blastomyces infection. The yeast morphology among the 3 Blastomyces species is similar [2]. The mycelia of B. dermatitidis and B. gilchristii are characterized by septate hyphae with a single conidium attached to a conidiophore, similar to a lollipop [3]. In contrast, B. percursus has more elaborate conidiation structures including primary and secondary conidiophores bearing conidia [2]. In addition to phenotypic differences, the genome size of B. percursus (32.2 Mb) is smaller than B. dermatitidis (66.6 Mb) and B. gilchristii (75.4 Mb) [2]. Thus, the B. percursus genome is similar to other dimorphic fungi including Histoplasma capsulatum, Coccidioides species, and Paracoccidioides species [2]. Despite differences in genome size, the 3 Blastomyces species contain a similar number of genes [2]. The mechanism underlying the differences in genome size and how this may contribute to virulence is unknown.

Geographic distribution and ecology
Blastomycosis is a predominantly North American disease. Blastomyces species are endemic to the Midwest, south-central, and southeast regions of the United States along the Ohio and Mississippi River valleys as well as U.S. states and Canadian provinces bordering the Great Lakes and St. Lawrence river [4]. Outbreaks of Blastomycosis associated with beaver dams and riverways within endemic areas have facilitated understanding of the specific ecological niche of Blastomyces, which is characterized by sandy soils with an acidic pH that are in forested areas with decaying vegetation and nearby fresh water [5,6]. Moreover, there are differences in the geographic distribution of B. dermatitidis and B. gilchristii in the endemic area. B. dermatitidis follows the traditionally accepted geographic range of blastomycosis from Canada to the southeastern United States. B. gilchristii is limited to Ontario, Saskatchewan, Alberta, Minnesota and Wisconsin [7].
Culture-proven cases of blastomycosis have been rarely reported outside of North America with approximately 100 cases from Africa and less than 10 cases from India [4]. Recently multilocus phylogenic analysis of Blastomyces species recovered from clinical specimens of three human cases outside North America (2 South Africa, 1 Israel) identified a new species, B. percursus, which is genetically distinct from B. dermatitidis and B. gilcrhsitii [2].
The majority of blastomycosis cases are sporadic; however, > 17 outbreaks have been reported since 1953 in the United States [4]. Activities that disrupt soil and aerosolize hyphal fragments or conidia can lead to infection. This includes construction of homes or roads, exploration of beaver dams, entering underground forts, forestry work, hunting, and water recreation (e.g., canoeing, tubing along a river) [4][5][6].

The phase transition
The reversible morphologic transition between hyphae and yeast, known as the phase transition, defines the biology of the dimorphic fungi [8]. In the soil (22-25 C), these fungi grow as septate hyphae that produce conidia. Disruption of soil by human activities can aerosolize conidia and hyphal fragments. When inhaled into the lungs of a mammalian host (37 C), these infectious particles undergo a morphologic switch to pathogenic yeast (or spherules for Coccidioides) to cause pneumonia. The yeast phase facilitates evasion of host immune defenses and allows for extrapulmonary dissemination [4].
A shared biology allows for parallels and contrasts to be drawn between Blastomyces and other dimorphic fungi. Although temperature is the predominant stimulus for the phase transition, there are additional influences including carbon dioxide (CO 2 ) tension, exogenous cysteine, and steroid hormones. Elevated CO 2 tension (5% CO 2 ) is required for Coccidioides arthroconidia to germinate into spherules and for optimal growth of H. capsulatum yeast in culture [9,10]. In Blastomyces, elevated CO 2 is dispensable for germination of conidia and growth as yeast in vitro. In contrast, the uptake of exogenous cysteine by Blastomyces as well as by H. capsulatum, and Paracoccidioides is required to restart mitochondrial respiration, which transiently stops following a shift in temperature from 25 o C to 37 o C [11,12]. Without exogenous cysteine, the morphologic switch to yeast cannot be completed [11,12]. For some dimorphic fungi, the sex steroid hormone 17b-estradiol influences morphologic development and immune responses in women. The growth of Coccidioides spherules is accelerated following binding of 17b-estradiol, which may explain why pregnant women have an increased risk of disseminated coccidioidomycosis [13]. For Paracoccidioides, the conversion from conidia or hyphae to pathogenic yeast is blocked following binding of 17b-estradiol. The defect in the morphologic switch to yeast is associated with decreased transcription of genes involved in cell signaling, heat shock proteins, and cell wall remodeling [14][15][16]. The inhibitory effect of estradiol may account for the higher incidence of paracoccidioidomycosis in men compared to women [14]. In contrast, Blastomyces has limited capacity to bind estradiol [17].

Morphologic conversion to the yeast phase promotes evasion of host immune defenses
The conversion to yeast is essential for Blastomyces and other dimorphic fungi to cause infection in mammalian hosts. Once inhaled into mammalian lungs, Blastomyces conidia are ingested by resident pulmonary macrophages and are capable of surviving intracellularly by converting to yeast and replicating [18]. Blastomyces yeast can also replicate extracellularly. This facultative intracellular lifestyle is not unique to Blastomyces and is important for the pathogenesis of H. capsulatum, Coccidioides, Paracoccidioides, T. marneffei, and S. schenckii [19][20][21][22][23]. For H. capsulatum and Cryptococcus neoformans, intracellular survival in macrophages promotes dissemination [24]. Whether Blastomyces uses a similar method for extrapulmonary dissemination is unknown. Approximately 25-40% of patients with blastomycosis have extrapulmonary dissemination, most often to the skin and bone [25].
During the phase transition to yeast, the thermally dimorphic fungi upregulate yeast phase-specific genes to actively subvert host immune defenses. B. dermatitidis and B. gilchristii increase the transcription of BAD1 (Blastomyces adhesin-1; formerly WI-1), which encodes a 120kDA multifunctional, secreted protein that promotes tissue adhesion and immune evasion (Table 1) [26][27][28][29][30][31]. In vivo transcriptional profiling during murine pulmonary infection demonstrated that BAD1 was the most upregulated gene in the trancriptome of B. dermatitidis [32]. Secreted BAD1 can remain soluble in the extracellular milieu or bind back to the yeast cell surface via interactions between the C-terminus of the BAD1 protein and chitin in the cell wall [27,29]. Cell surface-bound BAD1 promotes adhesion of yeast to host cells by binding heparin sulfate and complement receptors (CR3) [27,33]. In addition to facilitating adhesion, cell surface-bound BAD1 inhibits TNF-a production by macrophages and neutrophils in a transforming growth factor-b (TNF-b) dependent manner [30,31,33]. Soluble BAD1 also blocks TNF-a production via a mechanism that is independent of TGF-b [30]. Neutralization of TNF-a in mice results in more severe infection [31]. The Food and Drug Administration (FDA) published a warning that persons receiving TNF-a inhibitors are at increased risk for blastomycosis, histoplasmosis, and coccidioidomycosis [34]. BAD1 also impairs the activation of CD4 + T lymphocytes, and in turn, decreases their production of IL-17 and INF-g [27]. BAD1 is essential for disease pathogenesis and deletion of the gene severely attenuates Blastomyces yeast during murine infection [26].
In addition to BAD1, the secretion of a serine protease known as dipeptidyl-peptidase IVA (DPPIVA) contributes to virulence in vivo (Table 1) [35]. B. dermatitidis DPPIVA silenced strains have reduced survival in the presence of activated innate immune cells and attenuated virulence in a murine model of pulmonary infection [35]. DPPIVA degrades granulocyte-macrophage colony stimulating factor (GM-CSF), a cytokine that activates neutrophils and macrophages to control fungal infection in vivo [35]. DPPIVA also impedes the recruitment of innate immune cells [35].  Involved in the biosynthesis of L-cysteine. Important for transition to yeast and maintenance of the yeast-phase growth. CDG (cysteine dioxygenase) Catabolic enzyme that catabolizes L-cysteine to L-cysteine sulfonic acid, which can further be broken down to sulfite.
In C. albicans deletion of CDG1 attenuates virulence during murine infection. Arthroderma benhamiae cdgΔ mutants have reduced growth on hair and nails. Potential to contribute to growth of Blastomyces on keratinized structures.

SSU1
Transmembrane sulfite efflux pump. Sulfite is toxic to cells and is secreted via the SSU1 efflux pump.
Arthroderma benhamiae ssu1Δ mutants have impaired growth on hair and nails. Potential to contribute to growth of Blastomyces on keratinized structures. PRA1, ZRT1, ZRT2 PRA1 encodes a zincophore. ZRT1 and ZRT2 encode high and low affinity zinc transporters, respectively. These genes are upregulated in yeast during pulmonary infection.
C. albicans pra1Δ mutants have decreased ability to lyse endothelial cells under zinc-poor conditions. Blastomyces pra1Δ and zrt1Δ mutants are impaired in establishing infection in mice.

NIC1
Transports nickel which is used as a cofactor for urease (Urea ! Ammonia + CO 2 ).
In C. neoformans, deletion of NIC1 results in decreased ability of yeast cells to enter CNS. Potential to contribute to Blastomyces virulence or dissemination.

DRK1
Essential for the temperature-dependent conversion to yeast at 37 o C. During phase transition, changes in cell wall carbohydrate composition may also contribute to virulence and immune evasion. During the conversion from mold to yeast, the content of cell wall a-(1,3)-glucan increases, whereas b-(1,3)-glucan decreases from 40% to less than 5% [36]. The reduction of b-(1,3)-glucan in the yeast cell wall may limit dectin-1-mediated recognition by host immune cells and mannose-binding lectins [37].
Although Blastomyces uses multiple strategies to subvert immune defenses to establish infection in healthy persons, these mechanisms are not infallible. The host can mount an immune response to halt the progression of infection. On the basis of outbreak investigations, an estimated 50% of persons exposed to Blastomyces develop symptomatic infection, most commonly pneumonia [5,6]. The remaining 50% of exposed persons will experience asymptomatic or subclinical infection [5,6].

Identification of genes via in vivo transcriptional profiling that have the potential to influence virulence in Blastomyces
Traditionally, novel virulence factors have been discovered using forward and reverse genetic approaches in vitro. The development of high-throughput RNA sequencing along with the ability to quickly isolate Blastomyces yeast from lung tissue has led to the ability to perform in vivo transcriptional profiling of yeast during murine pulmonary infection [32,38]. To uncover genes potentially important for pathogenesis and virulence, RNA seq analysis was used to compare B. dermatitidis yeast harvested from murine lung tissue versus yeast grown in vitro at 37 o C (with and without macrophages) and hyphae at 22 C [32]. K-means cluster analysis identified 72 genes that were upregulated >2-fold during pulmonary infection and independent of temperature, cocultivation with macrophages, and media conditions [32]. Upregulated genes of interest include those involved in amino acid metabolism and metal uptake ( Table 1). In this gene set, the two most highly upregulated genes were involved in cysteine metabolism (cysteine synthase) and exogenous zinc uptake (PRA1) [32]. Recent molecular developments exploiting CRISPR/Cas9 for high frequency gene targeting and editing in Blastomyces have revealed that the secreted zincophore PRA1 and its transporter ZRT1 are required for disease pathogenesis in a murine model of infection (Table 1) [39].
Cysteine synthase is involved in biosynthesis of L-cysteine from acetyl-L-serine. As previously mentioned, cysteine is important for the phase transition and maintenance of the yeast phase. In addition to cysteine synthase, other genes involving cysteine metabolism were upregulated including cysteine dioxygenase (CDG) and a transmembrane sulfite efflux pump (SSU1) [32]. CDG encodes a catabolic enzyme that converts L-cysteine to L-cysteine sulfonic acid, which can be further broken down to pyruvate and sulfite. Sulfite accumulation is toxic to cells and is secreted into the extracellular milieu via the Ssu1 efflux pump. In C. albicans, deletion of CDG1 null mutants (cdg1D) attenuates virulence during murine infection [40]. In Arthoderma benhamiae, a dermatophyte, the catabolism of cysteine to sulfite and its efflux promotes fungal growth on keratinized structures [41]. Sulfite breaks down keratin by cleaving disulfide bonds [41]. Cysteine dioxygenase and SSU1 null mutants exhibit reduced growth on hair and nails [41]. There is potential that cysteine biosynthesis and catabolism followed by sulfite secretion could promote the growth of Blastomyces yeast in skin, the most common site for extrapulmonary dissemination.
During in vivo infection, B. dermatitidis yeast upregulate genes important for exogenous zinc uptake including a zincophore encoded by PRA1, a high-affinity zinc transporter ZRT1, and a low affinity zinc transporter ZRT2 [32]. In C. albicans, the PRA1 zincophore is secreted in the extracellular environment, binds zinc, and brings it back to it to the fungal cell via its interaction with ZRT1 at the plasma membrane [42]. In addition to zinc uptake, B. dermatitidis increases the transcription of a nickel transporter, NIC1 [32]. Nickel serves as a cofactor for urease, which catalyzes the conversion of urea to ammonia and CO 2 . In mammalian tissues, catabolism of purine nucleotides results in the production of urea. In C. posadasii, deletion of the urease gene attenuates virulence of the ureD mutant in murine model of pulmonary infection [43]. Similarly, deletion of NIC1 and URE1 in C. neoformans results in decreased ability of yeast cells to enter the central nervous system [44].

Molecular regulation of the phase transition
The development of molecular tools to dissect the pathogenesis of the thermally dimorphic fungi has led to the discovery of genes and gene networks that regulate the phase transition. The discovery of DRK1, which encodes an intracellular group III hybrid histidine kinase, demonstrated that the morphologic switch to yeast at 37 o C is required for virulence of Blastomyces and Histoplasma (Table 1) [45,46]. DRK1 insertional mutants, null mutants (drk1Δ), and RNA interference (RNAi) strains fail to convert to yeast, remain constitutively hyphal at 37 o C, do not upregulate important yeast-phase specific virulence genes including Blastomyces BAD1 and Histoplasma CBP1 (calcium-binding protein-1), and display defects in cell wall integrity [45]. DRK1-silenced strains of Blastomyces and Histoplasma are avirulent in a murine model pulmonary infection following intratracheal instillation of conidia [45]. The deletion mutants were unable to be tested in vivo because drk1Δ strains produce few conidia [45]. The function of DRK1 is conserved in other thermally dimorphic fungi. Deletion of drkA, a homolog of DRK1 in T. marneffei, impairs the germination of conidia to yeast during co-cultivation with macrophages [47]. Transcriptional analysis has demonstrated upregulation of drkA in T. marneffei yeast at 37 o C. Similarly, DRK1 transcript is upregulated in yeast (versus hyphae) in Paracoccidioides and Sporothrix schenckii [48,49]. DRK1 is postulated to participate in the high-osmolarity glycerol (HOG1) signaling pathway. The HOG1 cascade facilitates adaptation to a variety of exogenous stimuli including temperature, oxidative stress, and osmotic stress. Under osmotic stress, DRK1 and drkA transcription is upregulated in Paracoccidioides and T. marneffei, respectively [47,49].
The temperature-dependent transition in the opposite direction, yeast to hyphae, is governed by a GATA transcription factored encoded by SREB in B. dermatitidis, and its homolog, SRE1, in H. capsulatum [58][59][60]. SREB deletion mutants (srebD) and SRE1-knockdown strains are unable to complete the conversion from yeast to hyphae following a drop-in temperature from 37 o C to 22-25 o C [58][59][60]. In B. dermatitidis srebΔ strains, the morphologic defect at 22 o C is temporally associated with the reduction in neutral lipid (ergosterol, triacylglycerol) biosynthesis and loss of lipid droplets [59]. The defects in phase transition and lipid droplet formation are partially restored by supplementing the media with exogenous saturated fatty acids [59]. In contrast, unsaturated fatty acids do not correct the defects in filamentous development and lipid droplets [59]. Collectively, these data suggest that neutral lipid metabolism contributes to filamentous development at ambient temperature in Blastomyces [59]. In contrast, the role of SREB (and SRE1) as a negative regulator of genes involved in iron assimilation appears to be independent of phase transition [59,60]. Nevertheless, iron has the potential to influence morphogenesis. Deletion of VMA1, which encodes a vacuolar ATPase important for iron homeostasis, renders H. capsulatum yeast incapable of converting to hyphae at room temperature [61]. However, the mechanism underlying the defect in the phase transition due to loss of VMA1 has yet to be characterized. In T. marneffei, hyphal development and maintenance of filamentous morphology is regulated by transcription factors encoded by hygrA and tupA, respectively [62,63].
Similar to C. albicans, exposure of Blastomyces and Histoplasma to N-acetylglucosamine (GlcNAc) accelerates the conversion from yeast to hyphae at ambient temperature and promotes hyphal growth [64]. This event is mediated by NGT1 and NGT2, which encode Nacetylglucosamine transporters 1 and 2, respectively [64]. Both of these transporters are functional and RNAi interference of NGT1 or NGT2 in H. capsulatum impairs the conversion to mycelia at room temperature [64]. The effect of GlcNAc on the phase transition is specific and does not occur with other carbohydrates such as fructose or glucosamine [64]. Moreover, GlcNAc does not alter the morphology or growth rate of yeast [64]. How GlcNAc induces temperature-mediated hyphal development is unknown; however, it is likely independent of GlcNAc metabolism because RNA silencing of HXK1, which encodes a kinase important for GlcNAc catabolism and glycan biosynthesis, does not alter the morphologic switch [64]. Whether NGT1 and NGT2 directly sense GlcNAc or import it for intracellular sensing remains to be determined [64].
The phase transition from Blastomyces yeast to hyphae following a drop-in temperature to 22-25 o C is important and contributes to pathogenesis. The hyphal form is critical for the production of conidia, which allows for thermally dimorphic fungi in soil to be transmitted to mammalian hosts. Yeast (and spherules) are not transmissible via the airborne or respiratory routes. Similarly, hyphal growth is essential for mating, which promotes genetic diversity in Blastomyces as well as other fungi [65,66]. Blastomyces mating occurs exclusively in the mycelial form when hyphal filaments of opposite mating types -MAT1-1 and MAT1-2join together to exchange genetic material [65]. Both mating types are capable of causing infection.

Host response
The host response to Blastomyces infection requires both innate and adaptive immune defenses. Although B. dermatitidis can germinate and replicate intracellularly, alveolar neutrophils and macrophages are capable of killing a large percentage of conidia [67]. Conidia that survive the initial host response can germinate to yeast, which are more challenging to kill. Yeast are relatively resistant to reactive oxygen species, do not induce an intense oxidative response, and are capable of suppressing macrophage nitric oxide production [68]. The adaptive immune response involves T-helper 17 and T-helper 1 cells releasing cytokines such as IL-17, TNF-a, and INF-g to promote macrophage fungicidal activity [69,70]. Following acute blastomycosis, cell-mediated immunity persists for at least 2 years and possibly longer [71]. This finding suggests that after recovery from blastomycosis, there is potential for protective immunity against re-infection; however, clinical studies in humans are lacking with regard to long-term protective immunity after infection.

Bench-to-bedside advances
Research discoveries over the past 2 decades have begun to influence the diagnosis of blastomycosis. Traditional serologic testing by immunodiffusion and complement fixation for the diagnosis of blastomycosis suffers from low sensitivity ( 28%) [72]. A new serologic assay to detect antibodies against BAD1 has enhanced sensitivity (87%) compared to the traditional assays and is highly specific (94-99%) [73]. Because BAD1 is unique to Blastomyces, the BAD1 assay can distinguish between blastomycosis and histoplasmosis [73]. Cross-reaction between existing antibody and antigen tests for Blastomyces and Histoplasma is common. Similarly, a real-time PCR assay that amplifies the BAD1 promoter has been developed. This is a highly specific diagnostic test that allows for rapid identification of B. dermatitidis in culture and in paraffin-embedded tissues [74]. The ability of PCR to identify Blastomyces in paraffin-embedded tissues can facilitate the diagnosis when there is a paucity of yeast in the sample or a lack of characteristic broad-based budding [74].
Although b-(1,3)-glucan assays and echinocandin antifungal drugs have respectively improved the diagnosis and treatment of many fungal infections, the low level of b-(1,3)-glucan in the Blastomyces yeast cell wall precludes the use of b-(1,3)-glucan assays for accurate diagnosis and renders the echinocandin antifungal drug class ineffective [4,25]. Therefore, effective treatment of blastomycosis is limited to the azole antifungals and polyene amphotericin B formulations [25]. Thus, the development of novel antifungal drugs is important. The group III hybrid histidine kinase is being investigated as a target for antifungal drug discovery [75,76]. Similarly, an attenuated B. dermatitidis yeast strain devoid of BAD1 (bad1Δ) is being used to uncover mechanisms important for fungal vaccine development. Mice vaccinated with bad1Δ yeast develop sterilizing immunity following a lethal experimental challenge with B. dermatitidis [77]. Research on bad1Δ vaccine-induced immunity has led to the discovery that calnexin has a conserved sequence among pathogenic ascomycetes that makes it a potential pan-fungal vaccine target [78], and that Blastomyces endoglucanase-2 (Bl-Eng2) represents a novel ligand for dectin-2 with potent adjuvant properties for the induction of Th17 immune cells [79].

Conclusion
Blastomyces species are ascomycete pathogens that are capable of infecting both immunocompromised and the immunocompetent persons. The phase transition from mycelia-to-yeast is essential for virulence. This morphologic switch results in the upregulation of virulence factors that promote adhesion to host tissues, growth in macrophages, dysregulation of the host cytokine response, and blunts the effectiveness of cell-mediated immune defenses. Modern molecular techniques have provided novel insight into the phylogeny and pathogenesis of Blastomyces. The discovery of virulence factors such as BAD1 and DRK1 have led to new diagnostic tests and formed a foundation for the development of new antifungal drugs and experimental vaccines.