A single SNP, G929T (Gly310Val), determines the presence of a functional and a non-functional allele of HIS4 in Candida albicans SC5314: detection of the non-functional allele in laboratory strains

Candida albicans is a diploid organism that exhibits high levels of heterozygosity. Although the precise manner by which this heterozygosity provides advantage for the commensal/pathogenic life styles of C. albicans is not known, heterozygous markers are themselves useful for studying genomic rearrangements, which occur frequently in C. albicans. Treatment of CAI-4 with UV light yielded histidine auxotrophs which could be complemented by HIS4, suggesting that strain CAI-4 is heterozygous for HIS4. These auxotrophs appeared to have undergone mitotic recombination and/or chromosome loss. As expected from a heterozygote, disruption of the functional allele of HIS4 resulted in a his4::hisG-URA3-hisG strain that is auxotrophic for histidine. Sequencing of random clones of the HIS4 ORF from CAI-4 and its precursor SC5314 revealed the presence of 11 SNPs, seven synonymous and four non-synonymous. Site-directed mutagenesis indicates that only one of those SNPs, T929G (Gly310Val), is responsible for the non-functionality of the encoded enzyme. HIS4 analysis of five commonly used laboratory strains is reported. This study provides a new, easily measured nutritional marker that can be used in future genetic studies in C. albicans.     

Comparative genome hybridization reveals widespread aneuploidy in Candida albicans laboratory strains

Clinical strains of Candida albicans are highly tolerant of aneuploidies and other genome rearrangements. We have used comparative genome hybridization (CGH), in an array format, to analyse the copy number of over 6000 open reading frames (ORFs) in the genomic DNA of C. albicans laboratory strains carrying one (CAI-4) to three (BWP17) auxotrophies. We find that during disruption of the HIS1 locus all genes telomeric to HIS1 were deleted and telomeric repeats were added to a 9 nt sequence within the transforming DNA. This deletion occurred in approximately 10% of transformants analysed and was stably maintained through two additional rounds of transformation and counterselection of the transformation marker. In one example, the deletion was repaired, apparently via break-induced replication. Furthermore, all CAI-4 strains tested were trisomic for chromosome 2 although this trisomy appears to be unstable, as it is not detected in strains subsequently derived from CAI-4. Our data indicate CGH arrays can be used to detect monosomies and trisomies, to predict the sites of chromosome breaks, and to identify chromosomal aberrations that have not been detected with other approaches in C. albicans strains. Furthermore, they highlight the high level of genome instability in C. albicans laboratory strains exposed to the stress of transformation and counterselection on 5-fluoro-orotic acid.     

Interaction between Mismatch Repair and Genetic Recombination in Saccharomyces Cerevisiae

The yeast Saccharomyces cerevisiae encodes a set of genes that show strong amino acid sequence similarity to MutS and MutL, proteins required for mismatch repair in Escherichia coli. We examined the role of MSH2 and PMS1, yeast homologs of mutS and mutL, respectively, in the repair of base pair mismatches formed during meiotic recombination. By using specifically marked HIS4 and ARG4 alleles, we showed that msh2 mutants displayed a severe defect in the repair of all base pair mismatches as well as 1-, 2- and 4-bp insertion/deletion mispairs. The msh2 and pms1 phenotypes were indistinguishable, suggesting that the wild-type gene products act in the same repair pathway. A comparison of gene conversion events in wild-type and msh2 mutants indicated that mismatch repair plays an important role in genetic recombination. (1) Tetrad analysis at five different loci revealed that, in msh2 mutants, the majority of aberrant segregants displayed a sectored phenotype, consistent with a failure to repair mismatches created during heteroduplex formation. In wild type, base pair mismatches were almost exclusively repaired toward conversion rather than restoration. (2) In msh2 strains 10-19% of the aberrant tetrads were Ab4:4. (3) Polarity gradients at HIS4 and ARG4 were nearly abolished in msh2 mutants. The frequency of gene conversion at the 3' end of these genes was increased and was nearly the frequency observed at the 5' end. (4) Co-conversion studies were consistent with mismatch repair acting to regulate heteroduplex DNA tract length. We favor a model proposing that recombination events occur through the formation and resolution of heteroduplex intermediates and that mismatch repair proteins specifically interact with recombination enzymes to regulate the length of symmetric heteroduplex DNA.     

Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions

Disruption of newly identified genes in the pathogen Candida albicans is a vital step in determination of gene function. Several gene disruption methods described previously employ long regions of homology flanking a selectable marker. Here, we describe disruption of C. albicans genes with PCR products that have 50 to 60 bp of homology to a genomic sequence on each end of a selectable marker. We used the method to disrupt two known genes, ARG5 and ADE2, and two sequences newly identified through the Candida genome project, HRM101 and ENX3. HRM101 and ENX3 are homologous to genes in the conserved RIM101 (previously called RIM1) and PacC pathways of Saccharomyces cerevisiae and Aspergillus nidulans. We show that three independent hrm101/hrm101 mutants and two independent enx3/enx3 mutants are defective in filamentation on Spider medium. These observations argue that HRM101 and ENX3 sequences are indeed portions of genes and that the respective gene products have related functions.     

Deletion of the Pichia pastoris KU70 homologue facilitates platform strain generation for gene expression and synthetic biology

Targeted gene replacement to generate knock-outs and knock-ins is a commonly used method to study the function of unknown genes. In the methylotrophic yeast Pichia pastoris, the importance of specific gene targeting has increased since the genome sequencing projects of the most commonly used strains have been accomplished, but rapid progress in the field has been impeded by inefficient mechanisms for accurate integration. To improve gene targeting efficiency in P. pastoris, we identified and deleted the P. pastoris KU70 homologue. We observed a substantial increase in the targeting efficiency using the two commonly known and used integration loci HIS4 and ADE1, reaching over 90% targeting efficiencies with only 250-bp flanking homologous DNA. Although the ku70 deletion strain was noted to be more sensitive to UV rays than the corresponding wild-type strain, no lethality, severe growth retardation or loss of gene copy numbers could be detected during repetitive rounds of cultivation and induction of heterologous protein production. Furthermore, we demonstrated the use of the ku70 deletion strain for fast and simple screening of genes in the search of new auxotrophic markers by targeting dihydroxyacetone synthase and glycerol kinase genes. Precise knock-out strains for the well-known P. pastoris AOX1, ARG4 and HIS4 genes and a whole series of expression vectors were generated based on the wild-type platform strain, providing a broad spectrum of precise tools for both intracellular and secreted production of heterologous proteins utilizing various selection markers and integration strategies for targeted or random integration of single and multiple genes. The simplicity of targeted integration in the ku70 deletion strain will further support protein production strain generation and synthetic biology using P. pastoris strains as platform hosts.     

Identification of Candida albicans genes that induce Saccharomyces cerevisiae cell adhesion and morphogenesis

Morphogenesis and adhesion to host tissues and medical devices contribute to the virulence of Candida albicans, the most common fungal pathogen isolated from humans. However, identification of molecular mechanisms of C. albicans adhesion and morphogenesis has been impaired by the lack of effective molecular and genetic tools available for this organism. Saccharomyces cerevisiae provides an attractive model system for studying C. albicans adhesion and morphogenesis because of its well-characterized genetics and gene expression systems. To gain insight into the genetic mechanisms of C. albicans adhesion and morphogenesis, we used a parallel plate flow chamber to screen and quantitatively characterize attachment to polystyrene of an adhesion-deficient nonfilamentous flo8Delta S. cerevisiae strain expressing a C. albicans genomic library. We identified six C. albicans genes that are capable of promoting cell adhesion and pseudohyphal development in S. cerevisiae. We also analyzed the ability of these adhesion-promoting genes to regulate the expression of FLO11, which encodes an endogenous S. cerevisiae adhesin. One C. albicans gene, EAP1, appears to directly mediate adhesion and morphogenesis while the remaining five (EAP2, SWI1, MSB1, AAF1, and TEC1) upregulate expression of endogenous S. cerevisiae adhesins. These results suggest that S. cerevisiae is a useful system for molecular characterization of factors that regulate C. albicans adhesion and morphogenesis and that parallel plate flow chamber-based adhesion assays can be used in conjunction with genetic screens to identify molecular mechanisms regulating fungal cell adhesion.     

Gene annotation and drug target discovery in Candida albicans with a tagged transposon mutant collection

Candida albicans is the most common human fungal pathogen, causing infections that can be lethal in immunocompromised patients. Although Saccharomyces cerevisiae has been used as a model for C. albicans, it lacks C. albicans' diverse morphogenic forms and is primarily non-pathogenic. Comprehensive genetic analyses that have been instrumental for determining gene function in S. cerevisiae are hampered in C. albicans, due in part to limited resources to systematically assay phenotypes of loss-of-function alleles. Here, we constructed and screened a library of 3633 tagged heterozygous transposon disruption mutants, using them in a competitive growth assay to examine nutrient- and drug-dependent haploinsufficiency. We identified 269 genes that were haploinsufficient in four growth conditions, the majority of which were condition-specific. These screens identified two new genes necessary for filamentous growth as well as ten genes that function in essential processes. We also screened 57 chemically diverse compounds that more potently inhibited growth of C. albicans versus S. cerevisiae. For four of these compounds, we examined the genetic basis of this differential inhibition. Notably, Sec7p was identified as the target of brefeldin A in C. albicans screens, while S. cerevisiae screens with this compound failed to identify this target. We also uncovered a new C. albicans-specific target, Tfp1p, for the synthetic compound 0136-0228. These results highlight the value of haploinsufficiency screens directly in this pathogen for gene annotation and drug target identification.     

Isolation of the Candida albicans histidinol dehydrogenase (HIS4) gene and characterization of a histidine auxotroph.

Genetic studies were done with Candida albicans CBS 562. Various auxotrophs were isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. SAG5 (his4C), a stable histidine auxotroph defective in histidinol dehydrogenase activity, was characterized and chosen for further molecular studies. Therefore, the C. albicans HIS4 gene was isolated. The gene was obtained from a genomic library of the wild-type strain, which was constructed in plasmid YEp24. The HIS4 gene was isolated by transformation of a Saccharomyces cerevisiae strain that carried a his4 mutation. The isolated C. albicans HIS4 gene complemented S. cerevisiae his4A, his4B, his4C, and his4ABC mutant strains, which indicates that the clone contains the entire HIS4 gene. The gene was isolated on plasmid pSTC7, whose physical map was constructed with BamHI, SalI, and EcoRV restriction endonucleases, locating the HIS4 gene on a 14-kilobase-pair DNA fragment. Hybridization experiments with HIS4 and C. albicans genomic DNA showed correspondence between the restriction patterns of the gene with that of the chromosomal DNA, indicating that the gene originates from C. albicans and appears in a single copy. Chromosomes of C. albicans CBS562 and four other strains were resolved by orthogonal-field alteration gel electrophoresis. The electrokaryotyping results showed heterogeneity in chromosomal sizes. The electrokaryotyping of CBS 562 showed a resolution of six chromosomal bands, three of which seemed to be doublets. The C. albicans HIS4 gene was located on the largest resolvable chromosome in all of the strains.     

Many of the genes required for mating in Saccharomyces cerevisiae are also required for mating in Candida albicans

Candida albicans is the single, most frequently isolated human fungal pathogen. As with most fungal pathogens, the factors which contribute to pathogenesis in C. albicans are not known, despite more than a decade of molecular genetic analysis. Candida albicans was thought to be asexual until the discovery of the MTL loci homologous to the mating type (MAT) loci in Saccharomyces cerevisiae led to the demonstration that mating is possible. Using Candida albicans mutants in genes likely to be involved in mating, we analysed the process to determine its similarity to mating in Saccharomyces cerevisiae. We examined disruptions of three of the genes in the MAPK pathway which is involved in filamentous growth in both S. cerevisiae and C. albicans and is known to control pheromone response in the former fungus. Disruptions in HST7 and CPH1 blocked mating in both MTLa and MTL(alpha) strains, whereas disruptions in STE20 had no effect. A disruption in KEX2, a gene involved in processing the S. cerevisiae pheromone Mf(alpha), prevented mating in MTL(alpha) but not MTLa cells, whereas a disruption in HST6, the orthologue of the STE6 gene which encodes an ABC transporter responsible for secretion of the Mfa pheromone, prevented mating in MTLa but not in MTL(alpha) cells. Disruption of two cell wall genes, ALS1 and INT1, had no effect on mating, even though ALS1 was identified by similarity to the S. cerevisiae sexual agglutinin, SAG1. The results reveal that these two diverged yeasts show a surprising similarity in their mating processes.     

Targeted gene disruption in Candida albicans wild-type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates

Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by active drug efflux out of the cells. In clinical C. albicans strains, fluconazole resistance frequently correlates with constitutive activation of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not expressed detectably in fluconazole-susceptible isolates. However, the molecular changes causing MDR1 activation have not yet been elucidated, and direct proof for MDR1 expression being the cause of drug resistance in clinical C. albicans strains is lacking as a result of difficulties in the genetic manipulation of C. albicans wild-type strains. We have developed a new strategy for sequential gene disruption in C. albicans wild-type strains that is based on the repeated use of a dominant selection marker conferring resistance against mycophenolic acid upon transformants and its subsequent excision from the genome by FLP-mediated, site-specific recombination (MPAR-flipping). This mutagenesis strategy was used to generate homozygous mdr1/mdr1 mutants from two fluconazole-resistant clinical C. albicans isolates in which drug resistance correlated with stable, constitutive MDR1 activation. In both cases, disruption of the MDR1 gene resulted in enhanced susceptibility of the mutants against fluconazole, providing the first direct genetic proof that MDR1 mediates fluconazole resistance in clinical C. albicans strains. The new gene disruption strategy allows the generation of specific knock-out mutations in any C. albicans wild-type strain and therefore opens completely novel approaches for studying this most important human pathogenic fungus at the molecular level.     

Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans

Candida albicans is the most common human fungal pathogen and causes significant morbidity and mortality worldwide. Nevertheless, the basic principles of C. albicans pathogenesis remain poorly understood. Of central importance to the study of this organism is the ability to generate homozygous knockout mutants and to analyze them in a mammalian model of pathogenesis. C. albicans is diploid, and current strategies for gene deletion typically involve repeated use of the URA3 selectable marker. These procedures are often time-consuming and inefficient. Moreover, URA3 expression levels-which are susceptible to chromosome position effects-can themselves affect virulence, thereby complicating analysis of strains constructed with URA3 as a selectable marker. Here, we describe a set of newly developed reference strains (leu2Delta/leu2Delta, his1Delta/his1Delta; arg4Delta/arg4Delta, his1Delta/his1Delta; and arg4Delta/arg4Delta, leu2Delta/leu2Delta, his1Delta/his1Delta) that exhibit wild-type or nearly wild-type virulence in a mouse model. We also describe new disruption marker cassettes and a fusion PCR protocol that permit rapid and highly efficient generation of homozygous knockout mutations in the new C. albicans strains. We demonstrate these procedures for two well-studied genes, TUP1 and EFG1, as well as a novel gene, RBD1. These tools should permit large-scale genetic analysis of this important human pathogen.     

Cloning and expression of the 3-isopropylmalate dehydrogenase gene from Candida albicans

Abstract A variety of Saccharomyces cerevisiae genes e.g. HIS3, LEU2, TRP1, URA3 , are expressed in Escherichia coli and have been isolated by complementation of mutations in the corresponding E. coli genes [1]. The LEU2 gene was one of the first S. cerevisiae genes to be isolated in this way [2], and its isolation led to the development of transformation systems for S. cerevisiae [3,4]. The leuB gene in E. coli [5] and the LEU2 gene in S. cerevisiae [6] both code for 3-isopropylmalate dehydrogenase (3-IMDH; EC 1.1.1.85) which is essential for the biosynthesis of leucine in both organisms. This paper describes the cloning of a fragment of C. albicans DNA carrying the gene for 3-IMDH which will be useful in the development of transformation methods in C. albicans .     

Cloning of the Candida albicans homolog of Saccharomyces cerevisiae GSC1/FKS1 and its involvement in beta-1,3-glucan synthesis.

Saccharomyces cerevisiae GSC1 (also called FKS1) and GSC2 (also called FKS2) have been identified as the genes for putative catalytic subunits of beta-1,3-glucan synthase. We have cloned three Candida albicans genes, GSC1, GSL1, and GSL2, that have significant sequence homologies with S. cerevisiae GSC1/FKS1, GSC2/FKS2, and the recently identified FKSA of Aspergillus nidulans at both nucleotide and amino acid levels. Like S. cerevisiae Gsc/Fks proteins, none of the predicted products of C. albicans GSC1, GSL1, or GSL2 displayed obvious signal sequences at their N-terminal ends, but each product possessed 10 to 16 potential transmembrane helices with a relatively long cytoplasmic domain in the middle of the protein. Northern blotting demonstrated that C. albicans GSC1 and GSL1 but not GSL2 mRNAs were expressed in the growing yeast-phase cells. Three copies of GSC1 were found in the diploid genome of C. albicans CAI4. Although we could not establish the null mutation of C. albicans GSC1, disruption of two of the three GSC1 alleles decreased both GSC1 mRNA and cell wall beta-glucan levels by about 50%. The purified C. albicans beta-1,3-glucan synthase was a 210-kDa protein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and all sequences determined with peptides obtained by lysyl endopeptidase digestion of the 210-kDa protein were found in the deduced amino acid sequence of C. albicans Gsc1p. Furthermore, the monoclonal antibody raised against the purified beta-1,3-glucan synthase specifically reacted with the 210-kDa protein and could immunoprecipitate beta-1,3-glucan synthase activity. These results demonstrate that C. albicans GSC1 is the gene for a subunit of beta-1,3-glucan synthase.     

mazF as a counter-selectable marker for unmarked genetic modification of Pichia pastoris

In this study, we demonstrate a novel method for unmarked genetic modification of the methylotrophic yeast Pichia pastoris , in which the Escherichia coli toxin gene mazF was used as a counter-selectable marker. mazF was placed under the tightly controlled AOX1 promoter, and the induced expression of MazF in P. pastoris halted cell growth. A modular plasmid was constructed in which mazF and a Zeocin resistance gene acted as counter-selectable and active-selectable markers, respectively, and the MazF-ZeoR cassette was flanked by two direct repeats for marker recycling. Linearized delivery vectors constructed from the modular plasmid were integrated into the P. pastoris genome via homologous recombination, introducing genetic modifications. Upon counter-selection with methanol medium, which induces the AOX1 promoter, the markers were recycled efficiently via homologous recombination between the direct repeats. We used this method successfully to knock-out the ARG1 and MET2 genes, knock-in a green fluorescent protein expression cassette, and perform site-directed mutagenesis on the ARG1 gene, all without introducing unwanted selection markers. The novel method allows repeated use of the selectable marker gene for multiple modifications and will be a useful tool for P. pastoris studies.     

Identification and characterization of the genes for N-acetylglucosamine kinase and N-acetylglucosamine-phosphate deacetylase in the pathogenic fungus Candida albicans.

Like bacteria and many fungi, the pathogenic fungus Candida albicans can utilize GlcNAc as a carbon source for growth. A cluster of six genes was identified in the C. albicans genome. One of the genes in the cluster was CaNAG1, which is responsible for GlcN6P deaminase and is therefore essential for GlcNAc-dependent growth. The other five genes were designated CaNAG2, CaNAG3, CaNAG4, CaNAG5 and CaNAG6. The mRNA levels of CaNAG1, CaNAG2 and CaNAG5 were significantly induced by GlcNAc, whereas those of CaNAG3, CaNAG4 and CaNAG6 were not. Neither CaNAG2 nor CaNAG5 was essential for growth, but disruption of CaNAG2 or CaNAG5 greatly retarded the growth of cells using GlcNAc as the sole carbon source. Although no homolog of CaNAG2 or CaNAG5 was found in the Saccharomyces cerevisiae genome, CaNag2p displayed sequence similarities to Escherichia coli nagA, and CaNag5p is homologous to a wide variety of hexose kinases. When expressed as a fusion protein with glutathione S-transferase (GST), CaNag5p produced GlcNAc-P from GlcNAc in the presence of ATP, whereas GST alone did not. Furthermore, the recombinant GST-CaNag2p fusion protein converted GlcNAcP, which was produced by CaNag5p, into GlcNP. These results clearly demonstrate that CaNAG2 and CaNAG5 encode GlcNAcP deacetylase and GlcNAc kinase, respectively. CaNag5p recognized glucose and mannose as substrates, whereas the recently identified human GlcNAc kinase was specific to GlcNAc. Deletion of CaNAG2 or CaNAG5 markedly, and that of CaNAG1 moderately, attenuated the virulence of C. albicans in a mouse systemic infection model. Thus, it appears that GlcNAc metabolism of C. albicans is closely associated with its virulence.