Structure and regulation of theCandida albicans ADH1 gene encoding an immunogenic alcohol dehydrogenase

Bertram, Gwyneth; Swoboda, Rolf; Gooday, Graham W.; Gow, Neil A. R.; Brown, Alistair J. P.

doi:10.1002/(sici)1097-0061(199602)12:2<115::aid-yea889>3.0.co;2-e

Cited by 84 publications

(63 citation statements)

References 78 publications

(83 reference statements)

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“…Similarly, the C. albicans HST7 expression plasmid pDH186 was constructed by subcloning a KpnI-SalI fragment of HST7 into pVEC. To construct plasmid pYPB1-ADHpt-HST7 carrying HST7 under control of the ADH1 promoter, the coding region of HST7 flanked by BamHI sites was amplified by PCR by using the oligodeoxynucleotide primers 5Ј-GCTCATTCATCA-TGGATCCAACAATGACAAG-3Ј and 5Ј-GTATATGTATA-TGGGATCCGTTTACACTTTGC-3Ј (the newly introduced BamHI sites are underlined), and cloned into the BglII site of the C. albicans expression vector pYPB1-ADHpt containing the C. albicans ADH1 promoter and terminator regions (14), an autonomously replicating sequence, C. albicans URA3 as selectable marker, and S. cerevisiae 2 m sequences (G. Bertram, I.D.B., P. J. F. Feldmann, and A.J.P.B., unpublished).…”

Section: Methodsmentioning

confidence: 99%

Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans

Leberer

Harcus

Broadbent

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

305

318

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The CST20 gene of Candida albicans was cloned by functional complementation of a deletion of the STE20 gene in Saccharomyces cerevisiae. CST20 encodes a homolog of the Ste20p͞p65 PAK family of protein kinases. Colonies of C. albicans cells deleted for CST20 revealed defects in the lateral formation of mycelia on synthetic solid ''Spider'' media. However, hyphal development was not impaired in some other media. A similar phenotype was caused by deletion of HST7, encoding a functional homolog of the S. cerevisiae Ste7p protein kinase. Overexpression of HST7 partially complemented the deletion of CST20. Cells deleted for CST20 were less virulent in a mouse model for systemic candidiasis. Our results suggest that more than one signaling pathway can trigger hyphal development in C. albicans, one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelial formation in asexual C. albicans.Candida albicans is the major fungal pathogen in humans, causing various forms of candidiasis. This fungus is diploid with no sexual cycle and is capable of a morphological transition from a unicellular budding yeast to a filamentous form. Extensive filamentous growth leads to the formation of a mycelium displaying hyphae with branches and lateral buds. In view of the observation that hyphae seem to adhere to and invade host tissues more readily than does the yeast form, the switch from the yeast to the filamentous form probably contributes to the virulence of this organism (for a review, see ref. 1).Like C. albicans, bakers yeast Saccharomyces cerevisiae is also a dimorphic organism capable of switching under certain nutritional conditions from a budding yeast to a filamentous form. Under the control of nutritional signals, diploid cells switch to pseudohyphal growth (2), and haploid cells to invasive growth (3).The similarities between the dimorphic switching of S. cerevisiae and C. albicans suggest that these morphological pathways may be regulated by similar mechanisms in both organisms. In S. cerevisiae, morphological transitions are controlled by signaling components that are also involved in the mating response of haploid cells (3, 4). The switch to pseudohyphal growth requires a transcription factor encoded by the STE12 gene, and a mitogenactivated protein (MAP) kinase cascade including Ste7p (a homolog of MAP kinase kinase or MEK), Ste11p (a MEK kinase homolog), and Ste20p (a MEK kinase kinase) (3, 4). The MAP kinases involved in this response are as yet unknown (3, 4).We have recently shown that C. albicans contains a functional homolog of Ste7p (Hst7p; ref. 5). Cph1p, a homolog of the Ste12p transcription factor, is required for hyphal growth of C. albicans under certain in vitro conditions (6). Here we show a similar requirement for Hst7p and Cst20p, a C. albicans homolog of the Ste20p protein kinase. We also show in a mouse model for systemic candidiasis that Cst20p plays a role in virulence, as judged from signifi...

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Section: Methodsmentioning

confidence: 99%

Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans

Leberer

Harcus

Broadbent

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

305

318

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“…To introduce the C. albicans ADH1 terminator after GFP, the ADH1 terminator in pYPB1-ADHp1 (Bertram et al, 1996) was isolated as a PstI-EcoRI fragment and ligated to PstI-EcoRI-cut pMG1506, between the GFP and URA3 sequences, to generate pGFP-URA3 (Figure 1). …”

Section: Plasmid Constructionsmentioning

confidence: 99%

Cassettes for PCR‐mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans

Gerami-Nejad

Berman

Gale

2001

Yeast

184

192

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We have developed a set of plasmids containing fluorescent protein cassettes for use in PCR-mediated gene tagging in Candida albicans. We engineered YFP and CFP variants of the GFP sequence optimized for C. albicans codon usage. The fluorescent protein sequences, linked to C. albicans auxotrophic marker sequences, were amplified by PCR and transformed directly into yeast. Gene-specific sequence was incorporated into the PCR primers, such that the tag-cassette integrates by homologous recombination at the 3k-end of the gene of interest. This technique was used to tag Cdc3 and Tub1 with GFP, YFP and CFP, which were readily visualized by fluorescence microscopy and localized as expected. In addition, Tub1-YFP and Cdc3-CFP were visualized in the same cells. Thus, this technique directs one-step construction of multiple fluorescent protein fusions, facilitating the study of protein co-expression and co-localization in C. albicans cells in vivo.

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“…We obtained an amplicon of 705 bp, from the ATG start codon to the TAA stop codon, which was subcloned into the commercial vector pGEM-T Easy (Promega). The amplicon was rescued by digestion with BglII/XhoI and ligated to BglII/XhoI-digested pADH (Bertram et al, 1996) to give plasmid pADH-CaSSR1, which was used to transform the null mutant. The resulting transformed strain (C. albicans 30543) was tested for CaSSR1 overexpression.…”

Section: Methodsmentioning

confidence: 99%

“…Overexpression of CaSSR1 was achieved by subcloning an amplicon containing CaSSR1 in a pADH episomal vector (Bertram et al, 1996). Overexpression was confirmed by Western blot analysis of zymolyase extracts (Fig.…”

Section: Study Of the Cassr1-overexpressing Strainmentioning

confidence: 99%

Identification and study of a Candida albicans protein homologous to Saccharomyces cerevisiae Ssr1p, an internal cell-wall protein

et al. 2003

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After screening of a Candida albicans genome database, the product of an ORF (IPF 3054) that has 62 % homology with Saccharomyces cerevisiae Ssr1p, an internal cell-wall protein, was identified and named CaSsr1p. The deduced amino acid sequence shows that CaSsr1p contains an N-terminal hydrophobic signal peptide, is rich in Ser and Thr amino acids and has a potential glycosylphosphatidylinositol-attachment signal. CaSsr1p is released following degradation of isolated cell walls by zymolyase (mainly a 1,3-b-glucanase) and therefore seems to be covalently linked to the b-glucan of the cell walls. Both disruption and overexpression of the CaSSR1 gene caused an increased sensitivity to calcofluor white, Congo red and zymolyase digestion. These results suggest that CaSsr1p has a structural role associated with the cell-wall b-glucan. INTRODUCTIONCandida albicans is an opportunistic pathogenic fungus in humans which can cause either septicaemic or mucosal infections (Odds, 1988(Odds, , 1994. The number of fungal infections caused by C. albicans has increased dramatically in the last few decades, due to the rise in the number of immunocompromised patients and their life expectancies (Fox, 1993). C. albicans is a dimorphic organism capable of reproducing by budding (yeast cells) or by producing germ tubes (mycelial cells) depending upon environmental factors (Odds, 1988); this morphological transition has been associated with pathogenicity (Calderone & Braun, 1991;Odds, 1988;Sentandreu et al., 1993). Because the cell wall of C. albicans is the fungal structure responsible for the initial interaction with the host and for the characteristic shape of each growth form, several studies have focused on its biosynthesis and function (Gozalbo et al., 1994;Sentandreu et al., 1993;Valentin et al., 2000).The cell wall of C. albicans is a complex biochemical entity composed mainly of three components, namely, glucans (1,3-b-and 1,6-b-glucan), mannoproteins and chitin (Fleet, 1991;Valentin et al., 2000). The b-glucans are the main components, accounting for 50-60 % by weight of the cell wall in C. albicans and other fungi. Chitin, a linear polymer of 1,4-b-linked N-acetylglucosamine units, is a relatively minor (1-10 %) but important constituent. In most fungi, b-glucans and chitin polymers account for the rigidity of the cell wall as well as its morphology . Mannoproteins represent 30-40 % of the total cell wall and determine the surface properties, enabling C. albicans cells to interact and adhere to host tissues (Chaffin et al., 1998).Cell-wall mannoproteins can be divided into three groups according to the methods used for their extraction. One group can be solubilized by detergents, such as SDS, or chaotropic agents, such as urea, and is formed by mannoproteins loosely associated with other components of the cell wall (Elorza et al., 1985;Valentin et al., 1984). The second group can be extracted by reducing agents, DTT or b-mercaptoethanol (Casanova et al., 1989;Orlean et al., 1986). The third group can be released from the ce...

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Structure and regulation of theCandida albicans ADH1 gene encoding an immunogenic alcohol dehydrogenase

Cited by 84 publications

References 78 publications

Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans

Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans

Cassettes for PCR‐mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans

Identification and study of a Candida albicans protein homologous to Saccharomyces cerevisiae Ssr1p, an internal cell-wall protein

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