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In the screening of natural plant extracts for antifungal activity, assessment of their effects on the growth of cells in suspension or in the wells of microtiter plates is expedient. However, microorganisms, including Candida albicans , grow in nature as biofilms, which are organized cellular communities with a complex architecture capable of conditioning their microenvironment, communicating, and excluding low- and high-molecular-weight molecules and white blood cells. Here, a confocal laser scanning microscopy (CLSM) protocol for testing the effects of large numbers of agents on biofilm development is described. The protocol assessed nine parameters from a single z-stack series of CLSM scans for each individual biofilm analyzed. The parameters included adhesion, thickness, formation of a basal yeast cell polylayer, hypha formation, the vertical orientation of hyphae, the hyphal bend point, pseudohypha formation, calcofluor white staining of the extracellular matrix (ECM), and human white blood cell impenetrability. The protocol was applied first to five plant extracts and derivative compounds and then to a collection of 88 previously untested plant extracts. They were found to cause a variety of phenotypic profiles, as was the case for 64 of the 88 extracts (73%). Half of the 46 extracts that did not affect biofilm thickness affected other biofilm parameters. Correlations between specific effects were revealed. The protocol will be useful not only in the screening of chemical libraries but also in the analysis of compounds with known effects and mutations.

Candida albicans forms two types of biofilm, depending upon the configuration of the mating type locus. Although architecturally similar, a/ α biofilms are impermeable, impenetrable, and drug resistant, whereas a/a and α/α biofilms lack these traits. The difference appears to be the result of an alternative matrix. Overexpression in a/a cells of BCR1 , a master regulator of the a/ α matrix, conferred impermeability, impenetrability, and drug resistance to a/a biofilms. Deletion of BCR1 in a/ α cells resulted in the loss of these a/ α-specific biofilm traits. Using BCR1 overexpression in a/a cells, we screened 107 genes of interest and identified 8 that were upregulated by Bcr1. When each was overexpressed in a/a biofilms, the three a/ α traits were partially conferred, and when each was deleted in a/ α cells, the traits were partially lost. Five of the eight genes have been implicated in iron homeostasis, and six encode proteins that are either in the wall or plasma membrane or secreted. All six possess sites for O-linked and N-linked glycosylation that, like glycosylphosphatidylinositol (GPI) anchors, can cross-link to the wall and matrix, suggesting that they may exert a structural role in conferring impermeability, impenetrability, and drug resistance, in addition to their physiological functions. The fact that in a screen of 107 genes, all 8 of the Bcr1-upregulated genes identified play a role in impermeability, impenetrability, and drug resistance suggests that the formation of the a/ α matrix is highly complex and involves a larger number of genes than the initial ones identified here.

Summary Efg1 is a central transcriptional regulator of morphogenesis and metabolism in Candida albicans. In vivo genome-wide ChIP chip and in vitro footprint analyses revealed the Efg1 recognition sequence (EGR-box) TATGCATA in the yeast growth form of this human fungal pathogen. Upstream regions of EFG1 and genes encoding transcriptional regulators of hyphal growth including TCC1, CZF1, TEC1, DEF1 and NRG1 contained EGR- and/or EGR-like boxes. Unexpectedly, after brief hyphal induction the genome-wide Efg1 binding pattern was completely altered and new binding sites of yet unknown specificity had appeared. Hyphal induction abolished Efg1 accumulation on EFG1 and TCC1 promoters and led to rapid decline of both transcripts, although the Efg1 protein persisted in cells. While EFG1 promoter activity in the yeast growth form did not depend on bound Efg1, its downregulation under hyphal induction depended on the presence of Efg1 and the protein kinase A isoform Tpk2. Deletion analyses of the EFG1 upstream region revealed that none of its resident EGR-boxes is uniquely responsible for EFG1 promoter downregulation. These results suggest different binding specificities of Efg1 in yeast growth and in hyphal induction and suggest a brief time window following hyphal induction, in which Efg1 exerts its repressive effect on target promoters.

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 MF α, and self-induction of α/α biofilms requires expression of the a -receptor gene STE3 and the a -pheromone gene MF a . 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.

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 alpha-pheromone receptor Ste2p, is required for the alpha-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.

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes. published

Candida glabrata undergoes reversible, high-frequency core switching between phenotypes that include dark brown (DB), light brown (LB) and white (Wh). These phenotypes in turn can switch to the irregular wrinkle (IWr) phenotype. Natural isolates, however, express predominantly the DB phenotype, leading to the hypothesis that it has a colonization advantage over the other switch phenotypes. Using the mouse model of systemic infection, results are presented which support this hypothesis. DB has an advantage over other switch phenotypes in colonizing the two major target organs in the mouse model, the spleen and liver. A time-course study reveals that colonization of the major target organs occurs very rapidly (within 2 h) after host injection, and that the DB advantage for spleen and liver colonization is immediate. The DB advantage is maintained during clearing from spleen, liver and kidneys, and during delayed transient brain colonization. These results demonstrate that DB has a colonization advantage over other switch phenotypes, and that the switch phenotype expressed by a colonizing population therefore plays a fundamental role in virulence. It is therefore essential that switching be considered in both in vivo and in vitro studies of C. glabrata virulence.

In the mouse model for systemic infection, natural a/alpha strains of C. albicans are more virulent and more competitive than their spontaneous MTL-homozygous offspring, which arise primarily by loss of one chromosome 5 homologue followed by duplication of the retained homologue (uniparental disomy). Deletion of either the a or alpha copy of the MTL locus of natural a/alpha strains results in a small decrease in virulence, and a small decrease in competitiveness. Loss of the heterozygosity of non-MTL genes along chromosome 5, however, results in larger decreases in virulence and competitiveness. Natural MTL-homozygous strains are on average less virulent than natural MTL-heterozygous strains and arise by multiple mitotic cross-overs along chromosome 5 outside of the MTL region. These results are consistent with the hypothesis that a competitive advantage of natural a/alpha strains over MTL-homozygous offspring maintains the mating system of C. albicans.

MTL -homozygous ( a / a or α/α) white cells form a complex sexual biofilm that exhibits the same architecture as that of MTL -heterozygous ( a /α) pathogenic biofilms. However, the former is regulated by the mitogen-activated protein (MAP) kinase pathway, while the latter is regulated by the Ras1/cyclic AMP (cAMP) pathway. We previously demonstrated that in the formation of an MTL -homozygous, mature (48 h) sexual biofilm in RPMI 1640 medium, the MAP kinase pathway targets Tec1 rather than Cph1, the latter of which is the target of the same pathway, but for the opaque cell mating response. Here we continued our analysis of the role of Tec1 by comparing the effects of deleting TEC1 on initial adhesion to silicone elastomer, high-resolution confocal microscopy assessments of the stages and cellular phenotypes during the 48 h of biofilm development, human white cell penetration, and biofilm fragility. We show that although Tec1 plays only a minor role in initial adhesion to the silicone elastomer, it does play a major role in the growth of the basal yeast cell polylayer, vertical extension of hyphae and matrix deposition in the upper portion of the biofilm, final biofilm thickness, penetrability of human white blood cells, and final biofilm integrity (i.e., resistance to fluid flow). These results provide a more detailed description of normal biofilm development and architecture and confirm the central role played by the transcription factor Tec1 in the biofilm model employed here.

In Candida albicans , the a 1-α2 complex represses white-opaque switching, as well as mating. Based upon the assumption that the a 1-α2 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 Shwachman-Bodian-Diamond syndrome (SBDS) is an autosomal disorder with multisystem defects. The Shwachman-Bodian-Diamond syndrome gene (SBDS), which contains mutations in a majority of SBDS patients, encodes a protein of unknown function, although it has been strongly implicated in RNA metabolism. There is also some evidence that it interacts with molecules that regulate cytoskeletal organization. Recently, it has been demonstrated by computer-assisted methods that the single behavioral defect of polymorphonuclear leukocytes (PMNs) of SBDS patients is the incapacity to orient correctly in a spatial gradient of chemoattractant. We considered using the social amoeba Dictyostelium discoideum, a model for PMN chemotaxis, an excellent system for elucidating the function of the SBDS protein. We first identified the homolog of SBDS in D. discoideum and found that the amino acids that are altered in human disease were conserved. Given that several proteins involved in chemotactic orientation localize to the pseudopods of cells undergoing chemotaxis, we tested whether the SBDS gene product did the same. We produced an SBDS-GFP chimeric in-frame fusion gene, and generated transformants either with multiple ectopic insertions of the fusion gene or multiple copies of a non-integrated plasmid carrying the fusion gene. In both cases, the SBDS-GFP protein was dispersed equally through the cytoplasm and pseudopods of cells migrating in buffer. However, we observed differential enrichment of SBDS in the pseudopods of cells treated with the chemoattractant cAMP, suggesting that the SBDS protein may play a role in chemotaxis. In light of these results, we discuss how SBDS might function during chemotaxis.

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 MTL α locus of C. dubliniensis was highly similar to that of C. albicans . Hybridization with the MTL a 1, MTL a 2, MTL α 1 , and MTL α 2 open reading frames of C. albicans further revealed that, as in C. albicans , natural strains of C. dubliniensis exist as a /α, a / a , and α/α, 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 α/α 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.

Candida glabrata switches spontaneously at high frequency among the following four graded phenotypes discriminated on agar containing 1 mM CuSO 4 : white, light brown, dark brown (DB), and very dark brown. C. glabrata also contains three mating type loci with a configuration similar to that of the Saccharomyces cerevisiae mating type cassette system, suggesting it may also undergo cassette switching at the expression locus MTL1 . To analyze both reversible, high-frequency phenotypic switching and mating type switching at sites of colonization, primary samples from the oral cavities and vaginal canals of three patients suffering from C. glabrata vaginitis were clonally plated on agar containing CuSO 4 . It was demonstrated that (i) in each vaginitis patient, there was only one colonizing strain; (ii) an individual could have vaginal colonization without oral colonization; (iii) phenotypic switching occurred at sites of colonization; (iv) the DB phenotype predominated at the site of infection in all three patients; (v) genetically unrelated strains switched in similar, but not identical, fashions and caused vaginal infection; (vi) different switch phenotypes of the same strain could simultaneously dominate different body locations in the same host; (vii) pathogenesis could be caused by cells in different mating type classes; and (viii) mating type switching demonstrated at both the genetic and transcription levels occurred in one host.

EFG1, which encodes a trans-acting factor, is expressed as a more abundant 3.2 kb transcript in the white phase and as a less abundant 2.2 kb transcript in the opaque phase of the white-opaque transition in Candida albicans. To understand how alternative phase-specific mRNAs are transcribed from the same gene locus, the 2320 bp upstream region of the gene was functionally characterized by analysing the -activity of deletion derivatives in a luciferase-based reporter system. The white phase-specific promoter contained three discrete sequences involved in white phase-specific activation, between -2022 and -1809 bp (AR1), between -1809 and -1727 bp (AR2) and between -922 and -840 bp (AR3). A higher resolution deletion and mutation analysis of AR2 revealed two regions between -1809 and -1787 bp and between -1764 and -1728 bp that are responsible for AR2 activation. Targeting of promoter constructs to the ectopic ADE2 genomic site and the 3' end of the EFG1 genomic site revealed a positional requirement for white phase-regulated activation specific for the AR2 region of the promoter. Gel mobility shift assays using AR2 revealed a white phase-specific activation complex. No discrete activation sequences were identified in the overlapping promoter of the opaque phase-specific EFG1 transcript. The strength of opaque phase activation was directly proportional to the length of the promoter. Northern analysis excluded the possibility of an opaque phase-specific repressor. These results demonstrate overlapping promoters for white and opaque phase-specific expression of the gene for the transcription factor Efg1, with distinctly different mechanisms of phase-specific activation.

Five histone deacetylase genes (HDA1, RPD3, HOS1, HOS2,andHOS3) have been cloned fromCandida albicansand characterized. Sequence analysis and comparison with 17 additional deacetylases resulted in a phylogenetic tree composed of three major groups. Transcription of the deacetylasesHDA1andRPD3is down-regulated in the opaque phase of the white-opaque transition in strain WO-1.HOS3is selectively transcribed as a 2.5-kb transcript in the white phase and as a less-abundant 2.3-kb transcript in the opaque phase.HDA1andRPD3were independently deleted in strain WO-1, and both switching between the white and opaque phases and the downstream regulation of phase-specific genes were analyzed. Deletion ofHDA1resulted in an increase in the frequency of switching from the white phase to the opaque phase, but had no effect on the frequency of switching from the opaque phase to the white phase. Deletion ofRPD3resulted in an increase in the frequency of switching in both directions. Deletion ofHDA1resulted in reduced white-phase-specific expression of theEFG13.2-kb transcript,but had no significant effect on white-phase-specific expression ofWH11or opaque-phase-specific expression ofOP4, SAP1,andSAP3.Deletion ofRPD3resulted in reduced opaque-phase-specific expression ofOP4, SAP1,andSAP3and a slight reduction of white-phase-specific expression ofWH11and 3.2-kbEFG1. Deletion of neitherHDA1norRPD3affected the high level of white-phase expression and the low level of opaque-phase expression of the MADS box protein geneMCM1, which has been implicated in the regulation of opaque-phase-specific gene expression. In addition, there was no effect on the phase-regulated levels of expression of the other deacetylase genes. These results demonstrate that the two deacetylase genesHDA1andRPD3play distinct roles in the suppression of switching, that the two play distinct and selective roles in the regulation of phase-specific genes, and that the deacetylases are in turn regulated by switching.

The 11 kb complex DNA fingerprinting probe Ca3 is effective both in cluster analyses of Candida albicans isolates and in identifying microevolutionary changes in the size of hypervariable genomic fragments. A 2.6 kb EcoRI fragment of Ca3, the C fragment, retains the capacity to identify these microevolutionary changes, and when the C fragment is cleaved with SacI, the capacity is retained exclusively by a 1 kb subfragment, C1, which contains a partial RPS repeat element. The microevolutionary changes identified by Ca3, therefore, may involve reorganization of RPS elements dispersed throughout the genome. To test this possibility, hypervariable fragments from several strains of C. albicans were sequenced and compared. The results demonstrate that the microevolutionary changes identified by Ca3 are due to the insertion and deletion of full-length tandem RPS elements at specific genomic sites dispersed throughout the C. albicans genome. The RPS elements at these dispersed sites are bordered by the same upstream and downstream sequences. The frequency of recombination was estimated to be one recombination per 1000 cell divisions by following RPS reorganization in vitro. The results are inconsistent with unequal recombination between homologous or heterologous chromosomes, but consistent with intrachromosomal recombination. Two alternative models of intrachromosomal recombination are proposed: unequal sister-chromatid exchange and slipped misalignment at the replication fork.

The majority of strains of Candida albicans can switch frequently and reversibly between two or more general phenotypes, a process now considered a putative virulence factor in this species. Candida albicans WO-1 switches frequently and reversibly between a white and an opaque phase, and this phenotypic transition is accompanied by the differential expression of white-phase-specific and opaque-phase-specific genes. In the opaque phase, cells differentially express the gene OP4 , which encodes a putative protein 402 amino acids in length that contains a highly hydrophobic amino-terminal sequence and a carboxy-terminal sequence with a pI of 10.73. A series of deletion constructs fused to the Renilla reniformis luciferase was used to functionally characterize the OP4 promoter in order to investigate how this gene is differentially expressed in the white-opaque transition. An extremely strong 17-bp transcription activation sequence was identified between −422 and −404 bp. This sequence contained a MADS box consensus binding site, most closely related to the Mcm1 binding site of Saccharomyces cerevisiae . A number of point mutations generated in the MADS box consensus binding site as well as a complete deletion of the consensus site further demonstrated that it was essential for the activation of OP4 transcription in the opaque phase. Gel mobility shift assays with the 17-bp activation sequence identified three specific complexes which formed with both white- and opaque-phase cell extracts. Competition with a putative MADS box consensus binding site from the promoter of the coordinately regulated opaque-phase-specific gene PEP1 (SAP1) and the human MADS box consensus binding site for serum response factor demonstrated that one of the three complexes formed was specific to the OP4 sequence.

SUMMARY: Using degenerate primers of highly conserved regions of two-component response regulators for PCR amplification, a two-component response regulator was cloned from Candida albicans that is homologous to nik-l+ of Neurospora crassa. This two-component hybrid kinase, CaNIKl, also shows features of bacterial two-component response regulators, including a putative unorthodox second histidine kinase motif at the carboxy-terminal end. CaNIKl was expressed at low levels in both the white and opaque switch phenotypes and in the bud and hyphal growth forms of C. albicans strain WO-1, but in both developmental programmes, the level of transcript was modulated (levels were higher in opaque cells and in hyphae). Partial deletion of both CaNIKl alleles, by which the histidine autokinase- and ATP-binding domains were removed, did not inhibit either high-frequency phenotypic switching or the bud-hypha transition in high salt concentrations, but in both cases the efficiency of the developmental process was reduced.

In the past few years, highly sensitive gene reporters have been developed for the infectious fungi including gene reporters with altered codon usage. The tools are, therefore, now at hand for functionally characterizing the promoters of genes regulated by the bud—hypha transition, high frequency switching and cues from the cellular environment.

Candida albicans strain WO-1 undergoes two developmental programs, the bud-hypha transition and high-frequency phenotypic switching in the form of the white-opaque transition. The WH11 gene is expressed in the white budding phase but is inactive in the white hyphal phase and in the opaque budding phase. WH11 expression, therefore, is regulated in the two developmental programs. Through fusions between deletion derivatives of the WH11 promoter and the newly developed Renilla reniformis luciferase, the WH11 promoter has been characterized in the two developmental programs. Three transcription activation sequences, two strong and one weak, are necessary for the full expression of WH11 in the white budding phase, but no negative regulatory sequences were revealed as playing a role in either the white hyphal phase or the opaque budding phase. These results suggest that regulation is solely through activation in the white budding phase and the same mechanism, therefore, is involved in regulating the differential expression of WH11 in the alternative white and opaque phases of switching and the budding and hyphal phases of dimorphism.

CkF1,2 has been reported as an effective DNA fingerprinting probe of Candida krusei. It is composed of two genomic EcoRI-restriction fragments, F1 and F2, which are approximately 5.4 and 5.2 kb, respectively. Sequence analysis of F1 reveals that it is 5261 bp-long, has a GC content of 42.2 mol%, and originates from the intergenic region of the ribosomal RNA cistrons (IGR). F1 comprises 488 bp of the 3' end of a 25s rRNA gene, a non-transcribed spacer region 1 (NTS1), a 5s gene (121 bp), and a major portion of the non-transcribed spacer region 2 (NTS2). A 1256 bp-long repeated sequence, CKRS-1, with a GC content of 35 mol%, has been identified in NTS2. CKRS-1 contains eight tandemly repeated sub-elements, kre-0 to kre-7. The first two, kre-0 and kre-1, are 164 bp-long, the next five sub-elements, kre-2 to kre-6, are 165 bp-long, and the last element, kre-7, is 103 bp-long. The eight sub-elements share nucleotide-sequence homologies between 66 to 100%, with kre-2, kre-3 and kre-4 identical, and kre-0 the most divergent. Shorter repeated sequences were also identified in three regions of F1, which were named domains "a", "b" and "c". Restriction mapping, cross hybridization, and direct comparison of sequences show that F1 and F2 are polymophic forms of the IGR and their size difference is due both to the number of kre sub-elements in CKRS-1 and to a 24-bp deletion in domain "b". While F1 contains eight kre sub-elements, F2 contains seven. In C. krusei strain K31, four polymorphic forms of CKRS-1 have been identified containing five, six, seven and eight kre sub-elements. CKRS-1 is dispersed on three of the chromosomes of highest molecular weights separated by transverse alternating-field electrophoresis. CKRS-1 does not hybridize significantly to any transcription product. Polymorphisms in single DNA fingerprints and differences between the DNA fingerprints of strains of C. krusei based upon CkF1,2 hybridization patterns therefore appear to be based, at least in part, on the variable number of tandemly repeated kre sub-elements in CKRS-1.

Candida albicans , like other pathogens, can form complex biofilms on a variety of substrates. However, as the number of studies of gene regulation, architecture, and pathogenic traits of C. albicans biofilms has increased, so have differences in results. This suggests that depending upon the conditions employed, biofilms may vary widely, thus hampering attempts at a uniform description. Gene expression studies suggest that this may be the case. To explore this hypothesis further, we compared the architectures and traits of biofilms formed in RPMI 1640 and Spider media at 37°C in air. Biofilms formed by a /α cells in the two media differed to various degrees in cellular architecture, matrix deposition, penetrability by leukocytes, fluconazole susceptibility, and the facilitation of mating. Similar comparisons of a/a cells in the two media, however, were made difficult given that in air, although a/a cells form traditional biofilms in RPMI medium, they form polylayers composed primarily of yeast cells in Spider medium. These polylayers lack an upper hyphal/matrix region, are readily penetrated by leukocytes, are highly fluconazole susceptible, and do not facilitate mating. If, however, air is replaced with 20% CO 2 , a/a cells make a biofilm in Spider medium similar architecturally to that of a /α cells, which facilitates mating. A second, more cursory comparison is made between the disparate cellular architectures of a/a biofilms formed in air in RPMI and Lee's media. The results demonstrate that C. albicans forms very different types of biofilms depending upon the composition of the medium, level of CO 2 in the atmosphere, and configuration of the MTL locus.

Candida albicans forms two types of biofilm in RPMI 1640 medium, depending upon the configuration of the mating type locus. In the prevalent a /α configuration, cells form a biofilm that is impermeable, impenetrable by leukocytes, and fluconazole resistant. It is regulated by the Ras1/cyclic AMP (cAMP) pathway. In the a/a or α/α configuration, white cells form a biofilm that is architecturally similar to an a /α biofilm but, in contrast, is permeable, penetrable, and fluconazole susceptible. It is regulated by the mitogen-activated protein (MAP) kinase pathway. The MTL -homozygous biofilm has been shown to facilitate chemotropism, a step in the mating process. This has led to the hypothesis that specialized MTL -homozygous biofilms facilitate mating. If true, then MTL -homozygous biofilms should have an advantage over MTL -heterozygous biofilms in supporting mating. We have tested this prediction using a complementation strategy and show that minority opaque a/a and α/α cells seeded in MTL -homozygous biofilms mate at frequencies 1 to 2 orders of magnitude higher than in MTL -heterozygous biofilms. No difference in mating frequencies was observed between seeded patches of MTL -heterozygous and MTL -homozygous cells grown on agar at 28°C in air or 20% CO 2 and at 37°C. Mating frequencies are negligible in seeded patches of both a /α and a/a cells, in contrast to seeded biofilms. Together, these results support the hypothesis that MTL -homozygous ( a/a or α/α) white cells form a specialized “sexual biofilm.”

Cells of Candida albicans WO-1 switch frequently, spontaneously and reversibly between a white and opaque phase. The white-opaque transition involves the regulation of phase-specific genes. In the white budding phase, cells express the white phase-specific gene WH11, which encodes a protein with homology to the heat shock protein Hsp 12 of Saccharomyces cerevisiae. A recombinant Wh11 protein has been synthesized, purified to apparent homogeneity and used to generate a rabbit polyclonal antiserum. The antiserum was used to localize the Wh11 protein in white phase cells. Wh11 is distributed throughout the cytoplasm but appears to be excluded from vesicles, plasma membrane and nucleus. An analysis by Western blotting of Wh11 expression in a number of C. albicans strains and related species suggests a correlation between round budding cell shape and expression.

Most strains of Candida albicans switch frequently and reversibly among a number of different phenotypes distinguishable by colony morphology. Previous experiments indicated that switching involved differential gene expression. Using the white–opaque transition as a model switching system, we have cloned two opaque-specific genes, PEP1 and OP4, and one white specific gene, WH11. Differential transcription of these genes suggested that switching involves the coordinate regulation of batteries of unlinked phase-specific genes. It has been demonstrated that the frequency of integration at phase specific loci is a function of the transcriptional state of the phase-specific genes. In addition, a functional dissection of the 5′-upstream region of the WH11 gene has identified two major domains containing cis-acting regulatory sequences that are involved in phase-specific transcription. Gel retardation experiments provide evidence for white phase-specific trans-acting factors which form complexes with both domains. The regulation of the switching event is discussed. Key words: Candida albicans, phenotypic switching, white–opaque transition, phase-specific genes, integrative transformation, promoter regions, WH11 gene.

A rDNA cistron of Candida albicans strain WO-1 was cloned and the ITS1, ITS2, 5.8 s rDNA and 25 s rDNA coding regions sequenced in their entirety. These sequences were compared to those of three related yeast species (Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Thermomyces lanuginosus), and the 5.8 s rDNA was compared to seven additional 5.8 s rDNAs from organisms ranging in complexity from D. discoideum to H. sapiens. The C. albicans ITS regions are shorter than those of most other eukaryotes. The 25 s and 5.8 s rDNA sequences were folded into a secondary structure model based on comparative methods. In a comparison of regional similarities between the large subunit rDNAs of C. albicans, the three related yeasts and other eukaryotes, it is demonstrated that the additional sequences not present in the E. coli 23 s rDNA are more variable than the regions present in both prokaryotes and eukaryotes.

Similar multicellular structures can evolve within the same organism that may have different evolutionary histories, be controlled by different regulatory pathways, and play similar but nonidentical roles. In the human fungal pathogen Candida albicans, a quite extraordinary example of this has occurred. Depending upon the configuration of the mating type locus (a/α versus a/a or α/α), C. albicans forms alternative biofilms that appear similar morphologically, but exhibit dramatically different characteristics and are regulated by distinctly different signal transduction pathways. Biofilms formed by a/α cells are impermeable to molecules in the size range of 300 Da to 140 kDa, are poorly penetrated by human polymorphonuclear leukocytes (PMNs), and are resistant to antifungals. In contrast, a/a or α/α biofilms are permeable to molecules in this size range, are readily penetrated by PMNs, and are susceptible to antifungals. By mutational analyses, a/α biofilms are demonstrated to be regulated by the Ras1/cAMP pathway that includes Ras1→Cdc35→cAMP(Pde2—|)→Tpk2(Tpk1)→Efg1→Tec1→Bcr1, and a/a biofilms by the MAP kinase pathway that includes Mfα→Ste2→ (Ste4, Ste18, Cag1)→Ste11→Hst7→Cek2(Cek1)→Tec1. These observations suggest the hypothesis that while the upstream portion of the newly evolved pathway regulating a/a and α/α cell biofilms was derived intact from the upstream portion of the conserved pheromone-regulated pathway for mating, the downstream portion was derived through modification of the downstream portion of the conserved pathway for a/α biofilm formation. C. albicans therefore forms two alternative biofilms depending upon mating configuration., Author Summary Single-celled microbes can form biofilms, or aggregates of cells that adhere to one another on a surface, in response to many environmental factors. Like many microbial pathogens, the yeast Candida albicans can form biofilms that normally provide protective environments against antifungals, antibodies, and white blood cells, thus ensuring higher rates of survival in response to assault by drugs or the human immune system. We report that while a majority (around 90%) of C. albicans strains form traditional biofilms that are impermeable to molecules of low and high molecular weight, and that are impenetrable to white blood cells, a minority (around 10%) form biofilms that are both permeable and penetrable. Formation of the minority-type alternative biofilms is dictated by a change at a single genetic locus, the mating type locus. Homozygous a/a or α/α cells are mating-competent, whereas the heterozygous a/α cells are mating-incompetent. Cells of the mating-incompetent a/α genotype form the impermeable, traditional biofilm, whereas the mating-competent a/a or α/α genotype forms the permeable biofilm. The characteristics of a/a and α/α biofilms are consistent with a suggested role in mating by facilitating the transfer of hormone signals through the permeable biofilm. The two types of biofilm are also regulated by different signal transduction pathways: the a/α form by the Ras1/cAMP pathway, and the a/a or α/α forms by the MAP kinase pathway. Components of the latter pathway suggest that its downstream portion evolved from the a/α pathway. C. albicans, therefore, forms two superficially similar biofilms, exhibiting very different permeability characteristics, regulated by different signal transduction pathways, dictated by different mating type locus configurations, and serving quite different purposes in its life history.

Among the hemiascomycetes, only Candida albicans must switch from the white phenotype to the opaque phenotype to mate. In the recent evolution of this transition, mating-incompetent white cells acquired a unique response to mating pheromone, resulting in the formation of a white cell biofilm that facilitates mating. All of the upstream components of the white cell response pathway so far analyzed have been shown to be derived from the ancestral pathway involved in mating, except for the mitogen-activated protein (MAP) kinase scaffold protein, which had not been identified. Here, through binding and mutational studies, it is demonstrated that in both the opaque and the white cell pheromone responses, Cst5 is the scaffold protein, supporting the evolutionary scenario proposed. Although Cst5 plays the same role in tethering the MAP kinases as Ste5 does in Saccharomyces cerevisiae, Cst5 is approximately one-third the size and has only one rather than four phosphorylation sites involved in activation and cytoplasmic relocalization., IMPORTANCE Candida albicans must switch from white to opaque to mate. Opaque cells then release pheromone, which not only induces cells to mate but also in a unique fashion induces mating-incompetent white cells to form biofilms that facilitate opaque cell mating. All of the tested upstream components of the newly evolved white cell pheromone response pathway, from the receptor to the mitogen-activated protein (MAP) kinase cascade, are the same as those of the conserved opaque cell response pathway. One key element, however, remained unidentified, the scaffold protein for the kinase cascade. Here, we demonstrate that Cst5, a homolog of the Saccharomyces cerevisiae scaffold protein Ste5, functions as the scaffold protein in both the opaque and the white cell pheromone responses. Pheromone induces Cst5 phosphorylation, which is involved in activation and cytoplasmic localization of Cst5. However, Cst5 contains only one phosphorylation site, not four as in the S. cerevisiae ortholog Ste5. These results support the hypothesis that the entire upper portion of the newly evolved white cell pheromone response pathway is derived from the conserved pheromone response pathway in the mating process.

Few mating-regulated genes have been characterized in Candida albicans. C. albicans FIG1 (Ca FIG1 ) is a fungus-specific and mating-induced gene encoding a putative 4-transmembrane domain protein that shares sequence similarities with members of the claudin superfamily. In Saccharomyces cerevisiae , Fig1 is required for shmoo fusion and is upregulated in response to mating pheromones. Expression of Ca FIG1 was also strongly activated in the presence of cells of the opposite mating type. CaFig1-green fluorescent protein (GFP) was visible only during the mating response, when it localized predominantly to the plasma membrane and perinuclear zone in mating projections and daughter cells. At the plasma membrane, CaFig1-GFP was visualized as discontinuous zones, but the distribution of perinuclear CaFig1-GFP was homogeneous. Exposure to pheromone induced a 5-fold increase in Ca 2+ uptake in mating-competent opaque cells. Uptake was reduced substantially in the fig1 Δ null mutant. CaFig1 is therefore involved in Ca 2+ influx and localizes to membranes that are destined to undergo fusion during mating.

Cells of Candida albicans WO-1 switch spontaneously and frequently between a white and an opaque CFU. Recently, an opaque-phase-specific cDNA, PEP1, was cloned and was demonstrated to code for a pepsinogen. By using a differential hybridization screen, a second opaque-phase-specific cDNA, Op4, has been isolated and its corresponding gene has been cloned. Op4 is coordinately regulated with PEP1 but resides on a different chromosome. During temperature-induced mass conversion from opaque to white, transcription of PEP1 and Op4 is immediately inhibited by the increase in temperature, but transcription of both genes can be rapidly reestablished by a downshift in temperature prior to phenotypic commitment. However, the capacity to rapidly induce both PEP1 and Op4 is lost coincidentally with the second semisynchronous round of cell division and phenotypic commitment during mass conversion. Op4 shows no significant base or amino acid sequence homology with a known gene or protein, respectively. However, the deduced Op4 protein exhibits several interesting characteristics, including a hydrophobic amino terminus with 26 amino acids, a pI of 10.73 for the last 100 amino acids, two serine repeats adjacent to alanine repeats, and the potential for alpha-helical conformation within the alanine-rich sequences. No genomic reorganization was evident in the proximity of Op4 during transcriptional activation and deactivation accompanying the white-opaque transition.

To mate, the fungal pathogen Candida albicans must undergo homozygosis at the mating-type locus and then switch from the white to opaque phenotype. Paradoxically, opaque cells were found to be unstable at physiological temperature, suggesting that mating had little chance of occurring in the host, the main niche of C. albicans. Recently, however, it was demonstrated that high levels of CO2, equivalent to those found in the host gastrointestinal tract and select tissues, induced the white to opaque switch at physiological temperature, providing a possible resolution to the paradox. Here, we demonstrate that a second signal, N-acetylglucosamine (GlcNAc), a monosaccharide produced primarily by gastrointestinal tract bacteria, also serves as a potent inducer of white to opaque switching and functions primarily through the Ras1/cAMP pathway and phosphorylated Wor1, the gene product of the master switch locus. Our results therefore suggest that signals produced by bacterial co-members of the gastrointestinal tract microbiota regulate switching and therefore mating of C. albicans., Author Summary To mate, the human fungal pathogen Candida albicans must undergo a complex phenotypic change from a round “white” to large, elongated “opaque” cell. This involves the regulation of approximately 5% of the organism's genes. Surprisingly, this complex transition is not required for mating in other related yeast. Even more surprisingly, it was found that in vitro the mating-competent opaque phenotype was unstable at 37°C, the temperature of the host body. This observation led to a paradox. If C. albicans lives primarily in an animal host, physiological temperature would thwart mating, so where does C. albicans mate? This led to the suggestion that some physiological condition in the host niche stabilizes the opaque phenotype or even induces switching from white to opaque, so cells can mate. Recently, we demonstrated that the high concentrations of CO2 found in tissue and the gastrointestinal tract induced switching from white to opaque and then stabilized the opaque phenotype. Here, we demonstrate that a second factor, N-acetylglucosamine (GlcNAc), a sugar released primarily by bacteria in the gastrointestinal tract, also induces the switch from white to opaque and stabilizes the opaque phenotype. We demonstrate by mutational analysis that GlcNAc induction is regulated primarily by the Ras1/cAMP pathway, which also regulates filamentation of C. albicans. This is perhaps not surprising given that white-opaque switching shares with filamentation several phenotypic characteristics. Finally, we show that induction by GlcNAc requires the phosphorylated master switch gene that regulates spontaneous switching, suggesting that it induces the switch from white to opaque by activating the gene product of the master switch gene. Together, our results suggest that multiple signals from bacterial co-members of the gastrointestinal tract microbiota regulate switching and, therefore, mating of C. albicans in the colonized host.

There were errors in the Author Contributions. The correct contributions are: Conceived and designed the experiments: GH DRS. Performed the experiments: GH SY NS KJD TS. Analyzed the data: GH DRS. Wrote the paper: GH DRS. Made the figures: GH KJD.

Cells of Candida albicans WO-1 spontaneously switch between a white and opaque CFU, and this phase transition involves a dramatic change in cellular phenotype. By using a differential hybridization screen, an opaque-specific cDNA, Op1a, which represents the transcript of a gene regulated by switching, has been isolated. The gene for Op1a is transcribed by opaque but not by white cells. The nucleotide sequence of the Op1a cDNA reveals over 99% base homology with an acid protease gene of C. albicans, and the predicted amino acid sequence demonstrates that the product of this gene is a member of the family of pepsinogens, which possess a hydrophobic leader sequence for secretion and two catalytic aspartate domains. Southern blots of both genomic DNA digested with 14 different endonucleases and electrophoretically separated chromosomes were probed with the Op1a cDNA. No polymorphisms were detected in either case between white and opaque cells, suggesting that no genomic reorganization occurs in the proximity of the gene during the white-opaque transition. Although transcription of Op1a correlates with the high levels of extracellular protease activity in opaque cell cultures and the absence of activity in white cell cultures, stimulation of extracellular protease activity by addition of serum albumin is not accompanied by Op1a transcription in cultures of WO-1 white cells or cultures of two additional clinical isolates of C. albicans, suggesting that expression of one or more other protease genes is stimulated in these cases. The results demonstrate that transcription of the Op1a gene is under the rigid control of switching in strain WO-1.

To mate, Candida albicans must undergo homozygosis at the mating type-like locus MTL[1, 2], then switch from the white to opaque phenotype [3, 4]. Paradoxically, when opaque cells are transferred in vitro to 37 degrees C, the temperature of their animal host, they switch en masse to white [5-7], suggesting that their major niche might not be conducive to mating. It has been suggested that pheromones secreted by opaque cells of opposite mating type [8] or the hypoxic condition of host niches [9, 10] stabilize opaque cells. There is, however, an additional possibility, namely that CO(2), which achieves levels in the host 100 times higher than in air [11-13], stabilizes the opaque phenotype. CO(2) has been demonstrated to regulate the bud-hypha transition in C. albicans[14, 15], expression of virulence genes in bacteria [16], and mating events in Cryptococcus neoformans[14, 17]. We tested the possibility that CO(2) stabilizes the opaque phenotype, and found that physiological levels of CO(2) induce white-to-opaque switching and stabilize the opaque phenotype at 37 degrees C. It exerts this control equally under anaerobic and aerobic conditions. These results suggest that the high levels of CO(2) in the host induce and stabilize the opaque phenotype, thus facilitating mating.

Candida albicans must undergo a switch from white to opaque to mate. Opaque cells then release mating type-specific pheromones that induce mating responses in opaque cells. Uniquely in C. albicans, the same pheromones induce mating-incompetent white cells to become cohesive, form an adhesive basal layer of cells on a surface, and then generate a thicker biofilm that, in vitro, facilitates mating between minority opaque cells. Through mutant analysis, it is demonstrated that the pathways regulating the white and opaque cell responses to the same pheromone share the same upstream components, including receptors, heterotrimeric G protein, and mitogen-activated protein kinase cascade, but they use different downstream transcription factors that regulate the expression of genes specific to the alternative responses. This configuration, although common in higher, multicellular systems, is not common in fungi, and it has not been reported in Saccharomyces cerevisiae. The implications in the evolution of multicellularity in higher eukaryotes are discussed.

The cDNAs coding for human chromosomal proteins HMG-14 and HMG-17 were cloned into yeast expression vector pBM150, under the control of the Gal10 promoter. Northern analysis of transformed yeast cells revealed that both cDNAs were efficiently transcribed. Western analysis indicated that the mRNAs were translated into authentic proteins. Expression of human HMG proteins in yeast cell did not produce detectable phenotypic changes, as measured by the growth rate of the yeast cells under a variety of conditions. The antibiotic resistance of the transfected cells was similar to that of control cells, suggesting that the presence of HMG did not affect the expression of actively transcribed genes. However, examination of the protein profile on two-dimensional polyacrylamide gel electrophoresis revealed differences between control and HMG-transfected Cells.

The evolutionary origins and common features of the genes coding for the HMG-14/-17 family of chromosomal proteins have been studied by isolating and sequencing the chicken HMG-14b gene, the true homolog of the human and calf HMG-14 gene. Comparison of the structure of this gene to that of the human HMG-14 gene and to the human and chicken HMG-17 genes indicates that the HMG-14 and HMG-17 genes evolved from a common ancestor. We postulate that the ancestral gene consisted of six exons. In all genes the first exon codes for the entire 5' untranslated region and for the first four amino acids, which are invariant among all the known members of the HMG-14/-17 protein family. The last exon codes for ten to 16 amino acids and for the entire 3' untranslated region, which, for each gene, constitutes over 70% of the transcript. The DNA-binding domain of the proteins is encoded by two distinct exons. The genes are characterized by 5' regions that are highly enriched in G + C residues and have features characteristic of "housekeeping" genes. The HMG-17 genes are distinct from the HMG-14 in that the 5' regulatory region of the former has two TATA boxes while the HMG-14 genes have no such regulatory element. The chicken HMG-14b gene is a single-copy gene and produces a unique transcript. In this gene, exons II and III are fused and intron 2 is missing. The fusion of the two exons produced a codon for valine in a position that, among all HMG-14/-17 proteins, is unique to HMG-14b. The possible consequences of a valine insertion at the N-terminal end of the DNA-binding domains are discussed. The HMG-14 proteins evolve significantly faster than HMG-17, suggesting that the proteins are subject to different evolutionary pressure. However, certain amino acids are conserved among all the known members of the HMG-14/-17 protein family, suggesting that they are part of the functional domain of this family of chromosomal proteins.