UnderstandingCandida albicans at the Molecular Level

Pla, Jesús; Gil, Concha; Monteoliva, Lucía; Navarro‐García, Federico; Sánchez, Miguel; Nombela, César

doi:10.1002/(sici)1097-0061(199612)12:16<1677::aid-yea79>3.0.co;2-u

Cited by 62 publications

(5 citation statements)

References 282 publications

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“…Identification of essential genes in C. albicans is vital to assess prospective drug targets (30) and to consider more-refined functional tests (1). C. albicans genes have been considered essential if one allele can be disrupted but the second cannot.…”

Section: Resultsmentioning

confidence: 99%

“…In the past, this analysis has required two successive transformations to create homozygous null mutations or conditionally expressed alleles (1,7,30,43). These methods allow construction of precisely defined mutant strains that can be characterized via a battery of refined phenotypic tests.…”

Section: Discussionmentioning

confidence: 99%

“…We refer to this test as the HT test. The methodology fills an important need for basic research, because it is otherwise laborious to determine whether a gene is essential (29,30), and current technology can yield conflicting results (5,16,22,38). The methodology also fills an important need for genome-based pharmaceutical research, in which one must evaluate a large number of gene products as prospective drug targets (23).…”

Section: Discussionmentioning

confidence: 99%

“…However, genetic methods are cumbersome for two reasons (30). First, C. albicans strains are diploid (or of higher ploidy) and there is no meiotic division, so that gene disruption mutants must be constructed through two successive transformations.…”

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confidence: 99%

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A Single-Transformation Gene Function Test in Diploid Candida albicans

2000

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The fungal pathogen Candida albicans is naturally diploid, and current gene disruption strategies require two successive transformations. We describe here a genetic construct (UAU1) for which two copies may be selected. Insertion of UAU1 into one genomic site, after a single transformation, allows selection for segregants with two copies of the insertion. Major classes of segregants are those carrying homozygous insertion mutations and allelic triplications, which have two insertion alleles and a wild-type allele. Thus nonessential and essential genes may be distinguished rapidly through PCR tests for homozygosis and triplication. We find that homozygous mutations may be isolated at three nonessential loci (ADE2, RIM20, and YGR189), while only allelic triplications were found at two essential loci (SNF1 and CDC28). We have unexpectedly isolated homozygous mutants with mutations at CDC25; they are viable but defective in filamentation on serum-containing medium. The UAU1 cassette is thus useful to assess rapidly the essentiality of C. albicans genes.Candida albicans is an opportunistic fungal pathogen. It has been of experimental interest for two main reasons. First, it is a significant pathogen that causes oral mucosal infections, vaginitis, nosocomial bloodstream infections, and a variety of deep tissue infections (26). Therefore, many experimental studies have focused on pathogenesis, drug resistance, and analysis of prospective drug targets. Second, it is a distant cousin of the most well-characterized unicellular eukaryote, Saccharomyces cerevisiae, so that the function of a C. albicans gene may be suggested by its role in S. cerevisiae. Yet, surprisingly, C. albicans may use gene products and regulatory pathways in novel ways (4,16,17,28,34,37). The contrast between C. albicans and S. cerevisiae can provide unique insight into regulatory mechanisms, interpathway relationships, and general aspects of eukaryotic biology.Molecular genetics has played an increasingly prominent role in studies of C. albicans, particularly with the partial genomic sequence as a tool for gene discovery. However, genetic methods are cumbersome for two reasons (30). First, C. albicans strains are diploid (or of higher ploidy) and there is no meiotic division, so that gene disruption mutants must be constructed through two successive transformations. Second, with current methods, disruption of one allele may be straightforward, but disruption of the second allele is infrequent if the second disruption construct is homologous to the first. These problems can make it difficult to construct a homozygous mutant and to determine whether a gene is essential for growth.We describe here a genetic strategy that circumvents these difficulties. It yields homozygous insertion mutations after a single transformation. The strategy provides a rapid test for essential genes and should thus accelerate drug target validation. In addition, the strategy can provide a preliminary assessment of mutant phenotypes, and it lends itself to large-scale analysi...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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A Single-Transformation Gene Function Test in Diploid Candida albicans

2000

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“…albicans strain SC5314 was used because of its widespread use in genetic and virulence studies [13]. Cytoplasmic extracts were obtained as described previously [6].…”

Section: Sample Preparation and 2-d Pagementioning

confidence: 99%

Cross-species identification of novelCandida albicans immunogenic proteins by combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry

et al. 2000

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We have previously reported the usefulness of two-dimensional gel electrophoresis followed by Western blotting with sera from patients with systemic candidiasis in the detection of the major Candida albicans antigens (Pitarch et al., Electrophoresis 1999, 20, 1001-1010). The identification of these antigens would be useful for the characterization of good markers for the disease, and for the development of efficient diagnostic strategies. In this work we have used nanoelectrospray tandem mass spectrometry to obtain amino acid sequence information from the immunogenic proteins previously detected. We report here the cross-species identification of these antigens by matching of tandem mass spectrometry data to Saccharomyces cerevisiae proteins. Using this approach, we unambiguously identified the four C. albicans immunogenic proteins analyzed, namely aconitase, pyruvate kinase, phosphoglycerate mutase and methionine synthase. Furthermore, we report for the first time that aconitase, methionine synthase and phosphoglycerate mutase have antigenic properties in C. albicans.

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