Cloning and sequencing of the Candida albicans homologue of SRB1/PSA1/VIG9, the essential gene encoding GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae

Warit, Saradee; Walmsley, Richard M.; Stateva, Lubomira

doi:10.1099/00221287-144-9-2417

Cited by 17 publications

(24 citation statements)

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“…Unincorporated radioactive nucleotides were separated using mini Quick Spin DNA columns (Boehringer Mannheim). Total RNA was isolated according to Schmitt et al (1990) and used for Northern analysis as described previously (Warit et al, 1998). Sequencing was carried out in-house with ABI PRISM and the dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase.…”

Section: Methodsmentioning

confidence: 99%

The cAMP phosphodiesterase encoded by CaPDE2 is required for hyphal development in Candida albicans

Jung

Stateva

2003

Microbiology

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The cAMP-dependent pathway, which regulates yeast-to-hypha morphogenesis in Candida albicans, is controlled by changes in cAMP levels determined by the processes of synthesis and hydrolysis. Both low-and high-affinity cAMP phosphodiesterases are encoded in the C. albicans genome. CaPDE2, encoding the high-affinity cAMP phosphodiesterase, has been cloned and shown to be toxic in Saccharomyces cerevisiae upon overexpression under pGAL1, but functional under the moderate pMET3. Deletion of CaPDE2 causes elevated cAMP levels and responsiveness to exogenous cAMP, higher sensitivity to heat shock, severe growth defects at 42 6C and highly reduced levels of EFG1 transcription. In vitro in hypha-inducing liquid medium CaPDE2, deletion prohibits normal hyphal, but not pseudohyphal growth. On solid medium capde2 mutants form aberrant hyphae, with fewer branches and almost no lateral buds, which are deficient in hypha-to-yeast reversion. The phenotypic defects of capde2 mutants show that the cAMP-dependent pathway plays specific roles in hyphal and pseudohyphal development, its regulatory role however, being greater in liquid than on solid medium in vitro. The increased expression of CaPDE2 after serum addition correlates well with a drop in cAMP levels following the initial rise in response to the hyphal inducer. These results suggest that Capde2p mediates a desensitization mechanism by lowering basal cAMP levels in response to environmental stimuli in C. albicans.

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Section: Methodsmentioning

confidence: 99%

The cAMP phosphodiesterase encoded by CaPDE2 is required for hyphal development in Candida albicans

Jung

Stateva

2003

Microbiology

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“…2 and CaSRB1, which encode proteins required for supplying the Golgi with GDP-mannose, the mannose donor, are essential in C. albicans, indicating the overall importance of glycosylation to cell viability (27)(28)(29)(30). However, to date, no single glycosylation event in the Golgi has been shown to be essential.…”

Section: Cavrg4mentioning

confidence: 99%

Candida albicans Pmr1p, a Secretory Pathway P-type Ca2+/Mn2+-ATPase, Is Required for Glycosylation and Virulence

Bates

MacCallum

Bertram

et al. 2005

Journal of Biological Chemistry

179

213

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The cell surface of Candida albicans is the immediate point of contact with the host. The outer layer of the cell wall is enriched in highly glycosylated mannoproteins that are implicated in many aspects of the host-fungus interaction. Glycosylation of cell wall proteins is initiated in the endoplasmic reticulum and then elaborated in the Golgi as the protein passes through the secretory pathway. Golgi-bound mannosyltransferases require Mn 2؉ as an essential cofactor. In Saccharomyces cerevisiae, the P-type ATPase Pmr1p transports Ca 2؉ and Mn 2؉ ions into the Golgi. To determine the effect of a gross defect in glycosylation on host-fungus interactions of C. albicans, we disrupted the PMR1 homolog, CaPMR1. This mutation would simultaneously inhibit many Golgi-located, Mn 2؉ -dependent mannosyltransferases. The Capmr1⌬ null mutant was viable in vitro and had no growth defect even on media containing low Ca 2؉ /Mn 2؉ ion concentrations. However, cells grown in these media progressively lost viability upon entering stationary phase. Phosphomannan was almost completely absent, and O-mannan was severely truncated in the null mutant. A defect in N-linked outer chain glycosylation was also apparent, demonstrated by the underglycosylation of surface acid phosphatase. Consistent with the glycosylation defect, the null mutant had a weakened cell wall, exemplified by hypersensitivity to Calcofluor white, Congo red, and hygromycin B and constitutive activation of the cell integrity pathway. In a murine model of systemic infection, the null mutant was severely attenuated in virulence. These results demonstrate the importance of glycosylation for cell wall structure and virulence of C. albicans.

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“…Psa1p, involved in cell wall maintenance and biogenesis (95,96), might participate in CWP mannose outer chain elongation at the cell surface. Ino1p is required for the biosynthesis of myo-inositol and phosphatidylinositol.…”

Section: Albicans Cell Wall Proteome Consists Of Highly Heterogenementioning

confidence: 99%

Sequential Fractionation and Two-dimensional Gel Analysis Unravels the Complexity of the Dimorphic Fungus Candida albicans Cell Wall Proteome

Pitarch

Sánchez

Nombela

et al. 2002

Molecular & Cellular Proteomics

240

246

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The cell wall proteins of Candida albicans play a key role in morphogenesis and pathogenesis and might be potential target sites for new specific antifungal drugs. However, these proteins are difficult to analyze because of their high heterogeneity, interconnections with wall polysaccharides (mannan, glucan, and chitin), low abundance, low solubility, and hydrophobic nature. Here we report a subproteomic approach for the study of the cell wall proteins (CWPs) from C. albicans yeast and hyphal forms. Most of the mannoproteins present in this compartment were extracted by cell wall fractionation according to the type of interactions that they establish with other structural components. CWPs were solubilized from isolated cell walls by hot SDS and dithiothreitol treatment followed by extraction either by mild alkali conditions or by enzymatic treatment with glucanases and chitinases. These highly enriched cell wall fractions were analyzed by two-dimensional PAGE, showing that a large number of proteins are involved in cell wall construction and that the wall remodeling that occurs during germ tube formation is related to changes in the composition of CWPs. We suggest that the CWP-chitin linkage is an important retention mechanism of CWPs in C. albicans mycelial forms. This article also highlights the usefulness of the combination of sequential fractionation and two-dimensional PAGE followed by Western blotting using specific antibodies against known CWPs in the characterization of incorporation mechanisms of such CWPs into the cell wall and of their interactions with other wall components. Mass spectrometry analyses have allowed the identification of several cell surface proteins classically associated with both the cell wall and other compartments. The physiological significance of the dual location of these moonlighting proteins is also discussed. This approach is therefore a powerful tool for obtaining a comprehensive and inte-

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Cloning and sequencing of the Candida albicans homologue of SRB1/PSA1/VIG9, the essential gene encoding GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae

Cited by 17 publications

References 50 publications

The cAMP phosphodiesterase encoded by CaPDE2 is required for hyphal development in Candida albicans

The cAMP phosphodiesterase encoded by CaPDE2 is required for hyphal development in Candida albicans

Candida albicans Pmr1p, a Secretory Pathway P-type Ca2+/Mn2+-ATPase, Is Required for Glycosylation and Virulence

Sequential Fractionation and Two-dimensional Gel Analysis Unravels the Complexity of the Dimorphic Fungus Candida albicans Cell Wall Proteome

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