Abstract

The ability of pathogenic fungi to switch between a multicellular hyphal and unicellular yeast growth form is a tightly regulated process known as dimorphic switching. Dimorphic switching requires the fungus to sense and respond to the host environment and is essential for pathogenicity. This review will focus on the role of dimorphism in fungi commonly called thermally dimorphic fungi, which switch to a yeast growth form during infection. This group of phylogenetically diverse ascomycetes includes Talaromyces marneffei (recently renamed from Penicillium marneffei), Blastomyces dermatitidis (teleomorph Ajellomyces dermatitidis), Coccidioides species (C. immitis and C. posadasii), Histoplasma capsulatum (teleomorph Ajellomyces capsulatum), Paracoccidioides species (P. brasiliensis and P. lutzii) and Sporothrix schenckii (teleomorph Ophiostoma schenckii). This review will explore both the signalling pathways regulating the morphological transition and the transcriptional responses necessary for intracellular growth. The physiological requirements of yeast cells during infection will also be discussed, highlighting recent advances in the understanding of the role of iron and calcium acquisition during infection.

Graphical Abstract Figure.

Fungi generate a variety of cellular morphologies to facilitate colonization of new environmental niches. Many pathogenic fungi switch from a multi-cellular to a unicellular growth form during infection and this permits adaptation to unique environmental niches within the host.

Graphical Abstract Figure.

Fungi generate a variety of cellular morphologies to facilitate colonization of new environmental niches. Many pathogenic fungi switch from a multi-cellular to a unicellular growth form during infection and this permits adaptation to unique environmental niches within the host.

INTRODUCTION

Fungi generate a variety of cellular morphologies to facilitate adaptation and colonization of new environmental niches. Highly polarized multicellular hyphae or unicellular yeast cells are the most commonly utilized cellular morphologies. However, fungi are also capable of producing specialized cell types with unique cellular morphologies during developmental pathways such as sexual or asexual reproduction.

For fungi that cause disease, infections are usually initiated by the inhalation of dormant spores (generally asexual conidia), produced outside of the host during the differentiation of the hyphal growth form (asexual or sexual development). In the lungs, host innate immune cells such as macrophages and neutrophils recognize these propagules. Specific fungal cell-wall components termed pathogen-associated molecular patterns (PAMPs) are recognized via membrane-associated pattern recognition receptors (PRRs) on macrophage membranes. Once recognized, fungal cells are phagocytosed and the phagocyte PRRs induce a variety of cellular responses (Gantner et al.2003; Bellocchio et al.2004; Roeder et al.2004). The phagocytes deploy mechansims to destroy the fungal cell through the phagolysosomal system by generating damaging reactive oxygen species (ROS), producing various hydrolytic enzymes and restricting nutrients (Fig. 1).

Figure 1.

Phagocytes of the host immune system destroy fungal cells through the phagolysosomal pathway. In the lungs of an immunocompetent host, the fungal infectious propagules (usually asexual spores) are recognized by host innate immune cells such as macrophages and neutrophils. Specific fungal cell-wall components termed PAMPs are recognized via membrane-associated PRRs on macrophage membranes. Once recognized, fungal cells are phagocytosed into an early phagosome. The early phagosome matures to a late phagosome by producing both ROS and RNS to damage the fungal cell from NADPH oxidase and iNOS. Lysosomes fuse with the late phagosome to produce the phagolysosome, a compartment with low pH and which contains hydrolytic enzymes to further damage the fungal cell. The MHC complex displays peptide fragments from the destroyed fungal cell for recognition by T cells. The activated macrophage releases cytokines to stimulate other cells of the immune system. Fungal cells may survive within the macrophage by the neutralization or adaptation to ROS and RNS production, by preventing the release of cytokines and by inducing genes allowing the acquisition of iron and calcium.

Figure 1.

Phagocytes of the host immune system destroy fungal cells through the phagolysosomal pathway. In the lungs of an immunocompetent host, the fungal infectious propagules (usually asexual spores) are recognized by host innate immune cells such as macrophages and neutrophils. Specific fungal cell-wall components termed PAMPs are recognized via membrane-associated PRRs on macrophage membranes. Once recognized, fungal cells are phagocytosed into an early phagosome. The early phagosome matures to a late phagosome by producing both ROS and RNS to damage the fungal cell from NADPH oxidase and iNOS. Lysosomes fuse with the late phagosome to produce the phagolysosome, a compartment with low pH and which contains hydrolytic enzymes to further damage the fungal cell. The MHC complex displays peptide fragments from the destroyed fungal cell for recognition by T cells. The activated macrophage releases cytokines to stimulate other cells of the immune system. Fungal cells may survive within the macrophage by the neutralization or adaptation to ROS and RNS production, by preventing the release of cytokines and by inducing genes allowing the acquisition of iron and calcium.

Some fungi have developed adaptations to circumvent the effectiveness of these host defence responses. A number of these fungi reside within phagocytotic cells of the host where they are shielded from the rest of the immune system. As protracted hyphal growth within phagocytes would lead to cell rupture, thus exposing the fungus to the host immune system, a number of fungi switch from the multicellular hyphal growth form found in the environment to a unicellular yeast growth form in a process known as dimorphic switching (Fig. 2). Other dimorphic fungi use the yeast cell form to avoid phagocytosis and the cytotoxic environment of the phagolysosomal system; instead, they are adapted to tolerating the adaptive immune responses. Thus, dimorphic switching allows for the colonization of unique environmental niches within the host and the failure to switch almost always attenuates pathogenicity in these fungi. In other dimorphic fungi which exist predominately as a yeast vegetative growth form outside the host, such as the plant pathogen Ustilago maydis and the human pathogen Candida albicans, the dimorphic switch from a yeast to a filamentous growth form can facilitate tissue penetration during infection (Liu 2002; Nadal, Garcia-Pedrajas and Gold 2008; Karkowska-Kulet, Rapala-Kozik and Kozik 2009). This review will focus on the role of dimorphism in pathogenic fungi which switch to a yeast growth form during infection. This group of phylogenetically diverse ascomycetes includes Talaromyces marneffei (recently renamed from Penicillium marneffei), Blastomyces dermatitidis (teleomorph Ajellomyces dermatitidis), Coccidioides species (C. immitis and C. posadasii), Histoplasma capsulatum (teleomorph Ajellomyces capsulatum), Paracoccidioides species (P. brasiliensis and P. lutzii) and Sporothrix schenckii (teleomorph Ophiostoma schenckii) (Fig. 2).

Figure 2.

Cellular morphologies of dimorphic human fungal pathogens. The growth morphologies of the dimorphic ascomycetes Histoplasma capsulatum (Ajellomyces capsulatum), Blastomyces dermatitidis (Ajellomyces dermatitidis), Talaromyces marneffei (Penicillium marneffei), Coccidioides immitis, Paracoccidioides brasiliensis and Sporothrix schenckii (Ophiostoma schenckii). In general, these fungi grow in a multicellular hyphal form at 25°C and switch to producing unicellular yeast growth forms at 37°C, with the exception of Coccidioides species that produce spherules. For a number of these fungi, the yeast form serves to accommodate intracellular growth within host phagocytes.

Figure 2.

Cellular morphologies of dimorphic human fungal pathogens. The growth morphologies of the dimorphic ascomycetes Histoplasma capsulatum (Ajellomyces capsulatum), Blastomyces dermatitidis (Ajellomyces dermatitidis), Talaromyces marneffei (Penicillium marneffei), Coccidioides immitis, Paracoccidioides brasiliensis and Sporothrix schenckii (Ophiostoma schenckii). In general, these fungi grow in a multicellular hyphal form at 25°C and switch to producing unicellular yeast growth forms at 37°C, with the exception of Coccidioides species that produce spherules. For a number of these fungi, the yeast form serves to accommodate intracellular growth within host phagocytes.

This group of fungi, with the exception of S. schenckii, show high endemicity and cause substantial morbidity and mortality in their respective regions (for review, see Sil and Andrianopoulos 2015). Despite this, their impact has only recently been appreciated and consequently incidence data lags behind that of other microbes, including fungi such as C. albicans, Cryptococcus neoformans and Aspergillus fumigatus. However, it is clear that together this group of fungal pathogens have a significant impact on human health, singlehandedly causing over a million new infections a year in the USA alone (Knox and Hage 2010; Lopez-Martinez and Mendez-Tovar 2012; Marques 2012; Nguyen et al.2013; Mahajan 2014).

The importance of these pathogens has lead to renewed interest in understanding how the key determinant of pathogenicity, dimorphic switching, is controlled and how these fungi cause disease. Identification of genes required during the dimorphic switching process has occurred predominantly using targeted candidate gene or genome-wide expression approaches. Techniques to genetically manipulate these fungi have been developed, including RNA interference and Agrobacterium-mediated transformation, but site-specific gene deletion and mutation is limited either due to non-integration of introduced DNA or non-homologous DNA integration. The exception is T. marneffei, which is highly amenable to genetic manipulation and for which strains have been generated containing mutations to increase the frequency of homologous integration (Bugeja et al.2012). The emergence of CRISPR may radically change the genetic malleability of these important pathogens.

Despite these limitations, significant progress has been made in understanding the molecular mechanisms controlling dimorphic switching. This review will explore both the signalling pathways regulating the morphological transition to the yeast growth form and the transcriptional and morphological processes required for intracellular growth in this group of fungal pathogens. In particular, recent advances in understanding the role of iron and calcium acquisition during infection will be highlighted. Where appropriate, reference will be made to model fungi where there is a deeper understanding of the molecular mechanisms that impinge on homologous morphogenetic processes and these will be related to what is known in the dimorphic fungi.

Signalling pathways required for dimorphic switching and adaptation to the host environment

A number of signalling pathways have been identified that induce the dimorphic switch (Fig. 3). Since intracellular growth also necessitates survival through the macrophage phagolysosomal system, these signalling pathways also frequently coregulate processes important for adaptation to this environment, such as adaptation to oxidative stress. These pathways include two-component and heterotrimeric G protein signalling systems as well as Ras and cAMP signalling and the downstream mitogen-activated protein kinase (MAPK) signalling cascades (Fig. 3).

Figure 3.

Model of the signalling pathways controlling dimorphic switching and yeast growth at 37°C. The colours indicate characterized roles in dimorphism: green, mutation or RNAi inhibition affects dimorphism; yellow, expression is induced during the dimorphic switch, blue, chemical inhibition or exogenous addition affects the dimorphic switch; and orange, mutation indicates no role in dimorphism. (A) Two-component signalling systems in fungi require four sequential phosphorylation events (H→D→H→D) and comprising one or multiple membrane-associated HHK, an intermediate histidine phosphotransfer protein (HPt) and two RRs. The HK perceives the environmental stimulus and is autophosphorylated at a conserved histidine in the kinase domain. The phosphate group is then transferred to a conserved aspartate in the HHK's RR domain and then to a conserved histidine in the HPt phosphotransfer protein (HPt). The HPt protein transfers the phosphate to an aspartate residue in the RR. The RR can either bind DNA directly or activate MAPK pathways to elucidate a morphological and transcriptional response. (B) Binding of the ligand to the G protein coupled receptor triggers GDP to GTP exchange in the Gα subunit of a heterotrimeric G protein which dissociates from the ß and γ subunits. The Gα subunit activates adenylate cyclase (AC) and subsequent cAMP/protein kinase A (PKA) signalling, in addition to, the Ras GTPase. Ras activates the Rho GTPase Cdc42, which in addition to controlling actin-mediated polarized growth, regulates the p21 activated kinases Ste20 and Cla4 to signal via MAPK pathways and regulate cellular division. (C) Entry of calcium (Ca2+) into the cell or release from internal stores acts as a transient intracellular signal. Binding of Ca2+ to calmodulin (CaM) enables it to activate the calmodulin-dependent kinases (CDKs) and the calcineurin phosphatase (CnaB/CnaA). Ca2+ binding to the regulatory subunit of calcineurin (CnaB) allows activation of the catalytic subunit (CnaA). CnaA dephosphorylates the CrzA transcription factor to allow entry into the nucleus to activate genes controlling processes such as cell-wall synthesis, germination of spores (conidia), ion homeostasis, pH adaptation, polarity and conidiophore development.

Figure 3.

Model of the signalling pathways controlling dimorphic switching and yeast growth at 37°C. The colours indicate characterized roles in dimorphism: green, mutation or RNAi inhibition affects dimorphism; yellow, expression is induced during the dimorphic switch, blue, chemical inhibition or exogenous addition affects the dimorphic switch; and orange, mutation indicates no role in dimorphism. (A) Two-component signalling systems in fungi require four sequential phosphorylation events (H→D→H→D) and comprising one or multiple membrane-associated HHK, an intermediate histidine phosphotransfer protein (HPt) and two RRs. The HK perceives the environmental stimulus and is autophosphorylated at a conserved histidine in the kinase domain. The phosphate group is then transferred to a conserved aspartate in the HHK's RR domain and then to a conserved histidine in the HPt phosphotransfer protein (HPt). The HPt protein transfers the phosphate to an aspartate residue in the RR. The RR can either bind DNA directly or activate MAPK pathways to elucidate a morphological and transcriptional response. (B) Binding of the ligand to the G protein coupled receptor triggers GDP to GTP exchange in the Gα subunit of a heterotrimeric G protein which dissociates from the ß and γ subunits. The Gα subunit activates adenylate cyclase (AC) and subsequent cAMP/protein kinase A (PKA) signalling, in addition to, the Ras GTPase. Ras activates the Rho GTPase Cdc42, which in addition to controlling actin-mediated polarized growth, regulates the p21 activated kinases Ste20 and Cla4 to signal via MAPK pathways and regulate cellular division. (C) Entry of calcium (Ca2+) into the cell or release from internal stores acts as a transient intracellular signal. Binding of Ca2+ to calmodulin (CaM) enables it to activate the calmodulin-dependent kinases (CDKs) and the calcineurin phosphatase (CnaB/CnaA). Ca2+ binding to the regulatory subunit of calcineurin (CnaB) allows activation of the catalytic subunit (CnaA). CnaA dephosphorylates the CrzA transcription factor to allow entry into the nucleus to activate genes controlling processes such as cell-wall synthesis, germination of spores (conidia), ion homeostasis, pH adaptation, polarity and conidiophore development.

In bacteria, two-component systems comprise a membrane-associated histidine kinase (HK) and a cytoplasmic response regulator (RR). The HK perceives the environmental stimulus and is autophosphorylated at a conserved histidine in the kinase domain. The phosphate group is then transferred to a conserved aspartate in the receiver domain of the RR, which mediates changes in gene expression (Histidine (H)→ Aspartate (D)). In fungi, the HK is fused to the RR (hybrid HK, HHK) and further phosphorelay occurs via an additional phosphotransfer protein (HPt) and second RR (H→D→H→D) (Fig. 3A). The RR either directly regulates gene expression or activates a MAPK pathway that in turn regulates gene expression (Fig. 3A). The model yeast Saccharomyces cerevisiae, which has a relatively simple life cycle but displays a dimorphic switch known as pseudohyphal development, has a single HHK (Sln1) and HPt (Ypd1) and two RRs (Ssk1 and Skn7). Ssk1 negatively regulates the high-osmolarity glycerol (HOG) MAPK signalling pathway in the absence of stress, whereas Skn7 binds directly to DNA to regulate changes in gene expression in response to oxidative stress (Posas et al.1996; Raitt et al.2000). Fungi with more complex life cycles have a greatly expanded set of HHKs that fall into 11 classes (I–XI) (Catlett, Yoder and Turgeon 2003). However, despite this expansion, the phosphorylation signal is still transmitted to only two RRs, orthologous to Ssk1 and Skn7, via a single HPt, orthologous to Ypd1. The roles of two-component signalling systems have been investigated extensively in the non-dimorphic Neurospora crassa and in fungal pathogens of plants (Ochiai et al.2001; Yoshimi, Tsuda and Tanaka 2004; Motoyama et al.2005; Viaud et al.2006; Rispail and Di Pietro 2010; Zhang et al.2010). In N. crassa, the class III HHK Os-1 activates the HOG MAPK signalling pathway and mutations result in increased resistance to dicarboximide and phenylpyrrole antifungal agents (Ochiai et al.2001; Yoshimi et al.2005). The fungicidal activity of these agents is achieved by Hog1-induced glycerol accumulation in the absence of high external osmolarity (Vetcher et al.2007). Mutations in Os-1 orthologues in plant pathogens also result in sensitivity to osmotic and oxidative stress and reduce pathogenicity (Rispail and Di Pietro 2010; Zhang et al.2010).

Recent characterization of the class III HHK (Os-1 orthologue) in human dimorphic pathogens has revealed conserved roles in fungicide resistance, osmotic stress resistance and pathogenicity, in addition to previously unidentified roles in cell-wall integrity, asexual development and dimorphism (Nemecek, Wuthrich and Klein 2006; Boyce et al.2011) (Table 1). The B. dermatitidis class III HHK encoded by DRK1 was identified in an insertional mutagenesis screen for regulators of the yeast phase-specific gene BAD1 (Nemecek, Wuthrich and Klein 2006). DRK1 mutants of B. dermatitidis, and the phylogenetically related H. capsulatum, fail to undergo the dimorphic switch to the yeast growth form at 37°C, consequently showing reduced pathogenicity (Nemecek, Wuthrich and Klein 2006). Deletion of the T. marneffei orthologue drkA also results in a failure to switch to the yeast growth form (Boyce et al.2011). Unlike B. dermatitidis and H. capsulatum, which lack a class VI HHK (Sln1 orthologue), T. marneffei also has a class VI (slnA) HHK (Boyce et al.2011). Deletion of either slnA or drkA in T. marneffei results in increased sensitivity to osmotic stress and both proteins regulate the HOG MAPK pathway by increasing or decreasing, respectively, phosphorylated SakA (MAPK orthologous to S. cerevisiae Hog1) (Boyce et al.2011). In addition, the ΔdrkA mutant, but not the ΔslnA mutant, is resistant to the dicarboximide and phenylpyrrole classes of fungicides, which perturb osmoregulation, suggesting that in this mutant the HOG pathway cannot be induced. Thus, these class III and VI HHKs are essential for morphogenesis and adaptation to high osmotic and oxidative stress environments, such as those encountered in the intracellular environment of the host macrophage.

Table 1.

Genes shown by mutagenesis or RNAi knockdown to have a role in pathogenesis, dimorphic switching and/or yeast growth in dimorphic fungal pathogens.

Species Gene Encodes Role Reference 
Histoplasma capsulatum DRK1 Class III Hybrid histidine kinase (HHK) Dimorphic switching, osmotic stress adaptation, cell wall integrity, asexual development, pathogenicity. Nemecek, Wuthrich and Klein (2006
 RYP1 Transcriptional regulator Dimorphic switching to yeast growth at 37°C. Repression of vegetative spore production in liquid (conidia). Regulates the expression of phase specific genes. Nguyen and Sil (2008
 RYP2 Developmental regulator (velvet family) Dimorphic switching to yeast growth at 37°C, appropriate sporulation and spore viability. Nguyen and Sil (2008); Webster and Sil (2008
 RYP3 Developmental regulator (velvet family) Dimorphic switching to yeast growth at 37°C, appropriate sporulation and spore viability. Nguyen and Sil (2008); Webster and Sil (2008
 RYP4 Zn(II)2Cys6 binuclear cluster transcription factor Dimorphic switching to yeast growth at 37°C and regulates the expression of phase specific genes. Beyhan et al. (2013
 AGS1 ∝(1,3)-glucan synthase Biosynthesis of a(1,3)-glucan in the cell wall. Pathogenesis. Rappleye, Engle and Goldman (2004
 AMY1 Glycosyl hydrolase Biosynthesis of a(1,3)-glucan in the cell wall. Pathogenesis. Marion et al. (2006
 HCL1 HMG CoA lyase Preventing phagosome acidification during growth in macrophages. Isaac et al. (2013
 SRE1 GATA transcription factor Regulates the expression of the genes required for siderophore biosynthesis, iron transport and utilization. Hwang et al. (2008
 SID1 L-ornithine monoxygenase Siderophore production, growth in limiting iron and in macrophages and pathogenesis. Hilty et al. (2011
 VMA1 Vacuolar ATPase Growth on iron limiting media, hyphal growth at 28°C and pathogenicity. Hilty, Smulian and Newman (2008
 CBP Calcium binding protein Growth in calcium limiting conditions and in macrophages and pathogenicity. Batanghari et al. (1998); Sebghati et al. (2000
Talaromyces marneffei drkA Class III HHK Dimorphic switching, osmotic stress adaptation (phosphorylation of the MAPK SakA), fungicide resistance, cell wall integrity (phosphorylation of the MAPK MpkA), asexual development, pathogenicity. Boyce et al. (2011
 slnA Class IV HHK Osmotic stress adaptation, germination of asexual spores (conidia) during macrophage infection. Boyce et al. (2011
 rasA Ras GTPase Germination of conidia, the onset of asexual development and morphogenesis of both hyphal and yeast cells. Boyce, Hynes and Andrianopoulos (2005
 abaA ATTS transcription factor Asexual development and yeast growth. Borneman, Hynes and Andrianopoulos (2000
 hgrA C2H2 transcription factor Yeast-to-hyphal dimorphic switch and cell wall integrity. Bugeja, Hynes and Andrianopoulos (2013
 cflA Rho GTPase Regulate conidial germination and the actin-mediated polarized growth and morphology of hyphae at 25°C and yeast cells at 37°C. Boyce, Hynes and Andrianopoulos (2001); Boyce, Hynes and Andrianopoulos (2005
 cflB Rho GTPase Actin-mediated polarized growth of hyphae and asexual development structures at 25°C. Germination of conidia in macrophages. Boyce, Hynes and Andrianopoulos (2003
 pakA p21 activated kinase (PAK) Conidial germination and polarised growth of yeast cells at 37°C. Boyce and Andrianopoulos (2007
 pakB p21 activated kinase (PAK) Regulates the expression of phase specific genes. Required to prevent inappropriate yeast-like cell production at 25°C. Cellular division of conidiophores and macrophage-engulfed yeast cells. Boyce, Schreider and Andrianopoulos (2009
 myoB Type II myosin Cellular division. Canovas, Boyce and Andrianopoulos (2011
 rfxA Transcriptional regulator Regulates the expression of cell cycle genes. Bugeja, Hynes and Andrianopoulos (2010
Blastomyces dermatitidis DRK1 Class III Hybrid histidine kinase (HHK) Dimorphic switching, osmotic stress adaptation, cell wall integrity, asexual development, pathogenicity. Nemecek, Wuthrich and Klein (2006
 CDC11 septin Budding of yeast cells. Krajaejun et al. (2007
 CDC3 septin Budding of yeast cells. Marty and Gauthier (2013
 CDC10 septin Budding of yeast cells. Marty and Gauthier (2013
 CDC12 septin Budding of yeast cells. Marty and Gauthier (2013
 sreB GATA transcription factor Required for the expression of genes required for siderophore biosynthesis and uptake. Yeast-to-hyphal dimorphic switch. Gauthier et al. (2010
 BAD1 Calcium binding protein Pathogenicity. Affects levels of pro-inflammatory cytokines Brandhorst et al. (1999
Paracoccidioides brasiliensis cdc42 Rho GTPase Cell size, budding, variability in cell shape and pathogenesis. Almeida et al. (2009
Species Gene Encodes Role Reference 
Histoplasma capsulatum DRK1 Class III Hybrid histidine kinase (HHK) Dimorphic switching, osmotic stress adaptation, cell wall integrity, asexual development, pathogenicity. Nemecek, Wuthrich and Klein (2006
 RYP1 Transcriptional regulator Dimorphic switching to yeast growth at 37°C. Repression of vegetative spore production in liquid (conidia). Regulates the expression of phase specific genes. Nguyen and Sil (2008
 RYP2 Developmental regulator (velvet family) Dimorphic switching to yeast growth at 37°C, appropriate sporulation and spore viability. Nguyen and Sil (2008); Webster and Sil (2008
 RYP3 Developmental regulator (velvet family) Dimorphic switching to yeast growth at 37°C, appropriate sporulation and spore viability. Nguyen and Sil (2008); Webster and Sil (2008
 RYP4 Zn(II)2Cys6 binuclear cluster transcription factor Dimorphic switching to yeast growth at 37°C and regulates the expression of phase specific genes. Beyhan et al. (2013
 AGS1 ∝(1,3)-glucan synthase Biosynthesis of a(1,3)-glucan in the cell wall. Pathogenesis. Rappleye, Engle and Goldman (2004
 AMY1 Glycosyl hydrolase Biosynthesis of a(1,3)-glucan in the cell wall. Pathogenesis. Marion et al. (2006
 HCL1 HMG CoA lyase Preventing phagosome acidification during growth in macrophages. Isaac et al. (2013
 SRE1 GATA transcription factor Regulates the expression of the genes required for siderophore biosynthesis, iron transport and utilization. Hwang et al. (2008
 SID1 L-ornithine monoxygenase Siderophore production, growth in limiting iron and in macrophages and pathogenesis. Hilty et al. (2011
 VMA1 Vacuolar ATPase Growth on iron limiting media, hyphal growth at 28°C and pathogenicity. Hilty, Smulian and Newman (2008
 CBP Calcium binding protein Growth in calcium limiting conditions and in macrophages and pathogenicity. Batanghari et al. (1998); Sebghati et al. (2000
Talaromyces marneffei drkA Class III HHK Dimorphic switching, osmotic stress adaptation (phosphorylation of the MAPK SakA), fungicide resistance, cell wall integrity (phosphorylation of the MAPK MpkA), asexual development, pathogenicity. Boyce et al. (2011
 slnA Class IV HHK Osmotic stress adaptation, germination of asexual spores (conidia) during macrophage infection. Boyce et al. (2011
 rasA Ras GTPase Germination of conidia, the onset of asexual development and morphogenesis of both hyphal and yeast cells. Boyce, Hynes and Andrianopoulos (2005
 abaA ATTS transcription factor Asexual development and yeast growth. Borneman, Hynes and Andrianopoulos (2000
 hgrA C2H2 transcription factor Yeast-to-hyphal dimorphic switch and cell wall integrity. Bugeja, Hynes and Andrianopoulos (2013
 cflA Rho GTPase Regulate conidial germination and the actin-mediated polarized growth and morphology of hyphae at 25°C and yeast cells at 37°C. Boyce, Hynes and Andrianopoulos (2001); Boyce, Hynes and Andrianopoulos (2005
 cflB Rho GTPase Actin-mediated polarized growth of hyphae and asexual development structures at 25°C. Germination of conidia in macrophages. Boyce, Hynes and Andrianopoulos (2003
 pakA p21 activated kinase (PAK) Conidial germination and polarised growth of yeast cells at 37°C. Boyce and Andrianopoulos (2007
 pakB p21 activated kinase (PAK) Regulates the expression of phase specific genes. Required to prevent inappropriate yeast-like cell production at 25°C. Cellular division of conidiophores and macrophage-engulfed yeast cells. Boyce, Schreider and Andrianopoulos (2009
 myoB Type II myosin Cellular division. Canovas, Boyce and Andrianopoulos (2011
 rfxA Transcriptional regulator Regulates the expression of cell cycle genes. Bugeja, Hynes and Andrianopoulos (2010
Blastomyces dermatitidis DRK1 Class III Hybrid histidine kinase (HHK) Dimorphic switching, osmotic stress adaptation, cell wall integrity, asexual development, pathogenicity. Nemecek, Wuthrich and Klein (2006
 CDC11 septin Budding of yeast cells. Krajaejun et al. (2007
 CDC3 septin Budding of yeast cells. Marty and Gauthier (2013
 CDC10 septin Budding of yeast cells. Marty and Gauthier (2013
 CDC12 septin Budding of yeast cells. Marty and Gauthier (2013
 sreB GATA transcription factor Required for the expression of genes required for siderophore biosynthesis and uptake. Yeast-to-hyphal dimorphic switch. Gauthier et al. (2010
 BAD1 Calcium binding protein Pathogenicity. Affects levels of pro-inflammatory cytokines Brandhorst et al. (1999
Paracoccidioides brasiliensis cdc42 Rho GTPase Cell size, budding, variability in cell shape and pathogenesis. Almeida et al. (2009

Mutations in DRK1 in B. dermatitidis and H. capsulatum and drkA in T. marneffei also result in cell-wall defects including abnormal chitin distribution and sensitivity to cell-wall-binding agents, suggesting that they play conserved roles in mediating cell-wall integrity (Nemecek, Wuthrich and Klein 2006; Boyce et al.2011). The integrity of the cell wall is crucial for surviving both ROS and hydrolysis in the host macrophage. In T. marneffei, both SlnA and DrkA regulate the phosphorylation state of MpkA, which is orthologous to the S. cerevisiae Slt2 MAPK that regulates cell-wall construction (Lee et al.1993). DrkA is essential for the increase in MpkA phosphorylation in response to cell-wall stress (Boyce et al.2011). Similar to the non-dimorphic fungi Botrytis cinerea and A. nidulans, mutations in B. dermatitidis DRK1, H. capsulatum DRK1 and T. marneffei drkA also show a reduction in asexual development (sporulation) and consequently the production of the infectious propagules (Viaud et al.2006; Vargas-Perez et al.2007; Nemecek, Wuthrich and Klein 2006; Boyce et al.2011). Importantly, the DRK1 orthologues in B. dermatitidis, H. capsulatum and T. marneffei are essential for the generation of yeast cells in vivo as well as in vitro (Nemecek, Wuthrich and Klein 2006; Boyce et al.2011) and T. marneffei slnA is required for the germination of asexual spores (conidia) in macrophages (Boyce et al.2011). Recent protein profiling has shown that Drk1 is a highly abundant, differentially expressed protein in the yeast growth phase of S. schenckii and expression of the orthologue in P. brasiliensis is upregulated during the hyphal-to-yeast dimorphic switch (Bastos et al.2007; Zhang et al.2011). Therefore, the DrkA-regulated two-component signalling pathway plays a conserved, essential role during the initiation of the dimorphic switch. It is now important to identify the downstream components of these pathways in order to understand the specific roles they play in morphogenesis, osmoregulation and cell-wall integrity.

Heterotrimeric G proteins and the downstream Ras and cAMP signalling pathways have also been shown to influence dimorphic switching and adaptation to oxidative stress in dimorphic fungi, in addition to regulating asexual development and conidial germination (Zuber, Hynes and Andrianopoulos 2002, 2003; Boyce, Hynes and Andrianopoulos 2005; Valentin-Berrios et al.2009; Perez-Sanchez et al.2010). Canonical heterotrimeric G proteins comprise three subunits (α, ß and γ) that transmit signals from cell surface receptors (Fig. 3B). Binding of the ligand to the receptor triggers Gα subunit GDP to GTP exchange, resulting in a conformational shift and dissociation from the ß/γ subunits. The α and ß/γ subunits interact with cytoplasmic effector proteins to elicit responses to the environmental stimulus, although how this is achieved remains unclear (Fig. 3B). Expression of the genes encoding the α and ß subunits in P. brasiliensis is induced during the hyphal-to-yeast dimorphic switch but there is no evidence at this point in time that it is required for switching (Nunes et al.2005). The S. schenckii Ssg-1 Gα subunit interacts with proteins required for survival under oxidative stress and iron acquisition and the Ssg2 Gα subunit interacts with cytosolic phospholipase A2 (Valentin-Berrios et al.2009; Perez-Sanchez et al.2010). Inhibition of cytosolic phospholipase A2 activity stimulates the yeast-to-hyphal dimorphic switch and inhibits re-entry into the yeast cell cycle, suggestive of a more direct role in dimorphic switching (Valentin-Berrios et al.2009). In T. marneffei, mutation of genes encoding Gα subunits, gasA and gasC, results in defects in the onset of asexual development, conidial yield and conidial germination but has no effect on dimorphic switching or maintenance of yeast growth at 37°C (Zuber, Hynes and Andrianopoulos 2002; Zuber, Hynes and Andrianopoulos 2003). Thus, the role of these signalling complexes, and their receptors that may sense the trigger to effect dimorphic switching does not appear to be highly conserved amongst the dimorphic fungi.

In response to stimulation from heterotrimeric G proteins, GTPase-activating proteins convert GTPases of the Ras superfamily from an inactive GDP-bound form to an active GTP-bound form (Fig. 3). Ras GTPases have been shown to control diverse processes including cAMP signalling, morphogenesis, differentiation, cell cycle progression and the expression of pathogenicity genes in a variety of fungi (Alspaugh et al.2000; Leberer et al.2001; Fortwendel et al.2005). In S. cerevisiae, there are two RAS genes, of which the Ras2 protein controls cAMP signalling through direct binding to adenylate cyclase (Colombo et al.1998). The Ras/cAMP link is conserved in C. albicans, where the Ras/cAMP/PKA pathway is essential for pathogenicity by promoting hyphal growth and for mediating changes in gene expression (reviewed in Hogan and Sundstrom 2009). Adenylate cyclase can bind directly to one of the three Gα subunits (Gpa1) and the Gß subunit (Gpb1) in P. brasiliensis and the addition of cAMP prevents the hyphal-to-yeast dimorphic switch (Chen et al.2007). Ras protein function requires farnesylation for correct membrane association, and the addition of farnesyltransferase inhibitors to P. brasiliensis promotes the yeast-to-hyphal dimorphic switch (Fernandes et al.2008). Therefore, a heterotrimeric G protein and Ras signalling pathway clearly influence dimorphic switching in P. brasiliensis. In T. marneffei, RasA is required for the germination of conidia, the onset of asexual development and morphogenesis of both hyphal and yeast cells but the evidence for the involvement of cAMP in dimorphic switching is not strong (Zuber, Hynes, Andrianopoulos 2003; Boyce, Hynes and Andrianopoulos 2005).

The transcriptional regulation of dimorphic switching

Host or temperature-derived signals trigger dimorphic switching and are likely to be transmitted via signalling pathways, ultimately culminating in changes in gene expression. Many transcriptome-based approaches have identified phase (yeast or hyphal) specific genes but little is known to date about how the expression of these genes is controlled.

The RYP1–4 transcription factors from H. capsulatum have recently been shown to regulate dimorphic switching. RYP1–3 were identified in an Agrobacterium-mediated insertional mutagenesis screen to isolate genes required for yeast growth at 37°C (Nguyen and Sil 2008; Webster and Sil 2008). In contrast to wild type, the original RYP1 insertional mutants and a knockdown mutant generated using RNAi grow as hyphae at 37°C. RYP1 mutants also inappropriately produce vegetative spores (conidia) in liquid cultures suggesting that Ryp1 regulates multiple developmental processes (Nguyen and Sil 2008). Comparison of the transcriptional profile of wild-type H. capsulatum versus the RYP1 mutant showed that the expression of most 37°C phase-specific genes, including the known pathogenicity factors CBP1 and YPS3, was no longer induced at 37°C (Keath and Abidi 1994; Batanghari et al.1998; Sebghati, Engle and Goldman 2000; Bohse and Woods 2007). In addition, genes usually downregulated at 37°C were inappropriately expressed in the mutant (Nguyen and Sil 2008). Ryp1 shows homology to a family of transcriptional regulators that function as master regulators of fungal morphogenesis. The S. cerevisiae orthologue, Mit1, regulates the transition to pseudohyphal growth, whereas, C. albicans Wor1 regulates white-opaque switching (Huang et al.2006; Srikantha et al.2006). Ryp1 is preferentially expressed in yeast cells at 37°C and, like Mit1 and Wor1, has been shown by chromatin immunoprecipitation (ChIP) to associate with DNA (Nguyen and Sil 2008). H. capsulatum Ryp1, C. albicans Wor1 and S. cerevisiae Mit1 can all bind to the Mit1-binding site identified in the S. cerevisiae FLO11 promoter (Cain et al.2012). Interestingly, despite this highly conserved recognition site, comparison of target genes for each regulator suggests that there is very little overlap. It has been suggested that the movement of genes in and out of the control of this conserved master regulator may be responsible for the differences in morphology amongst the different species (Cain et al.2012).

Like RYP1 mutants, RYP2 and RYP3 mutants cannot grow as yeast at 37°C and inappropriately sporulate (Webster and Sil 2008). Ryp2 and Ryp3 have homology to the velvet family of developmental regulators first identified in A. nidulans (Mooney and Yager 1990; Bayram and Braus 2012). Ryp2 is orthologous to A. nidulans VosA, which regulates the production and viability of asexual spores (conidia), whereas Ryp3 shows homology to A. nidulans VelB, which is required for sexual spore formation (Ni and Yu 2007; Bayram et al.2008). Like the A. nidulans mutants, both the RYP2 and RYP3 mutants display defects in spore viability (Webster and Sil 2008). A fourth transcriptional regulator, a Zn(II)2Cys6 zinc binuclear cluster domain protein, Ryp4, was recently identified as a shared target of Ryp1/2/3 and is also required for yeast growth at 37ºC (Beyhan et al.2013). Ryp1, Ryp2 and Ryp3 physically interact and directly bind DNA (Beyhan et al.2013). Ryp1 binds to a conserved cis-acting regulatory sequence, whereas Ryp2 and Ryp3 bind together to a second cis-acting regulatory sequence in the upstream regulatory regions of core pathogenicity genes including CBP1 and SOD3 (Beyhan et al.2013).

Orthologues of these global regulators of morphogenesis are conserved across the fungi kingdom and it is clear that they are intimately linked to the regulation of morphogenetic processes such as dimorphic switching and asexual development, and processes such as secondary metabolism that are intimately associated with morphogenesis and differentiation. What remains to be elucidated is the link between the signalling pathways known to regulate the dimorphic transition and these core transcription factors.

Control of yeast cellular morphology, polarity and division in dimorphic fungal pathogens

The switch from hyphal to yeast growth requires changes in polarized growth and cellular morphology. The Rho GTPases (Cdc42, Rac and Rho) are members of the Ras GTPase superfamily that play conserved roles in regulating morphogenesis by controlling actin-mediated polarized growth and signalling pathways required for morphological responses (Fig. 3). In S. cerevisiae, Cdc42 acts upstream of two p21-activated kinases (PAK); Ste20 to regulate activation of the MAPK cascades controlling mating, osmoregulation and morphological switching and Cla4 to regulate cytokinesis via the phosphorylation of myosins and septins. Cdc42 is also required for recruitment of actin and polarisome components to the sites of polarized growth (reviewed in Johnson 1999). RNAi-mediated silencing of P. brasiliensis cdc42 resulted in a yeast cell population with a more homogenous morphology compared to wild type and decreased the average bud size (Almeida et al.2009). RNAi cdc42 knockdown strains were more efficiently phagocytosed by macrophages and displayed decreased pathogenicity, demonstrating that the large, multibudded state of P. brasiliensis plays an important role during pathogenesis by inhibiting phagocytosis (Almeida et al.2009). In T. marneffei, the Cdc42 orthologue, CflA, acts downstream of RasA to regulate conidial germination and actin-mediated polarized growth and morphology of both hyphal cells at 25°C and yeast cells at 37°C (Boyce, Hynes and Andrianopoulos 2001, 2005). The related Rac GTPase, CflB, is required for actin-mediated polarized morphogenesis of asexual development structures (conidiophores) but not for yeast morphogenesis at 37°C in vitro (Boyce, Hynes and Andrianopoulos 2003). However, CflB is required for the germination of conidia in macrophages (Boyce, Schreider and Andrianopoulos 2009). The Ste20 orthologue in T. marneffei, pakA, is essential for conidial germination and polarised growth of yeast cells at 37°C (Boyce and Andrianopoulos 2007). Interestingly, deletion of the second PAK, pakB (Cla4 orthologue), results in inappropriate yeast-like cell production at 25°C, as well as division defects during conidiophore production and in macrophage-engulfed yeast cells (Boyce, Schreider and Andrianopoulos 2009). Concomitant with the inappropriate yeast cell production at 25°C, the expression of hyphal-specific genes is decreased while yeast-specific gene expression is elevated, suggesting that PakB is regulating signalling pathways that control phase-specific gene expression (Boyce, Schreider and Andrianopoulos 2009). Deletion of the central regulator of conidiation, brlA, in ΔpakB strains results in suppression of the inappropriate yeast cell production at 25°C suggesting that the observed yeast cells arise from conidiophore cell types (Boyce, Schreider and Andrianopoulos 2009). This suggests that the developmental pathways regulating asexual development at 25°C and yeast cell production at 37°C share regulatory components. This is supported by the dual roles of the T. marneffei transcriptional regulator abaA and HHKs encoded by drkA and slnA in both conidiation and dimorphic growth (Borneman, Hynes and Andrianopoulos 2000; Boyce et al.2011). This link has also been suggested for H. capsulatum and B. dermatitidis (Krajaejun et al.2007; Nguyen and Sil 2008; Webster and Sil 2008). Interestingly, neither of these fungi have an abaA orthologue. Furthermore, pakB is strongly upregulated in T. marneffei upon phagocytosis by macrophages and is essential for yeast morphogenesis in macrophages but not during in vitro growth at 37°C (Boyce, Schreider and Andrianopoulos 2009). This implies that PakB is part of a signalling pathway that responds to host cell inductive signals, not temperature. Whether this mechanism is conserved in other intracellular pathogens such as H. capsulatum remains to be determined.

The correct execution of budding division is also essential for yeast morphogenesis. Septins are GTP-binding cytoskeleton components which form heterooligomeric complexes, such as filaments and rings, that are essential for cellular division. RNAi knockdown of the septin encoding genes (CDC11, CDC3, CDC10 and CDC12) in B. dermatitidis resulted in hyphal and yeast cells with aberrant morphology but did not affect the ability to undergo the dimorphic switch (Krajaejun et al.2007; Marty and Gauthier 2013). Deletion of the gene encoding the type II myosin in T. marneffei, myoB, which is essential for cellular division, results in defects in hyphal morphology, developmental progression during asexual development and yeast cell production at 37°C (Canovas, Boyce and Andrianopoulos 2011).

Remodelling of the fungal cell wall during dimorphic switching and host colonization

One common survival strategy used by dimorphic fungi is to rapidly remodel the cell wall during infection in order to prevent recognition by phagocytic cell PRRs. The fungal cell wall is composed of an outer layer of heavily glycosylated N- and O- linked mannoproteins which are attached by glycoslyphosphatidylinositol anchors to an inner layer of ß(1,6)-glucan, ß(1,3)-glucan, α(1,3)-glucan and chitin (Klis, De Groot and Hellingwerf 2001; Rappleye, Eissenberg and Goldman 2007; Puccia et al.2011). P. brasiliensis and B. dermatitidis decrease the ß(1,3)-glucan content and increase the α(1,3)-glucan content of the cell wall during infection and the hyphal-to-yeast dimorphic switch (Rappleye, Engle and Goldman 2004; Rappleye, Eissenberg and Goldman 2007; Sorais et al.2010). Consistent with this, in P. brasiliensis the two subunits of the ß(1,3)-glucan synthase complex, the Rho1 GTPase and Fks1, are expressed specifically in hyphae but not yeast, whereas the two subunits of the α(1,3)-glucan synthase complex, the Rho2 GTPase and Ags1, are specifically expressed during yeast growth (Sorais et al.2010). This may prevent recognition of ß(1,3)-glucan, which is hidden by layer of α(1,3)-glucan, by the dectin-1 PPR (Brown 2006; Rappleye, Eissenberg and Goldman 2007). Disruption or RNAi silencing of AGS1, encoding α(1,3)-glucan synthase, or AMY1, encoding a glycosyl hydrolase (required in the biosynthesis of α(1,3)-glucan), attenuates the ability of H. capsulatum to kill macrophages and colonize mice lungs (Rappleye, Engle and Goldman 2004; Marion et al.2006). In addition, levels of α(1,3)-glucan in P. brasiliensis, B. dermatitidis and some H. capsulatum chemotypes correlate with virulence (San-Blas, San-Blas and Cova 1976; Hogan and Klein 1994; Edwards, Alore and Rappleye 2011). Expression of the Chs3 chitin synthase is induced in P. brasiliensis and T. marneffei yeast cells and chitin content of P. brasiliensis yeast is increased compared to hyphae (Barreto et al.2010; Pasricha et al.2013). In C. albicans, chitin blocks the recognition of C. albicans via the dectin-1 receptor (Mora-Montes et al.2011). However, chitin does not bind the dectin-1 receptor directly suggesting that the presence of chitin influences the engagement of dectin-1 with ß-glucans in the fungal cell wall (Mora-Montes et al.2011). Thus, masking or obscuring cell-wall components detected by the host is a common mechanism of avoiding the full induction of the host's defence mechanisms.

Fungal adaptation or neutralization of the macrophage lysosome environment during host colonization

Phagocytes produce ROS and reactive nitrogen species (RNS) to damage microbes. In the phagosomal system, this is accompanied by the release of hydrolytic enzymes and toxic metabolites into the phagosome and lowering of the phagolysomal pH to aid in pathogen killing. Proteins required for oxidative stress resistance were recently found to be components of the extracellular proteome of H. capsulatum yeast cells (Holbrook et al.2011). H. capsulatum secretes catalase B (CatB), a Cu/Zn-type superoxide dismutase (Sod3) and a thioredoxin-like protein (Trl3) which are proposed to eliminate the oxidative stress present in the macrophage phagolysosome derived from NADPH oxidase (Holbrook et al.2011). Superoxide dismutase converts superoxide radicals into oxygen and hydrogen peroxide thus neutralizing ROS (Hwang et al.2002; Cox et al.2003). Expression of genes encoding superoxide dismutase is also induced when P. brasiliensis and T. marneffei grown in macrophages (Tavares et al.2007; Thirach et al.2007). In order to neutralize or prevent RNS production, H. capsulatum, P. brasiliensis, C. immitis and B. dermatitidis either detoxify nitric oxide (NO) or interfere with the enzymatic activity of the inducible nitric oxide synthase (iNOS) (Flavia Popi, Lopes and Mariano 2002; Hung, Xue and Cole 2007; Chao, Rine and Marletta 2008; Gonzalez, Hung and Cole 2011; Rocco, Carmen and Klein 2011). H. capsulatum contains a nitric oxide reductase cytochrome P450, encoded by nor1, which is proposed to detoxify NO (Chao, Rine and Marletta 2008). B. dermatitidis, P. brasiliensis and C. immitis yeast cells reduce nitric oxide levels by interference with iNOS enzymatic activity (Flavia Popi, Lopes and Mariano 2002; Hung, Xue and Cole 2007; Gonzalez, Hung and Cole 2011; Rocco, Carmen and Klein 2011). In addition, P. brasiliensis secretes a mannose glycoprotein that prevents the release of NO from macrophages and stimulates release of the cytokine IL-10 that reduces iNOS expression (Flavia Popi, Lopes and Mariano 2002). C. immitis infection elevates the production of host arginase I, which competes with iNOS for the common substrate L-arginine, thus reducing NO production (Hung, Xue and Cole 2007). Macrophage vaculolar ATPases pump protons into the phagolysosome to lower the pH, however, phagolysosomes containing H. capsulatum fail to acidify (Strasser et al.1999). HCL1, encoding HMG CoA lyase, was identified in an insertional mutagenesis screen aiming at identifying genes required for lysis of bone marrow-derived macrophages (BMDM) (Isaac et al.2013). The HCL1 mutant shows a severe growth defect in BMDMs and is critical for macrophage colonization and pathogenicity. Interestingly, increased phagosome acidification was observed in macrophages infected with the HCL1 mutant (Isaac et al.2013) but it is unclear if this relates to an inability of H. capsulatum mutants to control phagolysosomal pH or if acidification is caused by H. capsulatum itself as a consequence of the metabolic block in leucine biosynthesis.

Colonization of a host requires dimorphic fungi to establish thermal tolerance

The establishment of thermal homeostasis requires heat shock proteins (HSPs) to respond to changes in environmental temperature. HSPs function as chaperones to regulate correct protein folding, transport and assembly of protein complexes. In response to an acute thermal upshift, HSP levels are increased via binding of the Hsf1 transcription factor to heat shock elements (HSEs) in promoters of target genes (reviewed in Nicholls et al.2009). Hsf1 from P. lutzii (formally P. brasiliensis isolate 01) can complement the S. cerevisiae hsf1 mutant and has 650 potential HSE-binding sites in the P. lutzii genome (Paes et al.2011). P. lutzii Hsf1 can bind to HSEs identified in the promoters of drk1 and ryp1 suggesting coregulation of these key regulators of dimorphic switching with the heat shock response (Paes et al.2011).

Hsp30, Hsp70, Hsp82 and Hsp104 are highly abundant during the yeast phase of P. brasiliensis (da Silva et al.1999; Goldman et al.2003; Marques et al.2004; Borges et al.2011). Likewise, hsp60, hsp70 and hsp82 are highly expressed during the hyphal-to-yeast dimorphic switch in H. capsulatum (Caruso et al.1987; Kamei et al.1992; Minchiotti, Gargano and Maresca 1992). Studies in T. marneffei have shown that several HSPs are highly expressed during the dimorphic switch (Kummasook, Pongpom and Vanittanakom 2007; Chandler et al.2008; Vanittanakom et al.2009). However, a recent genome-wide expression study states that none of the HSPs or associated factors are highly overexpressed at 37ºC and only 6% of predicted heat response genes are overexpressed at 37ºC, which is significantly fewer than expected (Yang et al.2013). It should be noted that these studies were conducted in two different T. marneffei isolates. The expression of hsp70 and its virulence and thermal tolerance was assessed by measuring the proportion of viable cells at increasing temperature increments, in a number of different H. capsulatum isolates (Caruso et al.1987). The results suggested a direct correlation between increased hsp70 expression, increased thermal tolerance and pathogenicity in H. capsulatum (Caruso et al.1987). However, this correlation is not observed in P. brasiliensis (Theodoro et al.2008). There have been extensive studies on HSPs in C. albicans due to their presence at the cell surface and subsequent immunogenic nature, roles in morphogenesis and in potentiating the rapid evolution of drug resistance (reviewed in Brown, Leach and Nicholls 2010; Shapiro and Cowen 2010). Hsp90 negatively regulates morphogenesis by repressing the Ras-PKA signalling pathway (Shapiro et al.2009). Inhibition of Hsp90 with geldanomycin and racidicol, or repression of hsp90 expression, can induce the yeast-to-hyphal dimorphic switch and reduces pathogenicity (Shapiro et al.2009). Hsp90 in S. schenckii interacts with the calmodulin kinase 1 and Hsp90 inhibition blocks yeast growth at 35°C, suggesting a link between calcium signalling, thermal tolerance and the dimorphic switch (Rodriguez-Caban et al.2011). Clearly, the role of the heat shock response in morphogenesis and pathogenicity for the dimorphic fungi is patchy and warrants further investigation.

The ability to acquire iron from the host is a critical requirement for intracellular growth

Surviving within the macrophage phagolysosome poses many metabolic challenges for intracellular dimorphic pathogens, including a lack of essential trace elements such as iron and zinc (reviewed in Schaible and Kaufmann 2004). Host cells restrict available iron through sequestration by high-affinity iron-binding proteins such as transferrin and ferritin to prevent intracellular microbial proliferation. Host T-cells produce the cytokine interferon gamma (IFNγ) which downregulates surface transferrin receptors and NO production by activated macrophages, which also restricts iron availability (Lane, Wu-Hsieh and Howard 1991; Lane et al.1994; Gonzalez, Restrepo and Cano 2007). IFNγ has been shown to inhibit H. capsulatum, P. brasiliensis and T. marneffei intracellular proliferation and this restriction can be reversed by the addition of iron-saturated transferrin in H. capsulatum (Lane, Wu-Hsieh and Howard 1991; Cain and Deepe 1998; Taramelli et al.2000; Gonzalez, Restrepo and Cano 2007). Intracellular yeast growth of H. capsulatum and P. brasiliensis is inhibited by the iron chelator deferoxamine and the addition of transferrin can abolish this inhibition (Cano et al.1994; Newman et al.1994; Dias-Melicio et al.2005). Recently, proteomic analysis of P. brasiliensis was used to identify proteins sensitive to low iron (Parente et al.2011). Under iron deprivation, the abundance of glycolytic pathway enzymes was increased, whereas enzymes of the tricarboxylic acid, glyoxylate and methylcitrate cycles and proteins involved in electron transport were decreased (Parente et al.2011). This suggests that P. brasiliensis prioritizes iron-independent metabolic pathways during infection to adapt to the low iron environment of the macrophage.

Under iron-limiting conditions, fungi utilize high-affinity iron uptake systems such as reductive iron assimilation (RIA) and non-reductive, siderophore-based iron assimilation (non-RIA) (Fig. 4). The RIA system requires ferric iron (Fe3+) to be reduced to ferrous (Fe2+) by metalloreductases and transported into the cell by the high-affinity ferrous transporter complex comprising the ferroxidase Fet3 and permease Ftr1 (Fig. 4). Non-RIA utilizes secreted siderophores, ferric iron chelators which scavenge free iron (Timmerman and Woods 1999, 2001; Howard et al.2000; Jung and Kronstad 2008; Hilty, George Smulian and Newman 2011; Parente et al.2011; Haas 2012) (Fig. 4). Fungi differ in their ability to utilize RIA and non-RIA systems. For example, C. albicans and C. neoformans cannot synthesize siderophores (non-RIA), whereas A. nidulans lacks the RIA uptake system (Jung and Kronstad 2008; Haas 2012). The genomes of most dimorphic fungi have both RIA and siderophore (non-RIA) encoding genes (Fig. 4). The exception is C. immitis and P. brasiliensis, which lack both ftrA and fetC homologues suggesting that they do not utilize the RIA uptake system. Iron starvation results in extensive transcriptional changes mediated by a GATA transcription factor SreA and the bZIP transcription factor, HapX. SreA represses iron uptake during iron sufficiency, whereas HapX represses iron-consuming pathways during iron starvation (Jung et al.2006, 2010; Jung and Kronstad 2008; Schrettl et al.2008; Schrettl et al.2010; Haas 2012).

Figure 4.

High-affinity iron uptake systems in dimorphic fungi. Most dimorphic fungi utilize both RIA and non-RIA systems to mediate high-affinity iron uptake when bioavailable iron is low, such as within host phagocytes. In the RIA system, ferric iron (Fe3+) is reduced to ferrous iron (Fe2+) by FreA (orthologous to S. cerevisiae Fet1). Subsequently, in a copper and oxygen-dependent reaction, ferrous iron (Fe2+) is oxidized by the multi-copper oxidase FetC (orthologous to S. cerevisiae Fet3) and the ferric iron (Fe3+) is transported across the membrane by the FtrA permease (orthologous to S. cerevisiae Ftr1). C. immitis and P. brasiliensis lack both ftrA and fetC homologues. fetC is upregulated during yeast growth in T. marneffei. Non-RIA iron uptake requires the biosynthesis of extracellular ferric iron (Fe3+) chelators called siderophores. Siderophores can provide scavenged ferric iron (Fe3+) to FreA for the RIA uptake system or be transported into the cell by siderophore complex transporters.

Figure 4.

High-affinity iron uptake systems in dimorphic fungi. Most dimorphic fungi utilize both RIA and non-RIA systems to mediate high-affinity iron uptake when bioavailable iron is low, such as within host phagocytes. In the RIA system, ferric iron (Fe3+) is reduced to ferrous iron (Fe2+) by FreA (orthologous to S. cerevisiae Fet1). Subsequently, in a copper and oxygen-dependent reaction, ferrous iron (Fe2+) is oxidized by the multi-copper oxidase FetC (orthologous to S. cerevisiae Fet3) and the ferric iron (Fe3+) is transported across the membrane by the FtrA permease (orthologous to S. cerevisiae Ftr1). C. immitis and P. brasiliensis lack both ftrA and fetC homologues. fetC is upregulated during yeast growth in T. marneffei. Non-RIA iron uptake requires the biosynthesis of extracellular ferric iron (Fe3+) chelators called siderophores. Siderophores can provide scavenged ferric iron (Fe3+) to FreA for the RIA uptake system or be transported into the cell by siderophore complex transporters.

Expression of the genes required for siderophore biosynthesis, transport and utilization in H. capsulatum (sid1, sid3, sid4, nps1, oxr1, msf1 and abc1) is induced under iron-limiting conditions and all of these genes, with the exception of msf1, are located within a genomic cluster (Hwang et al.2008). The Sre1 GATA transcription factor binds a consensus site 5-(G/A)ATC(T/A)GATAA-3 found in the promoter region of all genes in the cluster (Hwang et al.2008). Mutation of H. capsulatum sid1, which encodes the L-ornithine monoxygenase, required for the catalysis of the first step in siderophore production, results in a growth defect in limiting iron, a failure to produce hydroxamate siderophores and reduced pathogenicity (Hwang et al.2008; Hilty, George Smulian and Newman 2011). Expression of sidA (sid1 orthologue), sit1 (msf1 orthologue) and the gene encoding the bZIP transcription factor hapX is increased during iron deprivation in P. brasiliensis (Parente et al.2011). sidF (sid3 orthologue) and sidD (nps1 orthologue) are highly expressed in T. marneffei yeast cells (Pasricha et al.2013). SreB in B. dermatitidis (sre1 orthologue) is also required for the expression of genes required for siderophore biosynthesis and uptake (Gauthier et al.2010). The ΔsreB mutant fails to repress dimerum acid and coprogen siderophore biosynthesis when iron is abundant. The expression of genes required for the biosynthesis (sidA and amcA) and uptake of siderophores (mirB and mirC) is induced in low iron and repressed when iron is abundant; however, the ΔsreB mutant exhibits depressed expression of these genes in abundant iron. Interestingly, in addition to this phenotype the mutant fails to complete the yeast-to-hyphal dimorphic switch when switched from 37 to 22°C (Gauthier et al.2010).

Physiological requirements for calcium during intracellular growth of yeast cells

Calcium signalling regulates a vast array of eukaryotic cellular processes, including ion homeostasis, pH adaptation, glucose metabolism, morphogenesis and development in fungi (Viladevall et al.2004; Soriani et al.2008; Ruiz, Serrano and Arino 2008; Spielvogel et al.2008; Cervantes-Chavez, Ali and Bakkeren 2011). Cytosolic Ca2+ concentrations are maintained at a low level by transporting excess Ca2+ into internal stores or outside of the cell. Internal or external signals result in uptake of Ca2+ from the environment or a release from internal stores to produce transient bursts of free Ca2+, which can act as intracellular signals (Fig. 3C). Ca2+ binds to the calmodulin kinase (CaM) that activates the catalytic and regulatory subunits of the calcineurin phosphatase (CnaA and CnaB). CnaA desphosphorylates the transcription factor CrzA that is subsequently transported into the nucleus where it can activate the expression of target genes (Yoshimoto et al.2002) (Fig. 3C). CaM can also activate calmodulin-dependent kinases to elicit responses. Expression of the genes encoding calmodulin and the calcineurin regulatory subunit are induced during the hyphal-to-yeast dimorphic switch in P. brasiliensis (Nunes et al.2005; Bastos et al.2007). In P. brasiliensis, inhibitors of calmodulin and calmodulin-depen-dent phosphodiesterase have been shown to inhibit the hyphal-to-yeast dimorphic switch, whereas addition of extracellular Ca2+ stimulated this transition (de Carvalho et al.2003; Campos et al.2008). In addition, the use of a membrane permeable Ca2+ fluorescent probe revealed a transient increase in cytoplasmic Ca2+ levels during the dimorphic switch (Campos et al.2008). Similarly, inhibitors of calmodulin and the calmodulin-dependent kinases in S. schenckii hindered the entry of yeast cells into the budding cycle and stimulated the yeast-to-hyphal switch (Valle-Aviles et al.2007). Reduction of calmodulin-dependent kinase 1 levels by RNAi inhibited S. schenckii yeast growth at 35°C (Rodriguez-Caban et al.2011). In the pathogenic zygomycete Mucor circinelloides, which switches from a hyphal growth form in aerobic conditions to a multibudded yeast growth form in anaerobic conditions, the addition of a calcineurin inhibitor or disruption of the gene encoding the calcineurin regulatory subunit (cnbR) resulted in exclusive multibudded yeast growth and decreased virulence (Lee et al.2013). Disruption of the gene encoding one of the calcineurin catalytic subunits (cnaA) resulted in hypersensitivity to calcineurin inhibitors, hyphal polarity defects, a mixture of hyphal and yeast cells under aerobic conditions and larger, more virulent spores (Lee et al.2013).

It has been known for some time that calcium plays an important role in H. capsulatum infection. Large amounts of calcium are essential for H. capsulatum hyphal but not yeast growth suggesting that yeast cells are adapted to surviving in low calcium environments such as the macrophage phagolysosome (Batanghari and Goldman 1997). Deletion of Cbp1, the major yeast-specific calcium-binding protein secreted during H. capsulatum infection, renders the fungus unable to kill macrophages in vitro or to undergo pulmonary colonization of the host (Batanghari et al.1998; Sebghati, Engle and Goldman 2000). The addition of exogenous Cbp1 results in increased cellular incorporation of calcium suggesting that Cbp1 may function to provide calcium in low calcium environments. Recent characterization of the protein structure suggests Cbp1 is a member of the saposin family of lipid-binding proteins which interact with cellular membranes (Beck et al.2009).

Bad1 (also called WI-1) is a yeast-specific calcium-binding protein which is essential for pathogenicity in B. dermatiditis (Brandhorst et al.1999). Bad1 contains 41 copies of a tandem repeat each with a calcium-binding EF-hand motif, facilitating calcium binding and growth in a poor calcium environment (Brandhorst et al.2005). Bad1 also acts as an adhesion, binding to chitin on the yeast cell wall and interacting with the complement type 3 receptors CR3 and CD14 on host macrophages and heparin (Newman, Chaturvedi and Klein 1995; Brandhorst et al.2013). Bad1 modulates the host's immune response by inhibiting T-cell activation via the surface protein CD47 and by affecting levels of proinflammatory cytokines TNF-α, TGF-ß, IL-17 and IFN-γ (Finkel-Jimenez, Wuthrich and Klein 2002; Brandhorst et al.2013).

CONCLUDING REMARKS

Genetic manipulation of dimorphic fungal pathogens has been previously limited due to a complete lack of DNA-mediated transformation, poor integration of introduced DNA or high frequencies of non-homologous DNA integration, making the generation of gene deletion or allelic replacement strains difficult. However, recent advances in genome-wide expression approaches, RNA inhibition and the development of strains containing mutations to increase the frequency of homologous integration have greatly accelerated our understanding of this group of medically relevant pathogens. Recent studies suggest that the core signalling pathways that sense and respond to the host are conserved in dimorphic species. Of significant interest is the central conserved role of the HHK of the two-component signalling system. Drk1 orthologues play conserved roles in regulating not only the dimorphic switch but also a number of traits known to be important for intracellular pathogenic growth. The future characterization of downstream components of this pathway and genome-wide expression analysis in mutants of these components will provide invaluable insight into the processes required for dimorphic switching and survival in the host. Additional pathways involving the p21-activated kinases are also likely to impinge on the dimorphic switch and the way temperature and host signals are interpreted by the cell. It is tempting to speculate that these pathways will culminate in the activation of orthologues of the RYP1–4 transcription factors in the various dimorphic fungi and these transcription factors will regulate not only genes required for the morphological transition but the additional mechanisms used to adapt to the host environment. This would include those required for the onset and extent of sporulation (the infectious agent), remodelling the fungal cell wall to avoid immune detection, adapting or neutralizing oxidative and osmotic stress and the physiological response to the host environment. Despite the emerging, albeit embryonic, convergence of factors that are involved in dimorphic switching across this group of fungi, there will no doubt be differences amongst them owing to their independent evolutionary histories. It is clear that dimorphic switching has polyphyletic origins but is one that rests on a core set of existing attributes that have been co-opted. Future investigations of the mechanisms by which dimorphic fungal pathogens sense and respond to the host environment will provide important insights into fungal pathogenesis and host–pathogen interactions.

Conflict of interest. None declared.

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