A histone deacetylation inhibitor and mutant promote colony-type switching of the human pathogen Candida albicans

Most strains of Candida albicans undergo high frequency phenotypic switching. Strain WO-1 undergoes the white-opaque transition, which involves changes in colony and cellular morphology, gene expression, and virulence. We have hypothesized that the switch event involves heritable changes in chromatin structure. To test this hypothesis, we transiently exposed cells to the histone deacetylase inhibitor trichostatin-A (TSA). Treatment promoted a dramatic increase in the frequency of switching from white to opaque, but not opaque to white. Targeted deletion of HDA1, which encodes a deacetylase sensitive to TSA, had the same selective effect. These results support the model that the acetylation of histones plays a selective role in regulating the switching process.     

A comparison of high frequency switching in the yeast Candida albicans and the slime mold Dictyostelium discoideum

Recently, high frequency switching systems have been identified in the infectious yeast Candida albicans and the cellular slime mold Dictyostelium discoideum. In C. albicans, cells can switch at spontaneous frequencies as high as 10(-2) between seven general colony morphologies in the case of strain 3153A or between two major phenotypes in the white-opaque transition in strain WO-1. In the latter system, dramatic changes occur in cellular phenotype as well. In D. discoideum, cells can switch at spontaneous frequencies of roughly 10(-2) between a number of colony phenotypes which include alterations in developmental timing, blocks at particular morphogenetic stages, morphological aberrations, and aggregation-minus. In the C. albicans and D. discoideum switching systems, the following characteristics are shared: 1) a limited number of switch phenotypes; 2) heritability; 3) high frequency reversibility; 4) low and high frequency modes of switching; and 5) ultraviolet (UV) stimulation of switching of cells in a low frequency mode of switching.     

Unique phenotype of opaque cells in the white-opaque transition of Candida albicans.

Select strains of Candida albicans switch reversibly and at extremely high frequency between a white and an opaque colony-forming phenotype, which has been referred to as the white-opaque transition. Cells in the white phase exhibit a cellular phenotype indistinguishable from that of most standard strains of C. albicans, but cells in the opaque phase exhibit an unusually large, elongate cellular shape. In comparing the white and opaque cellular phenotypes, the following findings are demonstrated. (i) The surface of the cell wall of maturing opaque cells when viewed by scanning electron microscopy exhibits a unique pimpled, or punctate, pattern not observed in white cells or standard strains of C. albicans. (ii) The dynamics of actin localization which accompanies opaque-cell growth first follows the pattern of budding cells during early opaque-bud growth and then the pattern of hypha-forming cells during late opaque-bud growth. (iii) A hypha-specific cell surface antigen is also expressed on the surface of opaque budding cells. (iv) An opaque-specific surface antigen is distributed in a punctate pattern.     

Analysis of phase-specific gene expression at the single-cell level in the white-opaque switching system of Candida albicans

The opportunistic fungal pathogen Candida albicans can switch spontaneously and reversibly between different cell forms, a capacity that may enhance adaptation to different host niches and evasion of host defense mechanisms. Phenotypic switching has been studied intensively for the white-opaque switching system of strain WO-1. To facilitate the molecular analysis of phenotypic switching, we have constructed homozygous ura3 mutants from strain WO-1 by targeted gene deletion. The two URA3 alleles were sequentially inactivated using the MPA(R)-flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid (MPA) resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. To investigate a possible cell type-independent switching in the expression of individual phase-specific genes, two different reporter genes that allowed the analysis of gene expression at the single-cell level were integrated into the genome, using URA3 as a selection marker. Fluorescence microscopic analysis of cells in which a GFP reporter gene was placed under the control of phase-specific promoters demonstrated that the opaque-phase-specific SAP1 gene was detectably expressed only in opaque cells and that the white-phase-specific WH11 gene was detectably expressed only in white cells. When MPA(R) was used as a reporter gene, it conferred an MPA-resistant phenotype on opaque but not white cells in strains expressing it from the SAP1 promoter, which was monitored at the level of single cells by a significantly enlarged size of the corresponding colonies on MPA-containing indicator plates. Similarly, white but not opaque cells became MPA resistant when MPA(R) was placed under the control of the WH11 promoter. The analysis of these reporter strains showed that cell type-independent phase variation in the expression of the SAP1 and WH11 genes did not occur at a detectable frequency. The expression of these phase-specific genes of C. albicans in vitro, therefore, is tightly linked to the cell type.     

Genetics of the white-opaque transition in Candida albicans: demonstration of switching recessivity and mapping of switching genes.

Spheroplast fusion has been used to analyze the genetics of the reversible phenotypic transition, white-opaque, in Candida albicans WO-1. This transition involves changes in cell shape, permeability, and colony morphology. Fusion of switching with nonswitching cells gave nonswitching fusants, suggesting that the white-opaque phenotype is recessive. Chromosome loss induced by heat shock gave segregants of the fusants which were able to undergo the transition, indicating that the repressor function is genetically defined and probably limited to one or two chromosomes. Chromosomes R, 1, 3, 4, and 7 were eliminated as unique sites for the repressor, leaving 2, 5, and 6 as possible locations. When a ura3 (chromosome 3) nonswitching strain was fused with a switching strain, all ura3 segregants induced by heat shock were incapable of the phenotypic transition. Therefore, some or all of the genes (called SWI genes) essential for the transition are located on chromosome 3. UV irradiation-induced recombination did give rise to Ura- switching progeny, showing that the failure to switch was not due to a side effect of the pyrimidine requirement. The failure to isolate normally switching ura3 progeny generated by UV irradiation suggests a close linkage between the two genes.     

Candida albicans MTLalpha tup1Delta mutants can reversibly switch to mating-competent, filamentous growth forms

Candida albicans strains that are homozygous at the mating type locus (MTLa or MTLalpha) can spontaneously switch from the normal round-to-oval yeast cell morphology to an elongated, so-called opaque cell form that can mate with opaque cells of the opposite mating type. In response to environmental signals, C. albicans also undergoes a transition from yeast to filamentous growth, which is negatively regulated by the general repressor Tup1p. Therefore, C. albicans mutants in which the TUP1 gene is inactivated grow constitutively in the filamentous form. We found that tup1Delta mutants of the MTLalpha strain WO-1 are still able to undergo phenotypic switching. Although the mutants had lost the capacity to grow in the normal yeast (white) or opaque forms, they could still reversibly switch between four different cell and colony phenotypes (designated as fuzzy, frizzy, wrinkled and smooth) at a frequency of about 10(-3) to 10(-4). Deletion of TUP1 resulted in deregulated expression of phase-specific genes. While the white-specific WH11 gene was constitutively expressed in all four cell types, the opaque-specific SAP1 gene remained repressed and the opaque-specific OP4 gene was weakly induced in all phase variants. In spite of the loss of white- and opaque-specific cell morphology and gene expression, the tup1Delta mutants retained an important characteristic of their wild-type parent, the ability to switch to a mating-competent form. The three filamentous phase variants (fuzzy, frizzy and wrinkled) all were able to mate and produce recombinant progeny with opaque cells of an MTLa strain at frequencies that were somewhat lower than those of normal opaque cells, whereas the smooth phase variant was unable to do so. Therefore, although deletion of TUP1 in C. albicans MTLalpha cells affects cellular morphology and gene expression patterns, the mutants can still reversibly switch between mating-competent and -incompetent cell types and mate with a partner of the opposite mating type.     

N-acetylglucosamine induces white-to-opaque switching and mating in Candida tropicalis, providing new insights into adaptation and fungal sexual evolution

Pathogenic fungi are capable of switching between different phenotypes, each of which has a different biological advantage. In the most prevalent human fungal pathogen, Candida albicans, phenotypic transitions not only improve its adaptation to a continuously changing host microenvironment but also regulate sexual mating. In this report, we show that Candida tropicalis, another important human opportunistic pathogen, undergoes reversible and heritable phenotypic switching, referred to as the "white-opaque" transition. Here we show that N-acetylglucosamine (GlcNAc), an inducer of white-to-opaque switching in C. albicans, promotes opaque-cell formation and mating and also inhibits filamentation in a number of natural C. tropicalis strains. Our results suggest that host chemical signals may facilitate this phenotypic switching and mating of C. tropicalis, which had been previously thought to reproduce asexually. Overexpression of the C. tropicalis WOR1 gene in C. albicans induces opaque-cell formation. Additionally, an intermediate phase between white and opaque was observed in C. tropicalis, indicating that the switching could be tristable.     

The white-phase-specific gene<Emphasis Type="Italic"> WH11</Emphasis> is not required for white-opaque switching in<Emphasis Type="Italic"> Candida albicans</Emphasis>

Phenotypic switching between white and opaque cells is important for adaptation to different host environments and for mating in the opportunistic fungal pathogen Candida albicans. Genes that are specifically activated in one of the two cell types are likely to be important for their phenotypic characteristics. The WH11 gene is a white-phase-specific gene that has been suggested to be involved in the maintenance of the white-phase phenotype. To elucidate the role of WH11 in white-opaque switching, we constructed mutants of the C. albicans strain WO-1 in which the WH11 gene was deleted. The wh11 mutants were still able to form both white and opaque cells whose cellular and colony phenotypes were indistinguishable from those of the wild type. Deletion of WH11 also did not affect the activation and deactivation of the white-phase-specific WH11 promoter and the opaque-phase-specific OP4 and SAP1 promoters in the appropriate cell type. Finally, switching from the white to the opaque phase and vice versa occurred with the same frequency in wild-type and wh11 mutants. Therefore, the WH11 gene is not required for phenotypic switching, and its protein product seems to have other roles in white cells, which are dispensable after the switch to the opaque phase.Communicated by E. Cerdá-Olmedo     

White-Opaque Switching of Candida albicans Allows Immune Evasion in an Environment-Dependent Fashion

Candida albicans strains that are homozygous at the mating type locus can spontaneously and reversibly switch from the normal yeast morphology (white) to an elongated cell type (opaque), which is the mating-competent form of the fungus. White-opaque switching also influences the ability of C. albicans to colonize and proliferate in specific host niches and its susceptibility to host defense mechanisms. We used live imaging to observe the interaction of white and opaque cells with host phagocytic cells. For this purpose, we generated derivatives of the switching-competent strain WO-1 that express green fluorescent protein from a white-specific promoter and red fluorescent protein from an opaque-specific promoter or vice versa. When mixed populations of these differentially labeled white and opaque cells were incubated with human polymorphonuclear neutrophils (PMNs) on a glass slide, the neutrophils selectively phagocytosed and killed white cells, despite frequent physical interaction with opaque cells. White cells were attacked only after they started to form a germ tube, indicating that the suppression of filamentation in opaque cells saved them from recognition by the PMNs. In contrast to neutrophils, dendritic cells internalized white as well as opaque cells. However, when embedded in a collagen matrix, the PMNs also phagocytosed both white and opaque cells with similar efficiency. These results suggest that, depending on the environment, white-opaque switching enables C. albicans to escape from specific host defense mechanisms.     

Bidirectional stimulation of the white-opaque transition of Candida albicans by ultraviolet irradiation

Most strains of Candida albicans are capable of switching spontaneously and at high frequency between a number of phenotypes distinguishable by colony morphology. The switching frequency of Candida albicans strain WO-1 between two predominant phenotypes, 'white' and 'opaque', and a minor phenotype, 'fuzzy', increased dramatically with low doses of ultraviolet irradiation that killed less than 20% of the population. The ultraviolet irradiation effect continued to be expressed over many generations as evidenced by stimulated sectoring. Ultraviolet irradiation stimulated switching in both the white-to-opaque and opaque-to-white direction, suggesting that a common mechanism functions in both directions.     

TOS9 regulates white-opaque switching in Candida albicans

In Candida albicans, the a1-alpha2 complex represses white-opaque switching, as well as mating. Based upon the assumption that the a1-alpha2 corepressor complex binds to the gene that regulates white-opaque switching, a chromatinimmunoprecipitation-microarray analysis strategy was used to identify 52 genes that bound to the complex. One of these genes, TOS9, exhibited an expression pattern consistent with a "master switch gene." TOS9 was only expressed in opaque cells, and its gene product, Tos9p, localized to the nucleus. Deletion of the gene blocked cells in the white phase, misexpression in the white phase caused stable mass conversion of cells to the opaque state, and misexpression blocked temperature-induced mass conversion from the opaque state to the white state. A model was developed for the regulation of spontaneous switching between the opaque state and the white state that includes stochastic changes of Tos9p levels above and below a threshold that induce changes in the chromatin state of an as-yet-unidentified switching locus. TOS9 has also been referred to as EAP2 and WOR1.     

The closely related species Candida albicans and Candida dubliniensis can mate

Because Candida dubliniensis is closely related to Candida albicans, we tested whether it underwent white-opaque switching and mating and whether white-opaque switching depended on MTL homozygosity and mating depended on switching, as they do in C. albicans. We also tested whether C. dubliniensis could mate with C. albicans. Sequencing revealed that the MTLalpha locus of C. dubliniensis was highly similar to that of C. albicans. Hybridization with the MTLa1, MTLa2, MTLalpha1, and MTLalpha2 open reading frames of C. albicans further revealed that, as in C. albicans, natural strains of C. dubliniensis exist as a/alpha, a/a, and alpha/alpha, but the proportion of MTL homozygotes is 33%, 10 times the frequency of natural C. albicans strains. C. dubliniensis underwent white-opaque switching, and, as in C. albicans, the switching was dependent on MTL homozygosis. C. dubliniensis a/a and alpha/alpha cells also mated, and, as in C. albicans, mating was dependent on a switch from white to opaque. However, white-opaque switching occurred at unusually high frequencies, opaque cell growth was frequently aberrant, and white-opaque switching in many strains was camouflaged by an additional switching system. Mating of C. dubliniensis was far less frequent in suspension cultures, due to the absence of mating-dependent clumping. Mating did occur, however, at higher frequencies on agar or on the skin of newborn mice. The increases in MTL homozygosity, the increase in switching frequencies, the decrease in the quality of switching, and the decrease in mating efficiency all reflected a general deterioration in the regulation of developmental processes, very probably due to the very high frequency of recombination and genomic reorganization characteristic of C. dubliniensis. Finally, interspecies mating readily occurred between opaque C. dubliniensis and C. albicans strains of opposite mating type in suspension, on agar, and on mouse skin. Remarkably, the efficiency of interspecies mating was higher than intraspecies C. dubliniensis mating, and interspecies karyogamy occurred readily with apparently the same sequence of nuclear migration, fusion, and division steps observed during intraspecies C. albicans and C. dubliniensis mating and Saccharomyces cerevisiae mating.     

近平滑念珠菌的形态转换观察

目的观察近平滑念珠菌在不同培养基的形态转换现象,以及温度对其形态转换的影响。方法收集近平滑念珠菌正常人皮肤携带株及临床致病株和标准株,接种于改良Lee培养基和含桃红B的YPD培养基,观察其不同形态转换,以及温度变化对光滑(W)与皱褶(O)形态转换的影响。结果近平滑念珠菌在Lee培养基和含桃红B的YPD培养基上,均可以出现多种形态以及一定频率W-O转换现象。在观察W向O形态转换过程中发现,与25℃培养温度相比,37℃条件下光滑菌落形态占更多的比例。结论近平滑念珠菌体外培养时存在形态转换及W-O转换现象,且于37℃时更易保持光滑形态。含桃红B的YPD培养基也可以用于基本的W-O形态转换观察。     

Opaque-white phenotype transition: a programmed morphological transition in Candida albicans.

This paper reports that the opaque and white phenotypes of Candida albicans constitute a true high-frequency reversible transition system. The rDNA restriction fragment and orthogonal field alternating gel electrophoresis profiles of opaque and white phenotypes are indistinguishable, and a genetic marker introduced into a white strain is present in all opaque derivatives of this strain. Opaque and white derivatives appear markedly different on a bismuth indicator medium and differ in a number of other respects. We have used bismuth medium to examine the spontaneous and temperature-induced frequencies of transition from opaque to white. The temperature-induced transition from opaque to white does not occur when opaque cells are held in water.