The histone deacetylase genes HDA1 and RPD3 play distinct roles in regulation of high-frequency phenotypic switching in Candida albicans.

Five histone deacetylase genes (HDA1, RPD3, HOS1, HOS2, and HOS3) have been cloned from Candida albicans and characterized. Sequence analysis and comparison with 17 additional deacetylases resulted in a phylogenetic tree composed of three major groups. Transcription of the deacetylases HDA1 and RPD3 is down-regulated in the opaque phase of the white-opaque transition in strain WO-1. HOS3 is 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. HDA1 and RPD3 were 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 of HDA1 resulted 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 of RPD3 resulted in an increase in the frequency of switching in both directions. Deletion of HDA1 resulted in reduced white-phase-specific expression of the EFG1 3.2-kb transcript, but had no significant effect on white-phase-specific expression of WH11 or opaque-phase-specific expression of OP4, SAP1, and SAP3. Deletion of RPD3 resulted in reduced opaque-phase-specific expression of OP4, SAP1, and SAP3 and a slight reduction of white-phase-specific expression of WH11 and 3.2-kb EFG1. Deletion of neither HDA1 nor RPD3 affected the high level of white-phase expression and the low level of opaque-phase expression of the MADS box protein gene MCM1, 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 genes HDA1 and RPD3 play 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.

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.

EFG1 null mutants of Candida albicans switch but cannot express the complete phenotype of white-phase budding cells.

The Candida albicans gene EFG1 encodes a putative trans-acting factor. In strain WO-1, which undergoes the white-opaque transition, EFG1 is transcribed as a 3.2-kb mRNA in white-phase cells and a less-abundant 2.2-kb mRNA in opaque-phase cells. cDNA sequencing and 5' rapid amplification of cDNA ends analysis demonstrate that the major difference in molecular mass of the two transcripts is due to different transcription start sites. EFG1 null mutants form opaque-phase colonies and express the opaque-phase cell phenotype at 25 degrees C. When shifted from 25 to 42 degrees C, mutant opaque-phase cells undergo phenotypic commitment to the white phase, which includes deactivation of the opaque-phase-specific gene OP4 and activation of the white-phase-specific gene WH11, as do wild-type opaque-phase cells. After the commitment event, EFG1 null mutant cells form daughter cells which have the smooth (pimpleless) surface of white-phase cells but the elongate morphology of opaque-phase cells. Taken together, these results demonstrate that EFG1 expression is not essential for the switch event per se, but is essential for a subset of phenotypic characteristics necessary for the full expression of the phenotype of white-phase cells. These results demonstrate that EFG1 is not the site of the switch event, but is, rather, downstream of the switch event.

Phenotypic switching in Candida glabrata involves phase-specific regulation of the metallothionein gene MT-II and the newly discovered hemolysin gene HLP.

Although Candida glabrata has emerged in recent years as a major fungal pathogen, there have been no reports demonstrating that it undergoes either the bud-hypha transition or high-frequency phenotypic switching, two developmental programs believed to contribute to the pathogenic success of other Candida species. Here it is demonstrated that C. glabrata undergoes reversible, high-frequency phenotypic switching between a white (Wh), light brown (LB), and dark brown (DB) colony phenotype discriminated on an indicator agar containing 1 mM CuSO(4). Switching regulates the transcript level of the MT-II metallothionein gene(s) and a newly discovered gene for a hemolysin-like protein, HLP. The relative MT-II transcript levels in Wh, LB, and DB cells grown in the presence of CuSO(4) are 1:27:81, and the relative transcript levels of HLP are 1:20:35. The relative MT-II and HLP transcript levels in cells grown in the absence of CuSO(4) are 1:20:30 and 1:20:25, respectively. In contrast, switching has little or no effect on the transcript levels of the genes MT-I, AMT-I, TRPI, HIS3, EPAI, and PDHI. Switching of C. glabrata is not associated with microevolutionary changes identified by the DNA fingerprinting probe Cg6 and does not involve tandem amplification of the MT-IIa gene, which has been shown to occur in response to elevated levels of copper. Finally, switching between Wh, LB, and DB occurred in all four clinical isolates examined in this study. As in Candida albicans, switching in C. glabrata may provide colonizing populations with phenotypic plasticity for rapid responses to the changing physiology of the host, antibiotic treatment, and the immune response, through the differential regulation of genes involved in pathogenesis. More importantly, because C. glabrata is haploid, a mutational analysis of switching is now feasible.

Misexpression of the opaque-phase-specific gene PEP1 (SAP1) in the white phase of Candida albicans confers increased virulence in a mouse model of cutaneous infection.

Candida albicans WO-1 switches reversibly and at high frequency between a white and an opaque colony-forming phenotype that includes dramatic changes in cell morphology and physiology. A misexpression strategy has been used to investigate the role of the opaque-phase-specific gene PEP1 (SAP1), which encodes a secreted aspartyl proteinase, in the expression of the unique opaque-phase phenotype and phase-specific virulence in two animal models. The PEP1 (SAP1) open reading frame was inserted downstream of the promoter of the white-phase-specific gene WH11 in the transforming vector pCPW7, and the resulting transformants were demonstrated to misexpress PEP1 (SAP1) in the white phase. Misexpression did not confer any of the unique morphological characteristics of the opaque phase to cells in the white phase and had no effect on the switching process. However, misexpression conferred upon white-phase cells the increased capacity of opaque-phase cells to grow in medium in which protein was the sole nitrogen source. Misexpression of PEP1 (SAP1) had no effect on the virulence of white-phase cells in a systemic mouse model, in which white-phase cells were already more virulent than opaque-phase cells. Misexpression did, however, confer upon white-phase cells the dramatic increase in colonization of skin in a cutaneous mouse model that was exhibited by opaque-phase cells. Misexpression of PEP1 (SAP1) conferred upon white-phase cells two dissociable opaque-phase characteristics: increased adhesion and the capacity to cavitate skin. The addition of pepstatin A to the cutaneous model inhibited the latter, but not the former, suggesting that the latter is effected by released enzyme, while the former is effected by cell-associated enzyme.

Microevolutionary changes in Candida albicans identified by the complex Ca3 fingerprinting probe involve insertions and deletions of the full-length repetitive sequence RPS at specific genomic sites.

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.

A MADS box protein consensus binding site is necessary and sufficient for activation of the opaque-phase-specific gene OP4 of Candida albicans.

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.

Advances in molecular genetics of Candida albicans and Candida glabrata.

Ten years ago, when molecular genetic methods were being applied vigorously to viruses, bacterial pathogens and eukaryotic parasites, there seemed to be a partial paralysis in applying them to infectious fungi; this state of affairs was more than apparent in the composition of the symposia at the ISHAM conference in 1987. Since then, however, things have changed. The ISHAM conference held in Italy in 1997 was replete with studies utilizing molecular genetic techniques to answer questions related to epidemiology, pathogenesis, drug development and typing. In the symposium Advances in Molecular Genetics of Fungal Pathogens, several new applications of molecular biology to fungal pathogenesis were reviewed. Although the presentations in this symposium covered only a fraction of the molecular methods now being applied to Candida pathogenesis, they nevertheless provided an intriguing view of what is in store for us in the coming years.

Reporters for the analysis of gene regulation in fungi pathogenic to man.

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.

Misexpression of the white-phase-specific gene WH11 in the opaque phase of Candida albicans affects switching and virulence.

Candida albicans WO-1 switches between a white- and an opaque-colony-forming phenotype. The gene WH11 is expressed differentially in the white phase. The WH11 open reading frame was inserted downstream of the promoter of the opaque-phase-specific gene OP4 in the transforming vector pCWOP16, and resulting transformants were demonstrated to misexpress WH11 in the opaque phase. Misexpression had no effect on the ability to switch from the white to the opaque or the opaque to the white phase, and it had no effect on the genesis of the unique opaque-phase cellular phenotype, even though the Wh11 protein was distributed throughout the cytoplasm in a manner similar to that observed for the endogenous gene product in the white phase. Misexpression did, however, increase the frequency of the opaque-to-white transition 330-fold and markedly increased the virulence of cells in the opaque phase in a mouse tail injection model.

The WH11 gene of Candida albicans is regulated in two distinct developmental programs through the same transcription activation sequences.

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.

A novel repeat sequence (CKRS-1) containing a tandemly repeated sub-element (kre) accounts for differences between Candida krusei strains fingerprinted with the probe CkF1,2.

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.

Cytoplasmic localization of the white phase-specific WH11 gene product of Candida albicans.

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 Hsp12 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.

The sea pansy Renilla reniformis luciferase serves as a sensitive bioluminescent reporter for differential gene expression in Candida albicans.

The infectious yeast Candida albicans progresses through two developmental programs which involve differential gene expression, the bud-hypha transition and high-frequency phenotypic switching. To understand how differentially expressed genes are regulated in this organism, the promoters of phase-specific genes must be functionally characterized, and a bioluminescent reporter system would facilitate such characterization. However, C. albicans has adopted a nontraditional codon strategy that involves a tRNA with a CAG anticodon to decode the codon CUG as serine rather than leucine. Since the luciferase gene of the sea pansy Renilla reinformis contains no CUGs, we have used it to develop a highly sensitive bioluminescent reporter system for C. albicans. When fused to the galactose-inducible promoter of GAL1, luciferase activity is inducible; when fused to the constitutive EF1 alpha 2 promoter, luciferase activity is constitutive; and when fused to the promoter of the white-phase-specific gene WH11 or the opaque-phase-specific gene OP4, luciferase activity is phase specific. The Renilla luciferase system can, therefore, be used as a bioluminescent reporter to analyze the strength and developmental regulation of C. albicans promoters.

Colonizing populations of Candida albicans are clonal in origin but undergo microevolution through C1 fragment reorganization as demonstrated by DNA fingerprinting and C1 sequencing.

The genetic homogeneity of nine commensal and infecting populations of Candida albicans has been assessed by fingerprinting multiple isolates from each population by Southern blot hybridization first with the Ca3 probe and then with the 0.98-kb C1 fragment of the Ca3 probe. The isolates from each population were highly related, demonstrating the clonal origin of each population, but each population contained minor variants, demonstrating microevolution. Variation in each case was limited to bands of the Ca3 fingerprint pattern which hybridized with the 0.98-kb C1 fragment. The C1 fragment was therefore sequenced and demonstrated to contain an RPS repetitive element. The C1 fragment also contained part or all of a true end of the RPS element. These results, therefore, demonstrate that most colonizing C. albicans populations in nonimmuno-suppressed patients are clonal, that microevolution can be detected in every colonizing population by C1 hybridization, and that C1 contains the repeat RPS element.

Functional analysis of the promoter of the phase-specific WH11 gene of Candida albicans.

Candida albicans WO-1 switches spontaneously, frequently, and reversibly between a hemispherical white and a flat gray (opaque) colony-forming phenotype. This transition affects a number of morphological and physiological parameters and involves the activation and deactivation of phase-specific genes. The WH11 gene is transcribed in the white but not the opaque phase. A chimeric WH11-firefly luciferase gene containing the 5' upstream region of WH11 was demonstrated to be under phase regulation regardless of the site of integration, and a series of promoter deletion constructs was used to delineate two white-phase-specific transcription activation domains. Gel retardation experiments with the individual distal or proximal domain and white-phase or opaque-phase protein extract demonstrated the formation of one distal white-phase-specific complex and two proximal white-phase-specific complexes. Specific subfragments were tested for their ability to compete with the entire domain in the formation of complexes with white-phase protein extract in order to map the proximal domain sequence involved in white-phase-specific complex formation. Our results indicate that white-phase-specific transcription of WH11 is positively regulated by trans-acting factors interacting with two cis-acting activation sequences in the WH11 promoter.

The frequency of integrative transformation at phase-specific genes of Candida albicans correlates with their transcriptional state.

The phase transition between the white and opaque phenotypes in the switching system of Candida albicans strain WO-1 is accompanied by the differential expression of the white-specific gene WH11 and the opaque-specific gene PEP1. The frequency of integrative transformation at the white-specific gene locus WH11 is between 4.5 and 7.0 times more frequent in white than in opaque spheroplasts, and the frequency of disruptive transformation at the opaque-specific gene locus PEP1 is 30.5 times more frequent in opaque spheroplasts than in white spheroplasts. In contrast, the frequencies of integrative transformation at the constitutively expressed loci ADE2 and EF1 alpha 2 are similar in the white and opaque phases. Therefore, the frequency of integration of linear plasmid DNA containing sequences of phase-specific genes correlates with the transcriptional state of the targeted locus.

Partial nucleotide sequence of a single ribosomal RNA coding region and secondary structure of the large subunit 25 s rRNA of Candida albicans.

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.

Developmental and molecular biology of switching in Candida albicans.

Candida albicans and related species switch frequently and reversibly between a number of general phenotypes usually discriminated by colony morphology and in some cases by cellular morphology. Switching has been demonstrated to affect a number of physiologic and architectural characteristics of single cells including most of the putative virulence factors of C.albicans. In the past few years, we have cloned several genes regulated by switching in the white-opaque transition of C.albicans strain WO-1. Two of the genes, PEP1 and Op4, are transcribed only in the opaque phase, and one of the genes, Wh11, is transcribed only in the white phase. These coordinately regulated genes are unlinked in the genome and do not undergo sequence reorganization in switching. With the identification of a cis-acting regulatory sequence in the five-prime flanking sequence of Wh11, we now believe that phase-specific genes are regulated by transacting factors and that these factors may be coded for or under the direct regulation of a single master regulatory gene at which site the basic switch event occurs.

A white-specific gene in the white-opaque switching system of Candida albicans.

The Candida albicans strain WO-1 cells switch spontaneously, frequently, and reversibly between white and opaque colony-forming units which differ dramatically in their budding phenotypes. By screening a subtracted white cDNA library, the first white-specific cDNA, cWh11, was isolated and sequenced. This, in turn, was used to clone the Wh11 gene. Wh11 shows significant homology to the glucose/lipid-regulated GLP1 gene of Saccharomyces cerevisiae. Upon temperature-induced mass conversion from opaque to white colonies, transcription of Wh11 is abruptly activated at the second cell doubling, concomitant with commitment to the white phenotype. Wh11 exhibits the unique characteristics of being regulated not only by switching, but also by the bud-hypha transition.