Regulation of phenotypic transitions in the fungal pathogen Candida albicans

Abstract

The human commensal fungus Candida albicans can cause not only superficial infections, but also life-threatening disease in immunocompromised individuals. C. albicans can grow in several morphological forms. The ability to switch between different phenotypic forms has been thought to contribute to its virulence. The yeast-filamentous growth transition and white-opaque switching represent two typical morphological switching systems, which have been intensively studied in C. albicans. The interplay between environmental factors and genes determines the morphology of C. albicans. This review focuses on the regulation of phenotypic changes in this pathogenic organism by external environmental cues and internal genes.

Keywords: :

• Candida albicans
• cAMP-PKA pathway
• filamentous development
• phenotypic transitions
• white-opaque switching

Introduction

The yeast Candida albicans is a harmless commensal in the oral cavity, digestive tract and genital region of healthy people, while it also causes superficial infections and life-threatening systemic disease.¹ With the increase of immunocompromised individuals due to HIV infection, organ transplantation and application of chemotherapy and indwelling devices, invasive candidiasis has become a serious public health problem in the recent several decades.² The switching from commensal to pathogenic phase has been widely thought to be associated with the phenotypic plasticity of C. albicans. It can grow in several morphological forms including unicellular yeast-form, elongated hyphae and pseudohyphae.³ In this review, hyphae and pseudohyphae are referred to as filamentous forms or cells. A plethora of external and internal factors regulate the switching between different phenotypes (Figs. 1 and 2A and Table 1).^3,4 The external factors include environmental cues, that the pathogen often confronts with during its life cycle and host infection, such as serum, high temperature (37°C), low levels of oxygen, high levels of CO₂, poor nutrition conditions and so on.^4,5 The internal genetic and epigenetic changes play a key role in the regulation of morphogenesis.^3-5 In recent years, great strides have been made in uncovering the underlying mechanisms of the morphologic regulation and the coordination and interplay between environmental factors and genes. Several signal transduction pathways and key transcription factors have been intensively investigated.^3-5 Another phenotypic switching system, referred to as white-opaque transition, has attracted increasing research interest in the past decade. The underlying molecular mechanisms have begun to be uncovered. This system was first identified in a clinical strain, WO-1, isolated from a transplant patient with a fatal blood stream infection.⁶ Although the white-opaque transition system gives distinguishable cellular and colony appearance, both the white and opaque forms are budding cells.⁷ The two types of cells also differ in virulence, sensitivity to immune cells, expression of a wide variety of genes and mating competence. In this review, I will focus on the molecular mechanisms involved in the regulation of yeast-filamentous growth switching as well as white-opaque transition in C. albicans.

Figure 1. Regulation of filamentous growth in C. albicans by multiple environmental cues and signal transduction pathways. The external inducers may function on the cell surface receptors or enter into the cell and directly bind the filamentous growth regulators. The transcription factors Flo8, Efg1 and Cph1 play a central role in the regulation of phenotypic transitions. Multiple signaling pathways converge on the three regulators. The cAMP/PKA pathway and its downstream regulators Flo8 and Efg1 can also play a negative role in filamentous development under embedded growth conditions. The general transcriptional repressor Tup1 is recruited by DNA-binding proteins Nrg1 and Rfg1 and targets on the promoters of hypha-specific genes.

Figure 2. Regulation of white-opaque transition in C. albicans. (A) Environmental factors regulate white-to-opaque and opaque-to-white transitions. (B) The cAMP/PKA pathway and Wor1 involved gene circuitry. The cAMP/PKA pathway regulates both CO₂ and GlcNAc induced opaque cell formation. There is also an unidentified pathway mediated CO₂ and GlcNAc sensing. The two pathways converge on the master regulator Wor1. The transcription factors Wor1, Wor2, Efg1 and Czf1 form a positive feedback loop controlling white-opaque switching. The inhibition of expression of WOR1 by the MTLa1/α2 heterozygous complex is also shown. The dashed line with an arrowhead represents the unidentified pathway involved in CO₂ and GlcNAc sensing.

Table 1. Environmental cues and pathways involved in filamentous growth regulation in C. albicans

	Environmental cues	Pathways and regulators	Reference(s)
Positive regulators	Serum, GlcNAc, starvation, poor nutrition (spider medium), glucose,	Ras1 → Cyr1, Bcy1 → cAMP → PKA (Tpk1 and Tpk2) → Efg1, Flo8 → hyphal specific genes (Hwp1, Ece1, etc.)	25, 41, 42, 47, 50, 52, 53, 55, 77, 89
	CO2	Cyr1, Nce103	13
	Serum, spider medium	Ras1 → Cst20 → Cst11 → Hst7 → Cek1, Cek2 → Cph1, Tec1; Efh1; Hgc1; Ssn6; Cph2 → Tec1	25, 27–31, 37, 57, 76–78, 81, 84, 92, 133, 134
	pH	Rim101, Phr1, Phr2	58–60, 63
	GlcNAc	Ngt1 → Hxk1 → Dac1 → Nag1	135–137
	Osmotic stress	Cst20 → Cst11 → Hst7 → Cek1, Hog1; Ssk1	70, 71, 73, 74
	Physical interaction	Czf1, Mkc1	21, 23
	Hypoxic conditions	Czf1, Efg1, Sch9	21, 138, 139
	Rapamycin	Tor1	140
Negative regulators	Farnesol	Ras1 → Cyr1, Bcy1 → cAMP → PKA (Tpk1 and Tpk2) → Efg1	141
	Hypoxic conditions	Cyr1, Efg1, Flo8	55
		Tup1, Nrg1, Rfg1, Rbf1	76–78, 82, 89

Regulation of Yeast-Filamentous Growth Transition

High-frequency phenotypic switching has been observed in C. albicans and other species in the Candida clade.¹ In 1985, Soll and colleagues reported that C. albicans can switch between at least seven colony phenotypes.⁸ The cellular morphologies in this switching system include the unicellular budding yeast and filamentous hyphal and pseudohyphal forms. Yeast-form cells are round and similar to diploid Saccharomyces cerevisiae cells. Hyphae consist of long tubes with no constrictions, while pseudohyphae consist of chains of elongated cells with constrictions between adjacent cells.⁹ The ability to switch between yeast and filamentous forms is thought to be tightly linked with virulence. Filamentous cells are more invasive and better at tissue penetration, while yeast cells are easy to be delivered and disseminated in the bloodstream.¹⁰ In infected tissues, both yeast-form and filamentous cells are found.¹ It seems that the ability of switching back and forth between the two forms is important for pathogenesis.

Environmental cues regulate yeast-filamentous transition

It is critical for all organisms to adapt to the changes in their environments. C. albicans can infect almost all human organs.¹ It encounters various microenvironmental factors that are unique to the different niches within the host. A range of factors reflecting the host microenvironments have been found to regulate the yeast-filamentous switching. For example, serum is one of the most powerful inducers of filamentous growth in C. albicans. Xu et al. have discovered that bacterial peptidoglycans in the host serum are the major component triggering C. albicans filamentous growth.¹¹ Since the human host cannot synthesize peptidoglycan molecules, it has been indicated that these molecules come from the commensal bacteria in the gastrointestinal tract (GI tract).¹¹ Another two host associated molecules regulating C. albicans yeast-filamentous transition are N-acetylglucosamine (GlcNAc) and CO₂.^12-16 GlcNAc is a component of the mucus of GI tract and bacterial cell wall, while CO₂ is a product of cellular respiration.^17,18 The level of CO₂ in blood is about 5%, which is much higher than that in ambient atmosphere (0.036%).¹⁹ The cAMP/PKA pathway is involved in GlcNAc and CO₂ induced filamentous growth.^12,13 C. albicans also undergoes morphological changes in response to the host temperature (37°C), neutral pH, nutrient limitation and low O₂ levels.^3-5,20-22 In vitro experiments demonstrate that the quorum sensing molecule, farnesol and physical interaction (e.g., growth in embedded matrix) regulate morphogenesis in C. albicans.^21,23 Interestingly, C. albicans can undergo filamentous growth under embedded conditions at relatively low temperature (25°C).²¹ During the past two decades, great progress has been made in understanding the underlying mechanisms regulating external factors induced morphogenesis in this fungal pathogen (Fig. 1). The following sections will focus on the major pathways and genes involved in regulation of morphogenesis in response to different environmental factors. Their roles in virulence will also be discussed.

Ras proteins

Ras is a member of a highly conserved family of small GTPases in eukaryotes ranging from yeast to humans.²⁴ Ras is activated when bound with GTP, while it is in an inactivated form when bound with GDP.²⁴ There are two Ras proteins in C. albicans, Ras1 and Ras2.^25,26 C. albicans Ras1 is a homolog of S. cerevisiae Ras2, which regulates the downstream MAPK and cAMP/PKA pathways.²⁷ C. albicans Ras2 belongs to a group of atypical Ras proteins and shares poor identity with C. albicans Ras1, S. cerevisiae Ras1 and Ras2.²⁶ Ras1 is required for filamentous growth and virulence in C. albicans. Deletion of RAS1 impairs serum induced hyphal growth, while ectopic expression of a dominant active form RAS1V13 has a promoting effect on hyphal development.²⁵ Supplementing the growth media with cAMP or overexpression of components of the MAPK cascade rescued the filamentous growth defect of the ras1Δ/Δ mutant, suggesting that C. albicans Ras1, like S. cerevisiae Ras2, is upstream of the cAMP and MAPK pathways.²⁷ In a systemic infection model, the ras1Δ/Δ mutant shows notably reduced virulence.²⁵ Ras2 has just been recently characterized in C. albicans. Deletion of RAS2 gene alone in a wild type strain has no notable effect, while disruption of it in a ras1Δ/Δ background mutant results in exacerbated hyphal growth defect. Although recombinant C. albicans Ras2 shows similar GTPase activity as does Ras1, Ras2 has an antagonizing effect on Ras1 at many aspects including regulation of cAMP level, stationary-phase entry and stress response.²⁶ The opposite roles of the two Ras proteins may fine-tune the downstream pathways in respond to different environmental changes.

Cst20-Cst11-Hst7-Cek1/2 mediated MAPK cascade

The roles of the MAPK pathway in morphogenesis and mating have been extensively studied in C. albicans.^28-32 The Rho-type GTPase Cdc42 and its exchange factor Cdc24 are required for normal budding, virulence and filamentous growth.^33-36 Cdc42, together with Cdc24, interacts with Ras1 to activate the MAPKKKK Cst20, which then triggers a subsequent phosphorylation of the MAPKKK (Ste11)-MAPKK (Hst7)-MAPK (Cek1/Cek2) cascade.^33-36 The cascade finally activates the downstream transcription factor Cph1,³⁷ a C. albicans homolog of S. cerevisiae Ste12.³⁸ Cph1 is required for hyphal growth on solid agar, but not in liquid media.³⁷ Cst20, a C. albicans homolog of S. cerevisiae Ste20 and a kinase of p65PAK family, is essential for hyphal development and virulence.^28,29 Although the function of C. albicans Ste11 in mating and filamentous growth has not been investigated, the kinase plays a critical role in pheromone induced biofilm formation.³⁹ Deletion of HST7, encoding a C. albicans homolog of S. serevisiae Ste7, results in defects in hyphal development. Overexpression of HST7 in the cst20Δ/Δ mutant can partially rescue its hyphal growth defect, suggesting Hst7 is downstream of Cst20.^28,29 The kinases Cek1 and Cek2 are homologous to S. cerevisiae Kss1 and Fus3, respectively.³⁰ The mutants of hst7Δ/Δ and cph1Δ/Δ, and the cek1Δ/Δ cek2Δ/Δ double mutant are completely defective in mating, while deletion of CST20, CEK1, and CEK2 genes only leads to partial defect.^30,31

cAMP/PKA signaling

The cAMP/PKA pathway is highly conserved in eukaryotes. In C. albicans, the cAMP/PKA pathway plays a critical role in morphogenesis.⁴⁰ CYR1 (also named as CDC35), encoding the only adenylyl cyclase in C. albicans, is required for hyphal development and virulence, although it is not essential for the basal level of growth.⁴¹ Deletion of CYR1 has a global impact on gene expression and results in many alterations in response to stresses and environmental cues.⁴² Cyr1 regulates serum and peptidoglycans induced filamentous growth in C. albicans.^11,41 Recently, Huang et al. have reported that Cyr1 plays a major role in GlcNAc induced white to opaque switching, but is not essential for CO₂ induction.^43,44 The upstream activator of Cyr1 is the small GTPase Ras1, which transduces the extracellular signals to Cyr1 and stimulates cAMP production.⁴⁰ cAMP binds to the protein kinase A (PKA) regulatory subunits and causes their dissociation, which then activates catalytic subunits to phosphorylate downstream transcription factors. There are two catalytic subunits of PKA, namely Tpk1 and Tpk2, in C. albicans.^45,46 The two isoforms play distinct and redundant roles in C. albicans. Inactivation of TPK1 gene causes defects in hyphal develpment on solid inducing media, but not in liquid media. In contrast, deletion of TPK2 only partially impairs hyphal growth on solid media, but has a remarkable effect in liquid media. Neither TPK1 nor TPK2 is essential for cell growth, whereas the tpk1Δ/Δ tpk2Δ/Δ double mutant is inviable.^45,46 The catalytic subunits Tpk1 and Tpk2 are regulated by the PKA regulatory subunit Bcy1.^46,47 In the absence of cAMP, two Bcy1 subunits bind to two catalytic subunits and inhibit PKA activity by forming an inactive heterotetrameric complex with Tpk1 or Tpk2. Null mutant of BCY1 is inviable, while it can be deleted in a tpk2/tpk2 mutant background strain.⁴⁷ In the presence of cAMP, binding of cAMP to Bcy1 leads to its dissociation from the complex and releases the catalytic subunits as active forms.⁴⁷ There are two genes, PDE1 and PDE2, encoding the low and high affinity phosphodiesterases, respectively, that keep the intracellular cAMP levels in check in C. albicans.^48-50 Pde2 is activated in response to intracellular acidification and downregulation of cAMP signaling induced by glucose addition. Pde2 plays a major role in regulation of the intracellular levels of cAMP. Deletion of PDE2 gene results in elevated cAMP levels and constitutive activation of the cAMP pathway. Pde2 is required for normal hyphal development, but not for pseudohyphal growth. Deletion of PDE2 actually causes hyperfilamentous growth and reduced virulence.^48-51

Efg1 and Flo8: downstream regulators of cAMP/PKA signaling

The cAMP/PKA pathway has been demonstrated to be mediated by the conserved APSES class protein Efg1 in C. albicans.^52,53 The potential PKA phosphorylation site, threonine-206 (T206) within the conserved APSES domain of Efg1, is important for hyphal formation. Alanine substitution of T206 leads to hyphal development defect both in liquid and solid media, while glutamic acid substitution of T206 results in hyperfilamentation.⁵³ The cells of the efg1Δ/Δ mutant can only slightly elongate in response to some stimuli.⁵² The double mutant of efg1Δ/Δ cph1Δ/Δ, representing inactivation of both the cAMP/PKA signal and the MAPK pathway, is completely locked in yeast phase under all conditions investigated.⁵² Recently, an in vivo genome-wide ChIP-chip and in vitro footprint analysis indicates that Efg1 recognizes a sequence motif TATGCATA (EGR-box). The promoters of transcriptional regulators of hyphal growth including EFG1 itself, TCC1, CZF1, TEC1, DEF1 and NRG1 contains EGR- and/or EGR-like boxes. Further analysis demonstrates different binding specificities of Efg1 in yeast growth and in hyphal induction.⁵⁴

Another transcription factor downstream of cAMP/PKA pathway is the LisH domain containing protein Flo8.⁵⁵ C. albicans Flo8 was first identified to be essential for filamentous growth by functional complementation of an S. cerevisiae flo8Δ mutant, which is defective in invasive growth.⁵⁶ The C. albicans flo8Δ/Δ showed remarkably reduced virulence possibly because of its filamentous development defect. By physically interacting with Efg1, Flo8 controls expression of a subset of Efg1 regulated genes such as the hyphal growth regulator HGC1, a gene encoding a hyphal-specific G1 cyclin-related protein.^55,57 Interestingly, the null mutants of FLO8, EFG1 and CDC35 display increased filamentation under microaerophilic conditions, suggesting that the cAMP/PKA pathway plays both positive and negative roles in regulation of morphogenesis.⁵⁵

pH signaling

C. albicans colonizes different niches with distinct ambient pH. For example, the normal pH of the vaginal tract is 4.5, while the pH of human blood is about 7.0. The ability of C. albicans to respond to pH changes is critical for successful colonization and infections. In C. albicans, the transcription factor Rim101 (initially named as Prr2) is the key regulator of the pH response pathway.^58-60C. albicans Rim101 is homologous to pacC, encoding a zinc-finger transcription factor that regulates pH-dependent gene expression in the model fungus Aspergillus nidulans.^61,62 Although deletion of RIM101 in C. albicans has no obvious effect on the cell growth at acidic or alkaline pH, the mutant shows filamentous growth defects in a number of media.^59,60 Expression of RIM101 is pH-dependent and also controlled by Rim8 (also named as Prr1), a homolog of A. nidulans palF that regulates pacC.⁶⁰ The rim101Δ/Δ mutant lost the ability of controlling the expression of alkaline and acid induced genes at alkaline pH. In vivo experiments suggest that the Rim101 pathway is required for pathogenesis. The rim101Δ/Δ and rim8Δ/Δ mutants show significantly reduced virulence in a systemic infection model.⁵⁸

Another two pH-regulated genes are PHR1 and PHR2,encoding two cell surface glycosidases required for proper cross-linking of β-1,3- and β-1,6-glucans.^63,64 PHR1 is expressed at pH 5.5 or higher, while PHR2 is expressed at an ambient pH below 5.5. PHR1 is upregulated in hyphal cells.⁶⁵ Deletion of PHR1 in C. albicans leads to growth and filamentous development defects at neutral to alkaline pHs. Conversely, deletion of PHR2 compromises filamentous growth at acidic pH.^63,66 Consistently, the virulence phenotypes of the phr1Δ/Δ and phr2Δ/Δ mutants parallel the pH dependence of their in vitro phenotypes. As mentioned above, the systemic pH is neutral, while the vaginal pH is acidic. The phr1Δ/Δ mutant is defective in a mouse model of systemic infection but not in a rat vaginal infection, while the virulence phenotype of the phr2Δ/Δ mutant is the inverse.⁶⁶ The transcription of PHR1 and PHR2 is also regulated by the components of endosomal sorting complexes (ESCRT).⁶⁷

Contact sensing

C. albicans contact-dependent responses, including invasive growth into the host tissues and development of biofilms on surfaces, are medically important. Mkc1 is a contact-activated MAPK with a role in the cell wall integrity pathway. C. albicans mkc1Δ/Δ mutant is defective in invasive hyphal growth and biofilm development, suggesting that the cell integrity pathway plays a role in contact sensing.²³ The zinc finger transcription factor Czf1 is also required for contact induced response. Deletion of CZF1 in C. albicans leads to filamentous growth defect of embedded cells. Deletion of CPH1 in a czf1Δ/Δ mutant enhances this defect.²¹ The Rho-type G protein Rac1 is highly similar to Cdc42 in protein sequence. But they have distinct roles in filamentous growth in response to different environmental conditions. In contrast to Cdc42, Rac1 is essential for filamentation induced under embedded conditions, but not required for hyphal induction by serum, GlcNAc and spider medium.^68,69 The similar responses to different stimuli in czf1Δ/Δ and rac1Δ/Δ mutants suggest Czf1 may function downstream of Rac1.

Osmotic sensing

The Hog1 MAPK pathway regulates not only osmotic stress but also oxidative stress induced responses in C. albicans.^70,71 This conserved signaling cascade includes three major components: the MAPKKK Ssk2, MAPKK Pbs2 and MAPK Hog1.⁷² Ssk2 phosphorylates and thereby activates Pbs2,⁷³ which then subsequently phosphorylates the MAPK Hog1. C. albicans Hog1 was first identified by functional complementation of the osmosenstitve phenotype in an S. cerevisiae hog1Δ mutant and proven to be involved in response to osmotic stress.⁷⁰ Microarray analysis shows that C. albicans Hog1 plays a global role in regulation of gene transcription in response to a variety of stresses.⁷⁴ Deletion of HOG1 in C. albicans results in abnormal filamentous growth as well as decreased virulence.⁷⁵ C. albicans Pbs2, a homolog to the S. cerevisiae MAPKK Pbs2, is required for stress regulation of Hog1p localization and activity. Similar to the hog1Δ/Δ mutant, the pbs2Δ/Δ mutant is sensitive to both osmotic and oxidative stresses.⁷³ Besides the MAPKKK Ssk2, C. albicans genome sequencing reveals the existence of the upstream components of the Hog1 cascade including Ssk1, Ypd1, Chk1 and Nik1, which are homologous to their counterparts in S. cerevisiae (CGD database, www.candidagenome.org).

Negative regulators of filamentous growth

Both filamentous and yeast cells of C. albicans have been found to be associated with tissue infections. It is more likely that one form may be better adapted than the other to survive in different host niches.¹ Therefore, the ability of interconversion between yeast and filamentous forms is critical for pathogenesis. As aforementioned, whereas quite a lot of effort has been put into understanding the mechanisms of yeast to filamentous conversion, only a few negative regulators have been intensively studied.

The general transcriptional repressor Tup1 controls filamentous formation in C. albicans under all conditions investigated.⁷⁶ It represses expression of numerous genes required for initiation and maintenance of filamentous growth. Tup1 is epistatic to the transcriptional activator Cph1 since the phenotype of the tup1Δ/Δ cph1Δ/Δ double mutant is indistinguishable from that of the tup1Δ/Δ mutant.⁷⁶ The zinc finger transcription factor Nrg1 is a DNA-binding protein associated with Tup1.^77,78 Deletion of NRG1 also leads to constitutive filamentous growth under all growth conditions tested. Consistently, overexpression of NRG1 repressed the yeast to filamentous transition.^77,78 Nrg1 is thought to act by recruiting Tup1 to the promoters of target genes. Microarray data indicates that a subset of Nrg1-regulated genes is controlled by Tup1.^79,80 Another transcriptional repressor in C. albicans is Ssn6, which acts together with Tup1. The Tup1-Ssn6 corepressor is conserved from yeast to human. In S. cerevisiae, Ssn6 forms a co-repressor complex with Tup1 and regulates a variety of genes involved in different biological processes.⁸⁰ Hwang et al. found that Ssn6 controls morphological conversion as well as virulence in C. albicans.⁸¹ The mutant of SSN6 displays increased filamentous growth ability in response to high temperature. Interestingly, overexpression of SSN6 leads to enhanced filamentous growth and reduced virulence. These results suggest that Ssn6 may function as a repressor as well as an activator for hyphal development.⁸¹ C. albicans ssn6Δ/Δ and tup1Δ/Δ mutants demonstrate distinct morphological and invasive growth phenotypes. Transcriptional profiling indicates that hypha-specific genes, which are targeted by Tup1 and Nrg1, are not derepressed in the ssn6Δ/Δ mutant, while expression of some white-opaque switching related genes (e.g., WH11) was increased.⁸⁰ Therefore, Ssn6 acts independent of Tup1 at least in some biological processes.

C. albicans Rfg1 is a homolog of S. cerevisiae Rox1, a key repressor of hypoxic genes. Rfg1 controls filamentous development and virulence in C. albicans, but does not appear to be required for the regulation of hypoxic genes.^82,83 Like Nrg1, Rfg1 is a sequence-specific DNA binding protein. In S. cerevisiae, Rox1 represses expression of hypoxic genes via recruitment of the Ssn6-Tup1 complex. Rfg1 may play a similar role in C. albicans. DNA microarray analysis demonstrated that 61 genes are induced significantly in response to exposure to serum and high temperature.⁸⁴ Half of these genes are repressed by the transcription factors, Rfg1, Nrg1, and Tup1. Deletion of RFG1 gene in C. albicans leads to constitutively filamentous growth, while overexpression of RFG1 does not inhibit hyphal formation either in vitro or in vivo.⁸⁴

Cell wall proteins

The cell wall of C. albicans contains about 60% of β-glucan and about 40% of mannoproteins and chitin.^85,86 It provides the cell with a scaffold and protection. The upstream signal transduction pathways and transcription factors finally target on the cell wall components including cell wall proteins. Cell wall proteins play critical roles in maintaining the integrity of the cell wall and sensing the external environmental cues and, therefore, are important for adaptation to the host and pathogenesis.

Hwp1 and Ywp1

Hwp1 (hyphal wall protein 1), a mannoprotein with a C-terminal GPI anchor, is exclusively expressed on the hyphal cell surface.^87,88 HWP1 is regulated by a variety of transcription factors including Tup1, Nrg1, Efg1 and Bcr1.^89,90 The protein Hwp1 is a substrate for the mammalian transglutaminase (TGase) and regulates covalent attachments between germ tubes and host epithelial cells.⁸⁷ Ywp1 (yeast wall protein 1) of C. albicans is a GPI protein containing an N-terminal secretion signal and a central region rich in serine and threonine.⁹¹ The protein Ywp1 has been found to be linked covalently to the wall matrix and to accumulate in liquid culture during stationary phase. Ywp1 is regulated by environmental pH, transcriptional activator Efg1 and Efh1.⁹² While Ywp1 is not essential for normal growth, hyphal development and virulence, deletion of YWP1 results in increased biofilm formation and adhesiveness.⁹¹

ALS family proteins

The ALS (agglutinin-like sequence) gene family of C. albicans encodes cell wall-bound adhesins that are critical for biolfilm development and tissue adherence during the process of infection.⁹³ ALS family proteins are large glycoproteins with a three-domain structure, including a conserved 5′ domain, a central region of 108 bp unit of a repeated motif and a variable 3′ domain with a serine-threonine-rich sequence. There are eight ALS genes (ALS1 to ALS7 and ALS9) in C. albicans.^93-95 ALS8 has been proven to be the same gene as ALS3. These ALS genes are located on three different chromosomes: Chr. 6 (ALS1, ALS2, ALS4 and ALS5), Chr. 3 (ALS6 and ALS7) and Chr. R (ALS3). ALS1 was first identified in C. albicans as a cell surface protein induced in response to high temperature and CO₂.⁹⁶ The expression of ALS1 is upregulated at neutral pH and is downregulated in ssk1/ssk1 mutants. Overexpression of ALS1 leads to extensive flocculation and aggregation. ALS1 functions downstream of cAMP/PKA pathway and is targeted by the transcription factor Efg1. Consistent with its role in adhesion, expression of ALS1 is induced in biofilm populations and overexpression of ALS1 in a bcr1/bcr1 mutant rescues its biofilm development defect.⁹⁰ Expression of ALS genes is also differentially regulated by culture conditions, morphological form and stage of growth.^97,98 The molecular features and roles of ALS proteins in adherence indicate their importance in virulence. Genome sequencing data show that ALS proteins also exist in other pathogenic Candida species including C. dubliniensis and C. tropicalis.⁹⁹

Regulation of White-Opaque Switching

The second high-frequency phenotypic switching system in C. albicans, namely white-opaque transition, has been extensively investigated during the past decade.¹⁰⁰ White and opaque phenotypes are heritable and bistable and show different cellular and colony appearances, gene expression profiles, mating ability and virulence.^6,7,101,102 Cells of each phase can maintain for many generations. White cells are relatively round and form smooth hemispherical colonies on solid media, while opaque cells are large and elongated and form flat and gray or “opaque” colonies.⁷ The two distinct types of cells express a set of phenotype-specific genes. For example, WH11 and the long transcription form of EFG1 are specifically expressed in white cells, whereas OP4 and SAP4 are enriched in opaque cells.^103-105 Remarkably, white cells express a fermentative profile of metabolism related genes, while opaque cells adopt an oxidative one.¹⁰¹ White and opaque cells also differ in virulence.¹⁰⁶ White cells are more virulent than opaque cells in a mouse systemic infection model and can rapidly colonize the host kidneys. In contrast, opaque cells have poor ability to colonize the host internal organs, but are better at cutaneous infection possibly due to the opaque-specific expression of secreted aspartyl proteinase (SAP) genes.

Mating type like locus (MTL)

Hull et al. discovered that C. albicans contained a mating type like locus in its genome.¹⁰⁷ The MTLa1 and MTLα2, homologous to the S. cerevisiae MATa1 and α2, have been identified on chromosome 5 in C. albicans. The MTL locus controls not only C. albicans mating but also white-opaque switching.¹⁰² The Mtla1/α2 heterozygous complex inhibits white-opaque switching via controlling the expression of the master regulator gene WOR1 (white-opaque regulator 1) (Fig. 2).^108-110 This finding explained why only a minor group of clinical strains could undergo white-opaque switching. This small group of strains has been proven to be homozygous at MTL locus.¹¹¹ In 2002, Miller et al. found that only opaque cells undergo efficient mating.¹⁰² Therefore, in order to mate, C. albicans cells first have to undergo homozygosis at the MTL and then switch from white to opaque phenotype.

The white-opaque switching regulatory gene circuitry

Although the MTL homozygous strains of C. albicans are switching-competent, switching from white to opaque phase is rare and stochastic. This fact suggests that the MTL locus is not the master regulator of this process. In 2006, three labs identified Wor1 as a master regulator of the complex switching system.^108-110 WOR1 is exclusively expressed in opaque cells and regulates its own expression by a positive feedback loop. Deletion of WOR1 locks the C. albicans cells in white phase, while overexpression of WOR1 leads to mass conversion of white to opaque form.^108-110 In a subsequent study, Zordan et al. identified an interlocking transcriptional circuit controlling white-opaque switching.¹¹² Wor1 occupies the central position of the gene circuit, which includes three other transcription factors, Efg1, Czf1 and Wor2 (Fig. 2). Wor1 binds to the promoter regions of Efg1, Czf1 and Wor2 and controls their expression. Efg1 is a negative regulator of white to opaque switching. Deletion of EFG1 almost completely locks C. albicans cells in opaque phase, while overexpression of EFG1 results in opaque to white switching.^92,105,112 Overexpression of CZF1 promotes opaque cell formation possibly because it inhibits EFG1 expression. Wor2 is required for opaque formation and maintenance of opaque phenotype. The wor2Δ/Δ mutant completely lost the white to opaque switching ability.¹¹² However, ectopic expression of WOR1 in the wor2/wor2 mutant induces opaque cell formation, suggesting that Wor1 is downstream of Wor2. Microarray and RNA-seq data indicate that more than 1,000 genes or non-coding RNAs are differentially expressed in white and opaque cells, suggesting the regulation of this transition process could be much more complex.^101,113,114 Several studies suggest that epigenetic modifications of the chromatin regulate white-opaque switching in C. albicans. Treatment of C. albicans cells with the histone deacetylase inhibitor trichostatin-A (TSA) promotes opaque cell formation. Consistently, deletion of HDA1, which encodes a deacetylase sensitive to TSA, leads to increased switching frequency from white to opaque.^115,116 Recently, Hnisz et al. have identified eight genes encoding histone-modifying enzymes as regulators of phenotypic switching.¹¹⁷ This study indicates that the conserved Set3/Hos2 histone deacetylase complex plays a key role in white-opaque regulation and links chromatin modification to the Wor1-Wor2-Efg1-Czf1 mediated transcriptional circuit.

Environmental cues regulate white-opaque switching

A plethora of environmental cues have been found to regulate white-opaque switching in C. albicans (Fig. 2A). Human or mammalian mucus is the natural niche for C. albicans, where the temperature is 37°C. In vitro experiments showed that opaque cells are extremely unstable and underwent mass conversion to white phase at this high temperature.⁶ Exposure of cells to low temperature also led to opaque to white switching, but had no obvious effect on white to opaque transition.⁶ Given opaque is the only mating competent form and is unstable at host temperature, how can C. albicans mate in the major niche of the mammalian host? Recently, Huang et al. found that two host environmental molecules, CO₂ and GlcNAc, not only promoted white to opaque switching, but also could stabilize the opaque phenotype at 37°C.^43,44 The cAMP signal, which has been proven to be essential for CO₂ induced filamentous growth in C. albicans, plays a minor role in CO₂ promoted white to opaque transition.⁴³ The major pathway controlling this process remains unclear. The carbonic anhydrase Nce103 is required for the low CO₂ level (1%) induced opaque cell formation, but not for the high CO₂ level (5%) induction.⁴³ The high levels of CO₂ in the host (4.5–30.0%) may play a critical role in C. albicans phenotypic switching and sexual reproduction.¹⁷ GlcNAc, a component of bacterial cell wall and GI tract mucus, is another inducer of white to opaque switching.⁴⁴ In contrast to CO₂, GlcNAc promotes opaque phenotype primarily via the Ras1-cAMP/PKA pathway. The mutants of RAS1 and CYR1 genes showed remarkably reduced response to GlcNAc. Activating the pathway by ectopic expression of RAS1V13, a constitutively activated form of Ras1,²⁵ resulted in hypersensitivity to GlcNAc stimulation. Consistently, deletion of the high affinity phosphodiesterase gene PDE2 had the similar effect. The activated cAMP signal finally targets on the master regulator Wor1, which contains a conserved PKA phosphorylation site (Fig. 2B).^44,108 Interestingly, GlcNAc and CO₂ have synergistic effect on induction of opaque phenotype, suggesting that these two molecules function via distinct major pathways.⁴⁴ Other factors, including oxidative stress, UV light and adenine, have also been found to regulate white-opaque switching, although the underlying mechanisms remain to be investigated.^117-119

Relationship between white-opaque switching and biofilm formation

Like S. cerevisiae MATa and α cells, C. albicans MTLa/a and α/α cells secrete a or α pheromones, respectively, and carry corresponding α-factor or a-factor receptors.^120-123 Although only opaque cells undergo efficient mating, α-pheromone induces expression of some mating-related genes, including STE2, CEK1, CEK2 and SST2, in both opaque and white cells.¹²⁴ The activation of the mating regulatory pathway in white cells leads to increased cohesiveness, adhesiveness and induces biofilm development. Daniels et al. demonstrate that a minority of opaque cells signal the majority of the white cell population to form biofilms, which provide an environment facilitating opaque cell mating.¹²⁴ The white cell response to pheromone has been shown to be a general feature of MTL-homozygous C. albicans strains via a pheromone-based paracrine system.^125,126 Interestingly, pheromones from related Candida species can induce C. albicans white cell response, indicating the plasticity of this signal.¹²⁷ Recently, the Soll group has found that the Ste11-Hst7-Cek1/2-Tec1 mediated MAPK pathway primarily controls the pheromone induced biofilm development in MTL homozygous strains, while the Ras-cAMP/PKA pathway governs the conventional biofilm formed by MTLa/α strains.³⁹ Tec1 plays a central role in biofilm development of white cells, while Cph1 controls the mating response of opaque cells.¹²⁸ We have recently reported that the GATA type zinc finger transcription factor Gat2 plays an important role in biofilm formation, filamentation and virulence in C. albicans. We also demonstrate that Gat2 may function downstream of Tec1.¹²⁹

Common and distinct mechanisms of white-opaque switching and filamentous growth regulation

Opaque cells share some common features with hyphae, including a big vacuole and cell surface antigens.⁷ It has been suggested that opaque phenotype represents a newly evolved biological process since it is unique to C. albicans and its closely related species Candida dubliniensis.¹³⁰ A lot of filamentous growth regulators, such as Efg1, Efh1, Czf1, Hda1 and Tup1, also control white to opaque switching.^{21,52,105,112,116,117,131} Remarkably, the conserved Ras-cAMP/PKA pathway regulates CO₂ and GlcNAc induced filamentous growth as well as opaque formation in C. albicans.^4,13,43,44 Other environmental cues including stresses, hypoxic conditions and UV, which play a critical role in filamentous growth induction, have also been found to regulate white-opaque switching.^{100,118,119,132} In some aspects, opaque cells are characterized by several unique features.⁷ First, opaque cells bud like white yeast cells although the dynamics of actin localization follow the hypha pattern later in the budding growth.⁷ Second, the surface of opaque cells exhibits unique pimples, which are not observed in white cells and hyphae.⁷ Third, white-opaque switching is specifically regulated by some phase related genes,^101,113,114 including the master regulator Wor1 and the zinc finger transcription factor Wor2.¹¹² Wor1 and Wor2 are not required for filamentous growth.^108,112 Fourthly, opaque cells switch to white quickly at high temperature (37°C) in vitro, while the high temperature facilitates filamentous growth.⁶ Finally, the white to opaque transition is a mating prerequisite in C. albicans.¹⁰² Only a minor part of natural strains, which are homozygous at the MTL locus, can undergo white to opaque switching.¹¹¹ Therefore, common and distinct mechanisms are involved in the regulation of the two phenotypic transition systems.

Conclusion

High-frequency phenotypic transition is a defining feature of the pathogen fungus C. albicans. The ability of switching between different morphological forms in response to environmental cues is widely thought to be associated with virulence. In this review, the underlying mechanisms controlling yeast-filamentous growth transition and white-opaque switching, which represent two typical phenotypic switching systems in C. albicans, have been reviewed. The complex interplay between internal genetic elements and external environments determines the morphological fate of this organism. The genes or pathways involved in phenotypic transition are often required for virulence, indicating the important link between morphogenesis and pathogenesis in C. albicans. Interestingly, a variety of environmental inducers and genes, which regulate filamentous growth, also control white-opaque switching, suggesting that the two switching systems are evolutionarily-related. The phenotypic plasticity of C. albicans enables the organism to rapidly adapt to the changing host environments, while the biological significance of switching from white to opaque state to mate remains unclear. Given the importance of morphological changes in C. albicans, more detailed and more extensive investigations will be needed for deeper insights into understanding its pathogenicity.

Acknowledgments

The author would like to apologize to all researchers whose important work cannot be cited because of the space restrictions. The author would like to thank Drs Song Yi, Nidhi Sahni and the Huang lab members for their comments on the manuscript. Work in the Huang lab is supported by the grant of “100 Talent Program” from Chinese Academy of Sciences and the Chinese National Natural Science Foundation Grant 31170086 to G.H.