Joy Sturtevant

The Candida albicans 14-3-3 gene, BMH1, is essential for growth

The 14‐3‐3 proteins are a family of conserved small acidic proteins that have been implicated in playing major roles in a wide variety of signalling cascades. In Saccharomyces cerevisiae, the 14‐3‐3 genes (BMH1 and BMH2) are essential for normal pseudohyphal induction and normal bud cell development. The Bmh proteins function in the cAMP‐dependent RAS/MAPK and rapamycin‐sensitive signalling cascades. Deletion of only one BMH gene demonstrates no phenotypic differences under normal growth conditions. Strains deleted of both BMH1 and BMH2 are either non‐viable or demonstrate sensitivity to environmental stresses. In Schizosaccharomyces pombe, the BMH homologues (RAD24 and RAD25) are essential for cell cycle control after DNA damage and deletion of both genes renders the cell inviable. The 14‐3‐3 gene in Candida albicans (BMH1) was identified using a novel adherence assay and differential display RT–PCR. Unlike other yeasts, C. albicans has only one 14‐3‐3 gene (BMH1). It was not possible to construct double knockouts by routine methods. These results suggested that the C. albicans BMH1 gene is essential. The essentiality of C. albicans BMH1 was confirmed by a PCR disruption technique. The C. albicans bmh1Δ/BMH1 heterozygotes exhibit growth and morphogenetic defects. Therefore, the BMH1 gene in C. albicans (Accession No. AF038154) isan excellent candidate to improve our understanding of the coordinate regulation of cell cycle and morphogenesis. Copyright

Applications of Differential-Display Reverse Transcription-PCR to Molecular Pathogenesis and Medical Mycology

The host-fungus interaction is characterized by changes in gene expression in both host and pathogen. Differential-display reverse transcription PCR (DDRT-PCR) is a PCR-based method that allows extensive analysis of gene expression among several cell populations. Several limitations and drawbacks to this procedure have now been addressed, including the large number of false-positive results and the difficulty in confirming differential expression. Modifications that simplify the reaction time, allow the use of minute quantities of RNA, or address unusual species- or gene-specific sequences have been reported. DDRT-PCR has been used to address biological questions in mammalian systems, including cell differentiation, cell activation, cell stress, and identification of drug targets. In microbial pathogenesis and plant pathogenesis, DDRT-PCR has allowed the identification of virulence factors, genes involved in cell death, and signaling genes. In Candida albicans, DDRT-PCR studies identified TIF-2, which may play a role in the upregulation of phospholipases, and the stress-related genes, CIP1 and CIP2. In Histoplasma capsulatum and C. albicans, genes involved in the host-pathogen interaction, including a member of the 100-kDa family in Histoplasma and an ALS and 14-3-3 gene in Candida, were potentially identified by DDRT-PCR. Although very few reports have been published in medical mycology, studies in mammalian, nonfungal microbial, and plant pathogen systems are easily applied to basic questions in fungal pathogenesis and antifungal therapeutics.

Disruption studies of a Candida albicans gene, ELF1 : a member of the ATP-binding cassette family

A 3.6 kb gene (ELF1) with homology to the ATP-binding cassette (ABC) gene family has been isolated from genomic libraries of Candida albicans. Members of this gene family include both membrane transport proteins which confer a drug-resistance phenotype, and proteins whose functions are associated with protein translation. ELF1 (Elongation Like Factor) showed greatest homology with a Saccharomyces cerevisiae ORF (YPL226W), whose function is unknown, and lower homology with fungal elongation factor 3 (EF-3) genes. In comparison, homology with a gene conferring a drug-resistant phenotype (CDR1) was low. To understand the function of ELF1 in C. albicans, gene-knockout experiments were conducted using the hisG-URA3-hisG disruption cassette. Both single-copy (heterozygote) and double-disrupted strains in ELF1 were isolated. Phenotypically, the disrupted strains grew more slowly than wild-type and produced a mixture of large, irregular cells and apparently normal cells.

Random mutagenesis of an essential Candida albicans gene

A method for the analysis of Candida albicans gene function, which involves random mutagenesis of the open reading frame, is described. This method is especially suited for the study of essential and multi-functional genes, with several advantages over regulatable promoters more commonly used to study essential gene function. These advantages include expression from the endogenous promoter, which should yield a more appropriate transcript expression and abrogate the need for shifts in carbon or amino acid sources necessary with the use of regulatable promoters. Furthermore, there is potential for isolating individual functions of multi-functional genes. To verify this experimental approach, we randomly mutated the essential C. albicans gene, BMH1. The resulting “pool” of putative mutant alleles was then introduced into a BMH1/bmh1Δ strain of C. albicans, such that only the mutagenized BMH1 sequences could be expressed. Transformants were screened for rapamycin sensitivity, defects in filamentation on M199 agar, and growth at 42°C. In this way, we identified six non-lethal mutant alleles of BMH1 with altered amino acid sequences. Further phenotypic analysis of these mutant strains enabled us to segregate individual functions of C. albicans BMH1. The relative merits of Escherichia coli versus PCR-mediated mutagenesis are discussed.

Reporter Gene Assays in Candida albicans

Reporter systems are used in Candida albicans in three major experimental areas. These include gene expression, promoter analysis, and protein expression/localization. Heterologous expression in C. albicans is either not effective or inefficient due to the alternative codon usage in Candida, particularly CTG. Consequently, several reporter genes have been constructed by optimizing codons for expression in Candida. The reporter systems include lacZ, luciferase, and GFP. Generally, PCR site directed mutagenesis has been used to construct the modified reporter. Reporter gene vectors are not commercially available for Candida, but they can normally be requested from the laboratories that developed the constructs.

Meeting Report: Candida and Candidiasis

Candida and Candidiasis Conference was a well organized meeting held in Austin, Texas in March 2004. The meeting began with comprehensive overviews of what we know about Candida in the areas of basic, clinical, and pharmaceutical research presented by Drs. Soll, Rex, and Hitchcock [1]. Throughout the 4 days, all aspects of present Candida research were covered. In order to emphasize current studies, only two speakers in each session were predetermined; the remaining three were selected from submitted abstracts. Three clinical presentations challenged basic researchers to redirect their experimental approaches to address existing problems in the wards. There was an outside the box session led by Drs. Casadevall and Haynes on ‘‘Candida and its hosts’’, which took the virulence session from the Conference in 2002 one step further. They challenged our concepts of how we classify ourselves as scientists (‘gene bashers’ or ‘host defenders’); and on why/when we use animals in experimentation. There were workshops from NICDR on funding, new developments by Merck, and a progress report on the Candida annotation working group. Just in case we weren’t learning enough about Candida there were three poster sessions, which included close to 250 presentations. Finally, there was plenty of opportunity to discuss Candida and all its aspects at lunch and down at the bar. Before I begin an overview of the scientific content of the meeting, I start with a disclaimer. It will be impossible to discuss, let alone mention every significant observation reported in this meeting. However, I will try to touch on the main topics that were discussed in the context of both Candida and candidiasis. Perhaps the greatest boost to our current research is the development of new tools to address old problems. However, we must remember to ask questions before deciding on corresponding experimental procedures. These questions should not only address biological, but also clinical significance. Nomodel will be perfect to answer all the mysteries of candidiasis but specific models will supply important information if the appropriate question(s) is asked. The clinical overviews presented by Drs. Ruhnke, Sobel, and Edwards, confirmed that more work needs to be done, and epidemiological studies also tell us there is still a problem. Those that research with C. glabrata and C. parapsilosis are certainly still in business. However, we are not much better off with treatment strategies and certainly no better off with diagnosis. At first glance of the program, one would think that there is not much work on the level of the host Mycopathologia 158: 141–146, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands. 141

Strategies for the Study of Gene Expression in Fungi

The occurrence of fungal infections has steadily escalated over the last two decades. This is due in large part to the fact that those immunocompromised by either disease or therapies are particularly at risk for such infections. Concurrently, there has been increased interest in defining those genes and gene products that contribute to pathogenesis of these organisms. Not only does analysis of such genes provide insight concerning the interaction of the fungal pathogen and its host, but determination of genes required for pathogenesis can also point to possible novel antifungal drug targets.

SRCI: An intron-containing yeast gene involved in sister chromatid segregation

Analysis of a three‐member gene family in the yeast Saccharomyces cerevisiae has allowed the discovery of a new gene that comprises two contiguous open reading frames previously annotated as YML034w and YML033w. The gene contains a small intron with two alternative 5′ splicing sites. It is specifically transcribed during G2/M in the cell cycle and after several hours of meiosis induction. Splicing of the mRNA is partially dependent on NAM8 but does not vary during meiosis or the cell cycle. Deletion of the gene induces a shortening of the anaphase and aggravates the phenotype of scc1 and esp1 conditional mutants, which suggests a direct role of the protein in sister chromatid separation. Copyright