A human-curated annotation of the Candida albicans genome

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications.     

Site-specific targeting of exogenous DNA into the genome of Candida albicans using the FLP recombinase

We have created a system that utilizes the FLP recombinase of Saccharomyces cerevisiae to reversibly introduce exogenous cloned DNA at defined locations into the Candida albicans genome. Recombination target (FRT) sites and the FLP gene can be introduced permanently at defined locations using homologous recombination. FLP recombinase is provided as needed through the regulated expression of its gene using the MAL2 promoter. Exogenous DNA is introduced on a cloning vector that is unable to replicate in C. albicans, and contains an FRT site and a selectable marker (URA3). Transformation by the lithium acetate or electroporation procedure is sufficient to obtain site-specific integration. This system permits rapid and precise excision of the introduced DNA when needed. It should facilitate studies on C. albicans genome structure and function, simplifying a wide range of chromosomal cloning applications, and generally enhancing the utility of C. albicans as a model organism for the study of fungal pathogenicity.     

Construction of an SfiI macrorestriction map of the Candida albicans genome.

The opportunistic fungal pathogen, Candida albicans, is diploid as usually isolated and has no apparent sexual cycle. Genetic analysis has therefore been very difficult. Molecular genetics has yielded important information in the past few years, but it too is hampered by the lack of a good genetic map. Using the well-characterized strain 1006 and strain WO-1, which undergoes the white-opaque phenotypic transition, we have developed a genomic restriction map of C. albicans with the enzyme SfiI. There are approximately 34 SfiI restriction sites in the C. albicans genome. Restriction fragments were separated by pulsed-field electrophoresis and were assigned to chromosomes by hybridization of complete and partial digests with known chromosome-specific probes as well as by digestion of isolated chromosomes. Telomeric fragments were identified by hybridization with a telomere-specific probe (C. Sadhu, M.J. McEachern, E.P. Rustchenko-Bulgac, J. Schmid, D.R. Soll, and J.B. Hicks, J. Bacteriol. 173:842-850, 1991). WO-1 differs from 1006 in that it has undergone three reciprocal chromosomal translocations. Analysis of the translocation products indicates that each translocation has occurred at or near an SfiI site; thus, the SfiI fragments from the two strains are similar or identical. The tendency for translocation to occur at or near SfiI sites may be related to the repeated sequence RPS 1, which contains four such sites and could provide homology for ectopic pairing and crossing over. The genome size of both strains is about 16 to 17 megabases, in good agreement with previous determinations.     

Infrequent genetic exchange and recombination in the mitochondrial genome of Candida albicans

Previous analyses of diploid nuclear genotypes have concluded that recombination has occurred in populations of the yeast Candida albicans. To address the possibilities of clonality and recombination in an effectively haploid genome, we sequenced seven regions of mitochondrial DNA (mtDNA) in 45 strains of C. albicans from human immunodeficiency virus-positive patients in Toronto, Canada, and 3 standard reference isolates of C. albicans, CA, CAI4, and WO-1. Among a total of 2,553 nucleotides in the seven regions, 62 polymorphic nucleotide sites and seven indels defined nine distinct mtDNA haplotypes among the 48 strains. Five of these haplotypes occurred in more than one strain, indicating clonal proliferation of mtDNA. Phylogenetic analysis of mtDNA haplotypes resulted in one most-parsimonious tree. Most of the nucleotide sites undergoing parallel change in this tree were clustered in blocks that corresponded to sequenced regions. Because of the existence of these blocks, the apparent homoplasy can be attributed to infrequent, past genetic exchange and recombination between individuals and cannot be attributed to parallel mutation. Among strains sharing the same mtDNA haplotypes, multilocus nuclear genotypes were more similar than expected from a random comparison of nuclear DNA genotypes, suggesting that clonal proliferation of the mitochondrial genome was accompanied by clonal proliferation of the nuclear genome.     

An electrophoretic karyotype for Candida albicans reveals large chromosomes in multiples

Summary Using field-inversion gel electrophoresis we defined an electrophoretic karyotype for the yeast, Candida albicans. The karyotype is distinct from other species of Candida and is species specific. A total of five distinct chromosomal mobility groups were observed, at least four of which are composed of a minimum of two fragments each. From the apparent sizes of these fragments relative to the large chromosomes of the morphologically related yeast Saccharomyces cerevisiae, together with the known genome size of this organism, we conclude that the karyotype is the result of the migration of intact chromosomes.     

Assignment of cloned genes to the seven electrophoretically separated Candida albicans chromosomes.

By using orthogonal-field alternating gel electrophoresis (OFAGE), field-inversion gel electrophoresis (FIGE), and contour-clamped homogeneous field gel electrophoresis (CHEF), we have clearly resolved 11 chromosomal bands from various Candida albicans strains. OFAGE resolves the smaller chromosomes better, while FIGE, which under our conditions causes the chromosomes to run in the reverse order of OFAGE, is more effective in separating the larger chromosomes. CHEF separates all chromosomes under some conditions, but these conditions do not often resolve homologs. The strains examined are highly polymorphic for chromosome size. Fourteen cloned Candida genes, isolated on the basis of conferral of new properties to or complementation of auxotrophic deficiencies in Saccharomyces cerevisiae, and three sequences of unknown function have been hybridized to Southern transfers of CHEF, FIGE, and OFAGE gels. Four sets of resolvable bands have been shown to be homologous chromosomes. On the basis of these data, we suggest that C. albicans has seven chromosomes. Genes have been assigned to the seven chromosomes. Two chromosomes identified genetically have been located on the electrophoretic karyotype.     

Assembly of a phased diploid Candida albicans genome facilitates allele-specific measurements and provides a simple model for repeat and indel structure

Background

Candida albicans is a ubiquitous opportunistic fungal pathogen that afflicts immunocompromised human hosts. With rare and transient exceptions the yeast is diploid, yet despite its clinical relevance the respective sequences of its two homologous chromosomes have not been completely resolved.

Results

We construct a phased diploid genome assembly by deep sequencing a standard laboratory wild-type strain and a panel of strains homozygous for particular chromosomes. The assembly has 700-fold coverage on average, allowing extensive revision and expansion of the number of known SNPs and indels. This phased genome significantly enhances the sensitivity and specificity of allele-specific expression measurements by enabling pooling and cross-validation of signal across multiple polymorphic sites. Additionally, the diploid assembly reveals pervasive and unexpected patterns in allelic differences between homologous chromosomes. Firstly, we see striking clustering of indels, concentrated primarily in the repeat sequences in promoters. Secondly, both indels and their repeat-sequence substrate are enriched near replication origins. Finally, we reveal an intimate link between repeat sequences and indels, which argues that repeat length is under selective pressure for most eukaryotes. This connection is described by a concise one-parameter model that explains repeat-sequence abundance in C. albicans as a function of the indel rate, and provides a general framework to interpret repeat abundance in species ranging from bacteria to humans.

Conclusions

The phased genome assembly and insights into repeat plasticity will be valuable for better understanding allele-specific phenomena and genome evolution.     

Simple rapid identification of Candida albicans with emphasis on the differentiation between Candida albicans and Candida stellatoidea

Summary Subcultures ofC. albicans, made from Sabouraud agar, grown at room temperature for 48 hours, were inoculated into a 10 times saline dilution of Sabouraud liquid medium and left in the incubator for 45–60 minutes at 37° C, transferred to corn meal agar plates and incubated at 37° C for 18–24 hours.Small portions of the surface agar containing the yeasts from these plates were pressed under cover glasses and examined under the oil immersion lens.Under these conditions,C. albicans cultures were observed to produce only yeast-like cells, whereasC. stellatoidea cultures contained predominantly abundant, long, thin mycelia.     

Circular mitochondrial genome of Candida albicans contains a large inverted duplication.

The mitochondrial DNA (mtDNA) of the dimorphic fungus Candida albicans has a molecular size of 41 kilobase pairs as judged by summation of the fragment sizes produced by digestion with restriction endonucleases EcoRI, PvuII, and a combination of both enzymes. Five of the six EcoRI fragments comprising the mitochondrial genome have been cloned into the plasmid vector, pBR322. Restriction mapping revealed a circular map as predicted by previous observations with the electron microscope. The use of nick-translated, purified mtDNA to probe digests of mtDNA from other strains of C. albicans revealed a common restriction pattern. Use of nick-translated, cloned EcoRI fragments to probe digests of mtDNA revealed a large (at least 5 kilobase pairs), inverted duplication as well as a smaller (less than 0.4 kilobase pairs) region of related sequences.     

白色念珠菌拮抗菌株的筛选

目的筛选对白色念珠菌具有明显拮抗作用的菌株.方法通过纸片琼脂扩散法(K-B法)观察各菌株对白色念珠菌的拮抗作用;再利用试管法观察有拮抗作用菌株对白色念珠菌的抑菌效果.结果 K-B法表明大肠埃希菌、甲型链球菌和卡他球菌对白色念珠菌无抑菌作用,表皮葡萄球菌、微球菌有抑菌作用,但抑菌环小;枯草杆菌有明显抑菌作用.试管法表明表皮葡萄球菌和枯草杆菌对白色念珠菌均有生物拮抗作用,其抑菌率分别为97%和89.7%.结论枯草杆菌是白色念珠菌的理想拮抗菌株.     

Insight into the antiadhesive effect of yeast wall protein 1 of Candida albicans

Ywp1 is a prominent glycosylphosphatidylinositol (GPI)-anchored glycoprotein of the cell wall of Candida albicans; it is present in the yeast form of this opportunistic fungal pathogen but absent from filamentous forms and chlamydospores. Yeast cells that lack Ywp1 are more adhesive and form thicker biofilms, implying an antiadhesive activity for Ywp1, with a possible role in yeast dispersal. The antiadhesive effect of Ywp1 is transplantable from yeast to hyphae, as hyphae that are forced to express YWP1 lose adhesion in an in vitro assay. Deletion of the GPI anchor results in loss of Ywp1 to the surrounding medium and reduction of the antiadhesive effect, implying an importance of time-dependent residency in the cell wall. Anchor-negative versions of Ywp1 possessing or lacking a C-terminal green fluorescent protein (GFP) tag were created in C. albicans and harvested from culture supernatants; in addition to serving as quantifiable markers for Ywp1 secretion, they revealed that the cleaved 11-kDa propeptide of Ywp1 remains strongly but noncovalently associated with the Ywp1 core. This association is resistant to highly acidic and basic solutions, 8 M urea, and 1% SDS (below 45°C). Above 50°C, SDS dissociates the isolated complex, but even higher temperatures are required to dissociate the propeptide from native Ywp1 that is anchored in a cell wall. This property has permitted detection, for the first time, of orthologs of Ywp1 in other members of the Candida clade. The cleaved propeptide, which carries the sole N-glycan of Ywp1, must participate in the antiadhesive effect of Ywp1.     

紫外线对白念珠菌的影响

紫外线作为重要的环境因子之一,能显著影响包括白念珠菌在内的多种生物的生长及生理过程.研究发现白念珠菌受紫外线照射后菌丝形成被抑制,孢子形成增多且没有向光性;脉冲紫外线辐射可通过多靶点程序使白念珠菌失活;rad 51缺陷株比rad 52缺陷株更易受紫外线损害,同时紫外线可导致白念珠菌杂合性丢失.研究还发现UVC治疗可明显减少烧伤后真菌微生物感染,核黄素/UVA治疗可明显抑制白念珠菌生长.因此紫外线对白念珠菌有一定的抑制作用.     

Effect of antifungal gels incorporated into a tissue conditioning material on the growth of Candida albicans

doi:10.1111/j.1741‐2358.2009.00337.x
Effect of antifungal gels incorporated into a tissue conditioning material on the growth of Candida albicans Objective: The aim of this study was to examine the effectiveness of antifungal gels incorporated into a tissue conditioner which inhibits the growth of Candida albicans in vitro. Background: The release of drugs from relining materials has been demonstrated earlier. However, the incorporation of antifungal agents in gel form has not yet been studied. Materials and methods: Visco‐gel^® tissue conditioner was prepared with chlorhexidine digluconate and miconazole in gel form in a concentration of 5, 10, 15, 20 and 25% by volume. Sample discs were prepared and placed on Sabouraud Dextrose Agar (SDA) plates which had been previously inoculated with C. albicans, and incubated aerobically at 37°C. To investigate antifungal activity over time, Visco‐gel discs containing 20%v/v miconazole were prepared and immersed in water for different time periods before being placed on SDA plates inoculated with C. albicans. Results: Chlorhexidine digluconate gel added to tissue conditioner had no inhibition effect on the growth of C. albicans. Incorporation of miconazole gave a dose‐related inhibitory effect on candidal growth. Immersion of the discs in water showed an inverse relationship between time of immersion and degree of inhibition. Conclusion: Miconazole added in gel form to Visco‐gel^® had an inhibitory effect on the growth of C. albicans in vitro.     

Molecular phylogenetics of Candida albicans

We analyzed data on multilocus sequence typing (MLST), ABC typing, mating type-like locus (MAT) status, and antifungal susceptibility for a panel of 1,391 Candida albicans isolates. Almost all (96.7%) of the isolates could be assigned by MLST to one of 17 clades. eBURST analysis revealed 53 clonal clusters. Diploid sequence type 69 was the most common MLST strain type and the founder of the largest clonal cluster, and examples were found among isolates from all parts of the world. ABC types and geographical origins showed statistically significant variations among clades by univariate analysis of variance, but anatomical source and antifungal susceptibility data were not significantly associated. A separate analysis limited to European isolates, thereby minimizing geographical effects, showed significant differences in the proportions of isolates from blood, commensal carriage, and superficial infections among the five most populous clades. The proportion of isolates with low antifungal susceptibility was highest for MAT homozygous a/a types and then alpha/alpha types and was lowest for heterozygous a/alpha types. The tree of clades defined by MLST was not congruent with trees generated from the individual gene fragments sequenced, implying a separate evolutionary history for each fragment. Analysis of nucleic acid variation among loci and within loci supported recombination. Computational haplotype analysis showed a high frequency of recombination events, suggesting that isolates had mixed evolutionary histories resembling those of a sexually reproducing species.