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.     

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.     

Delayed hypersensitivity to Candida albicans

Conditions and factors influencing the formation of delayed hypersensitivity (DH) to yeast-like fungi of the species C. albicans under experimental conditions have been studied. The intensity of this reaction has been found to depend on the dose and method used for infecting mice, the time of the test and the qualitative state of fungal cells. As the result of this study, the infectious model of DH to C. albicans has been proposed. This model may be used for the study of the influence of different exogenous and endogenous factors of cell-mediated immune response to candidiasis.     

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.     

The slow road to the eukaryotic genome

The eukaryotic genome is a mosaic of eubacterial and archaeal genes in addition to those unique to itself. The mosaic may have arisen as the result of two prokaryotes merging their genomes, or from genes acquired from an endosymbiont of eubacterial origin. A third possibility is that the eukaryotic genome arose from successive events of lateral gene transfer over long periods of time. This theory does not exclude the endosymbiont, but questions whether it is necessary to explain the peculiar set of eukaryotic genes. We use phylogenetic studies and reconstructions of ancestral first appearances of genes on the prokaryotic phylogeny to assess evidence for the lateral gene transfer scenario. We find that phylogenies advanced to support fusion can also arise from a succession of lateral gene transfer events. Our reconstructions of ancestral first appearances of genes reveal that the various genes that make up the eukaryotic mosaic arose at different times and in diverse lineages on the prokaryotic tree, and were not available in a single lineage. Successive events of lateral gene transfer can explain the unusual mosaic structure of the eukaryotic genome, with its content linked to the immediate adaptive value of the genes its acquired. Progress in understanding eukaryotes may come from identifying ancestral features such as the eukaryotic splicesome that could explain why this lineage invaded, or created, the eukaryotic niche.     

The long hard road to the doability of interdisciplinary research projects: the case of biosocial criminology

This paper combines Bourdieu’s Field Theory with the concept of “doability” to investigate an interdisciplinary scientific project, namely biosocial criminology. Biosocial criminologists seek to incorporate genetic and neuroscientific findings in criminology. Drawing on literature analysis and semi-structured interviews, I show how the doability of this interdisciplinary research project, which is mainly determined by considerations surrounding data and technologies, is a function of biosocial researchers’ position within the scientific field. This is made apparent at two different levels. First, the availability of genetic data, which is attributable to behavior geneticists’ “generosity” and inter-field exchanges, largely explains why biosocial criminologists mostly focused on genetic factors of crime and neglected neuroscience, a more costly area of endeavor. Second, the problem of doability prevents biosocial criminologists from performing any kind of genetic research. Because behavior geneticists’ generosity has its limits too, biosocial criminologists cannot resort to the most advanced methodological designs.     

The long and arduous road to CRAC

Store-operated calcium (SOC) entry is the major route of calcium influx in non-excitable cells, especially immune cells. The best characterized store-operated current, I(CRAC), is carried by calcium release activated calcium (CRAC) channels. The existence of the phenomenon of store-operated calcium influx was proposed almost two decades ago. However, in spite of rigorous research by many laboratories, the identity of the key molecules participating in the process has remained a mystery. In all these years, multiple different approaches have been adopted by countless researchers to identify the molecular players in this fundamental process. Along the way, many crucial discoveries have been made, some of which have been summarized here. The last couple of years have seen significant breakthroughs in the field-identification of STIM1 as the store Ca(2+) sensor and CRACM1 (Orai1) as the pore-forming subunit of the CRAC channel. The field is now actively engaged in deciphering the gating mechanism of CRAC channels. We summarize here the latest progress in this direction.     

Phenotypic evaluation to differentiate Candida albicans from Candida dubliniensis

This study evaluated the phenotypic tests used to differentiate Candida albicans from Candida dubliniensis. A total of 55 isolates from vaginal secretions, oral cavity and hemoculture were studied. They were originally identified as C. albicans, based on their morphological and physiological characteristics. These isolates were tested for colony color development on CHROMagar Candida medium, growth at 45 degrees C on Sabouraud Dextrose agar, lipolytic activity on Tween 80 Agar medium and colony morphology and chlamydoconidia formation on Staib agar medium. Of the 55 isolates studied, seven yielded one or more phenotypic characteristics suggestive of Candida dubliniensis. These isolates were tested by PCR with specific primers for Candida dubliniensis and API ID 32. The seven isolates were confirmed as Candida albicans. All of these finding indicate that DNA based tests should be used for definitive identification of Candida dubliniensis.     

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.     

The parasexual lifestyle of Candida albicans

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The ultrastructure of Candida albicans infections

Scrapings of Candida albicans plaques from the tongue and buccal mucosa of patients with oral candidiasis were examined by electron microscopy. In addition, urine sediment from patients with infection of their catheterized urinary tracts was similarly examined. Three types of C. albicans-oral epithelial cell interactions were noted: a loose adherence apparently mediated by a ruthenium red positive matrix, a "tight" adherence where no space could be seen between the host and yeast cell. and invasion of host cells by yeast hyphal elements. Adhesion of Candida blastospores to hyphal elements and adhesion of bacteria to Candida cells was also frequently observed. Urine sediments from patients with mixed bacteria-yeast infections demonstrated adhesion of the bacteria to the yeast cells. This phenomenon was also demonstrated in in vitro experiments and fibrous ruthenium red material invariably occupied the zone of adhesion. Phagocytosis of yeast by polymorphonuclear leukocytes was found in urinary, but not in oral. candidiasis. Our in vivo and in vitro observations indicate that a ruthenium red positive matrix covers the surfaces involved in the yeast to yeast, yeast to host, and yeast to bacteria adhesion.     

The contribution of the S-phase checkpoint genes MEC1 and SGS1 to genome stability maintenance in Candida albicans

Genome rearrangements, a common feature of Candida albicans isolates, are often associated with the acquisition of antifungal drug resistance. In Saccharomyces cerevisiae, perturbations in the S-phase checkpoints result in the same sort of Gross Chromosomal Rearrangements (GCRs) observed in C. albicans. Several proteins are involved in the S. cerevisiae cell cycle checkpoints, including Mec1p, a protein kinase of the PIKK (phosphatidyl inositol 3-kinase-like kinase) family and the central player in the DNA damage checkpoint. Sgs1p, the ortholog of BLM, the Bloom's syndrome gene, is a RecQ-related DNA helicase; cells from BLM patients are characterized by an increase in genome instability. Yeast strains bearing deletions in MEC1 or SGS1 are viable (in contrast to the inviability seen with loss of MEC1 in S. cerevisiae) but the different deletion mutants have significantly different phenotypes. The mec1Δ/Δ colonies have a wild-type colony morphology, while the sgs1Δ/Δ mutants are slow-growing, producing wrinkled colonies with pseudohyphal-like cells. The mec1Δ/Δ mutants are only sensitive to ethylmethane sulfonate (EMS), methylmethane sulfonate (MMS), and hydroxyurea (HU) but the sgs1Δ/Δ mutants exhibit a high sensitivity to all DNA-damaging agents tested. In an assay for chromosome 1 integrity, the mec1Δ/Δ mutants exhibit an increase in genome instability; no change was observed in the sgs1Δ/Δ mutants. Finally, loss of MEC1 does not affect sensitivity to the antifungal drug fluconazole, while loss of SGS1 leads to an increased susceptibility to fluconazole. Neither deletion elevated the level of antifungal drug resistance acquisition.     

In-vitro susceptibility of Candida albicans and Candida stellatoidea to sulfamethoxazole

The susceptibility of organisms of the speciesCandida to sulfamethoxazole were tested in-vitro using the disc sensitivity method.Under specified conditions a zone of growth inhibition surrounding sulfamethoxazole tablet sensi disc was consistently produced in cultures ofCandida albicans, Candida stellatoidea andSaccharomyces cervisiae.Diameters of growth inhibition zone were measured and found to be greatest in cultures ofCandida stellatoidea andSaccharomyces, but growth inhibition inCandida albicans cultures was observed to a lesser degree.The correlation between in vivo pathogenicity of organisms of the speciesCandida, and degree of growth inhibition by sulfamethoxazole sensi disc presents an interesting relationship.