White Cells Facilitate Opposite- and Same-Sex Mating of Opaque Cells in Candida albicans

Modes of sexual reproduction in eukaryotic organisms are extremely diverse. The human fungal pathogen Candida albicans undergoes a phenotypic switch from the white to the opaque phase in order to become mating-competent. In this study, we report that functionally- and morphologically-differentiated white and opaque cells show a coordinated behavior during mating. Although white cells are mating-incompetent, they can produce sexual pheromones when treated with pheromones of the opposite mating type or by physically interacting with opaque cells of the opposite mating type. In a co-culture system, pheromones released by white cells induce opaque cells to form mating projections, and facilitate both opposite- and same-sex mating of opaque cells. Deletion of genes encoding the pheromone precursor proteins and inactivation of the pheromone response signaling pathway (Ste2-MAPK-Cph1) impair the promoting role of white cells (MTL a) in the sexual mating of opaque cells. White and opaque cells communicate via a paracrine pheromone signaling system, creating an environment conducive to sexual mating. This coordination between the two different cell types may be a trade-off strategy between sexual and asexual lifestyles in C. albicans.     

An expanded regulatory network temporally controls Candida albicans biofilm formation

Candida albicans biofilms are composed of highly adherent and densely arranged cells with properties distinct from those of free‐floating (planktonic) cells. These biofilms are a significant medical problem because they commonly form on implanted medical devices, are drug resistant and are difficult to remove. C. albicans biofilms are not static structures; rather they are dynamic and develop over time. Here we characterize gene expression in biofilms during their development, and by comparing them to multiple planktonic reference states, we identify patterns of gene expression relevant to biofilm formation. In particular, we document time‐dependent changes in genes involved in adhesion and metabolism, both of which are at the core of biofilm development. Additionally, we identify three new regulators of biofilm formation, Flo8, Gal4, and Rfx2, which play distinct roles during biofilm development over time. Flo8 is required for biofilm formation at all time points, and Gal4 and Rfx2 are needed for proper biofilm formation at intermediate time points.     

Defining pheromone-receptor signaling in Candida albicans and related asexual Candida species

Candida albicans is an important human fungal pathogen in which sexual reproduction is under the control of the novel white-opaque switch. Opaque cells are the mating-competent form, whereas white cells do not mate but can still respond to pheromones, resulting in biofilm formation. In this study, we first define the domains of the α-pheromone receptor Ste2 that are necessary for signaling in both white and opaque forms. Both cell states require the IC loop 3 (IC3) and the C-terminal tail of Ste2 for the cellular response, whereas the first IC loop (IC1) of Ste2 is dispensable for signaling. To also address pheromone-receptor interactions in related species, including apparently asexual Candida species, Ste2 orthologues were heterologously expressed in Candida albicans. Ste2 receptors from multiple Candida clade species were functional when expressed in C. albicans, whereas the Ste2 receptor of Candida lusitaniae was nonfunctional. Significantly, however, expression of a chimeric C. lusitaniae Ste2 receptor containing the C-terminal tail of Ste2 from C. albicans generated a productive response to C. lusitaniae pheromone. This system has allowed us to characterize pheromones from multiple Candida species and indicates that functional pheromone-receptor couples exist in fungal species that have yet to be shown to undergo sexual mating.     

Comparison of Switching and Biofilm Formation between MTL-Homozygous Strains of Candida albicans and Candida dubliniensis

Candida albicans and Candida dubliniensis are highly related species that share the same main developmental programs. In C. albicans, it has been demonstrated that the biofilms formed by strains heterozygous and homozygous at the mating type locus (MTL) differ functionally, but studies rarely identify the MTL configuration. This becomes a particular problem in studies of C. dubliniensis, given that one-third of natural strains are MTL homozygous. For that reason, we have analyzed MTL-homozygous strains of C. dubliniensis for their capacity to switch from white to opaque, the stability of the opaque phenotype, CO₂ induction of switching, pheromone induction of adhesion, the effects of minority opaque cells on biofilm thickness and dry weight, and biofilm architecture in comparison with C. albicans. Our results reveal that C. dubliniensis strains switch to opaque at lower average frequencies, exhibit a far lower level of opaque phase stability, are not stimulated to switch by high CO₂, exhibit more variability in biofilm architecture, and most notably, form mature biofilms composed predominately of pseudohyphae rather than true hyphae. Therefore, while several traits of MTL-homozygous strains of C. dubliniensis appear to be degenerating or have been lost, others, most notably several related to biofilm formation, have been conserved. Within this context, the possibility is considered that C. dubliniensis is transitioning from a hypha-dominated to a pseudohypha-dominated biofilm and that aspects of C. dubliniensis colonization may provide insights into the selective pressures that are involved.     

The White Cell Response to Pheromone Is a General Characteristic of Candida albicans Strains

For Candida albicans, evidence has suggested that the mating pheromones activate not only the mating response in mating-competent opaque cells but also a unique response in mating-incompetent white cells that includes increased cohesion and adhesion, enhanced biofilm formation, and expression of select mating-related and white cell-specific genes. On the basis of a recent microarray analysis comparing changes in the global expression patterns of white cells in two strains in response to α-pheromone, however, skepticism concerning the validity and generality of the white cell response has been voiced. Here, we present evidence that the response occurs in all tested media (Lee's, RPMI, SpiderM, yeast extract-peptone-dextrose, and a synthetic medium) and in all of the 27 tested strains, including a/a and α/α strains, derivatives of the common laboratory strain SC5314, and representatives from all of the five major clades. The white cell response to pheromone is therefore a general characteristic of MTL-homozygous strains of C. albicans.     

Self-induction of a/a or alpha/alpha biofilms in Candida albicans is a pheromone-based paracrine system requiring switching

Like MTL-heterozygous (a/α) cells, white MTL-homozygous (a/a or α/α) cells of Candida albicans, to which a minority of opaque cells of opposite mating type have been added, form thick, robust biofilms. The latter biofilms are uniquely stimulated by the pheromone released by opaque cells and are regulated by the mitogen-activated protein kinase signal transduction pathway. However, white MTL-homozygous cells, to which opaque cells of opposite mating type have not been added, form thinner biofilms. Mutant analyses reveal that these latter biofilms are self-induced. Self-induction of a/a biofilms requires expression of the α-receptor gene STE2 and the α-pheromone gene MFα, and self-induction of α/α biofilms requires expression of the a-receptor gene STE3 and the a-pheromone gene MFa. In both cases, deletion of WOR1, the master switch gene, blocks cells in the white phenotype and biofilm formation, indicating that self-induction depends upon low frequency switching from the white to opaque phenotype. These results suggest a self-induction scenario in which minority opaque a/a cells formed by switching secrete, in a mating-type-nonspecific fashion, α-pheromone, which stimulates biofilm formation through activation of the α-pheromone receptor of majority white a/a cells. A similar scenario is suggested for a white α/α cell population, in which minority opaque α/α cells secrete a-pheromone. This represents a paracrine system in which one cell type (opaque) signals a second highly related cell type (white) to undergo a complex response, in this case the formation of a unisexual white cell biofilm.     

Carbon source‐induced reprogramming of the cell wall proteome and secretome modulates the adherence and drug resistance of the fungal pathogen Candida albicans

The major fungal pathogen Candida albicans can occupy diverse microenvironments in its human host. During colonization of the gastrointestinal or urogenital tracts, mucosal surfaces, bloodstream, and internal organs, C. albicans thrives in niches that differ with respect to available nutrients and local environmental stresses. Although most studies are performed on glucose‐grown cells, changes in carbon source dramatically affect cell wall architecture, stress responses, and drug resistance. We show that growth on the physiologically relevant carboxylic acid, lactate, has a significant impact on the C. albicans cell wall proteome and secretome. The regulation of cell wall structural proteins (e.g. Cht1, Phr1, Phr2, Pir1) correlated with extensive cell wall remodeling in lactate‐grown cells and with their increased resistance to stresses and antifungal drugs, compared with glucose‐grown cells. Moreover, changes in other proteins (e.g. Als2, Gca1, Phr1, Sap9) correlated with the increased adherence and biofilm formation of lactate‐grown cells. We identified mating and pheromone‐regulated proteins that were exclusive to lactate‐grown cells (e.g. Op4, Pga31, Pry1, Scw4, Yps7) as well as mucosa‐specific and other niche‐specific factors such as Lip4, Pga4, Plb5, and Sap7. The analysis of the corresponding null mutants confirmed that many of these proteins contribute to C. albicans adherence, stress, and antifungal drug resistance. Therefore, the cell wall proteome and secretome display considerable plasticity in response to carbon source. This plasticity influences important fitness and virulence attributes known to modulate the behavior of C. albicans in different host microenvironments during infection.     

pH Regulates White-Opaque Switching and Sexual Mating in Candida albicans

As a successful commensal and pathogen of humans, Candida albicans encounters a wide range of environmental conditions. Among them, ambient pH, which changes frequently and affects many biological processes in this species, is an important factor, and the ability to adapt to pH changes is tightly linked with pathogenesis and morphogenesis. In this study, we report that pH has a profound effect on white-opaque switching and sexual mating in C. albicans. Acidic pH promotes white-to-opaque switching under certain culture conditions but represses sexual mating. The Rim101-mediated pH-sensing pathway is involved in the control of pH-regulated white-opaque switching and the mating response. Phr2 and Rim101 could play a major role in acidic pH-induced opaque cell formation. Despite the fact that the cyclic AMP (cAMP) signaling pathway does not play a major role in pH-regulated white-opaque switching and mating, white and opaque cells of the cyr1/cyr1 mutant, which is defective in the production of cAMP, showed distinct growth defects under acidic and alkaline conditions. We further discovered that acidic pH conditions repressed sexual mating due to the failure of activation of the Ste2-mediated α-pheromone response pathway in opaque a cells. The effects of pH changes on phenotypic switching and sexual mating could involve a balance of host adaptation and sexual reproduction in C. albicans.     

Opaque cells signal white cells to form biofilms in Candida albicans

Upon homozygosis from a/alpha to a/a or alpha/alpha, Candida albicans must still switch from the 'white' to 'opaque' phenotype to mate. It was, therefore, surprising to discover that pheromone selectively upregulated mating-associated genes in mating-incompetent white cells without causing G1 arrest or shmoo formation. White cells, like opaque cells, possess pheromone receptors, although their distribution and redistribution upon pheromone treatment differ between the two cell types. In speculating about the possible role of the white cell pheromone response, it is hypothesized that in overlapping white a/a and alpha/alpha populations in nature, rare opaque cells, through the release of pheromone, signal majority white cells of opposite mating type to form a biofilm that facilitates mating. In support of this hypothesis, it is demonstrated that pheromone induces cohesiveness between white cells, minority opaque cells increase two-fold the thickness of majority white cell biofilms, and majority white cell biofilms facilitate minority opaque cell chemotropism. These results reveal a novel form of communication between switch phenotypes, analogous to the inductive events during embryogenesis in higher eukaryotes.     

A Candida albicans-specific region of the α-pheromone receptor plays a selective role in the white cell pheromone response

Candida albicans strains homozygous at the mating type locus can switch from white to opaque, and must do so to mate. Opaque cells then secrete mating pheromones that stimulate opaque cells of opposite mating type to undergo mating. These same pheromones stimulate mating-incompetent white cells to become cohesive and adhesive, and enhance white cell biofilm development, a pathogenic trait. Stimulation is mediated through the same receptor, G protein complex and mitogen-activated protein kinase pathway. Here we present evidence that a C. albicans -specific 55-amino-acid region of the first intracellular loop, IC1, of the α-pheromone receptor Ste2p, is required for the α-pheromone response of white cells, but not that of opaque cells. This represents a unique regulatory configuration in which activation of a common pathway by the same ligand, the same receptor and the same signal transduction pathway is dependent on a unique region of an intracellular loop of the common receptor in one of the two responding phenotypes.     

A constitutively active GPCR governs morphogenic transitions in Cryptococcus neoformans

Sex in fungi is driven by peptide pheromones sensed through seven‐transmembrane pheromone receptors. In Cryptococcus neoformans, sexual reproduction occurs through an outcrossing/heterothallic a ‐ sexual cycle or an inbreeding/homothallic – unisexual mating process. Pheromone receptors encoded by the mating‐type locus ( MAT ) mediate reciprocal pheromone sensing during opposite‐sex mating and contribute to but are not essential for unisexual mating. A pheromone receptor‐like gene, CPR2 , was discovered that is not encoded by MAT and whose expression is induced during a ‐ mating. cpr2 mutants are fertile but have a fusion defect and produce abnormal hyphal structures, whereas CPR2 overexpression elicits unisexual reproduction. When heterologously expressed in Saccharomyces cerevisiae , Cpr2 activates pheromone responses in the absence of any ligand. This constitutive activity results from an unconventional residue, Leu²²², in place of a conserved proline in transmembrane domain six; a Cpr2^L222P mutant is no longer constitutively active. Cpr2 engages the same G‐protein activated signalling cascade as the Ste3 a /α pheromone receptors, and thereby competes for pathway activation. This study established a new paradigm in which a naturally occurring constitutively active G protein‐coupled receptor governs morphogenesis in fungi.