A neutral trehalase gene from Candida albicans: molecular cloning, characterization and disruption

A multicopper oxidase gene from the human pathogenic yeast Candida albicans was isolated and characterized. An open reading frame of 1872 bp, designated CaFET3, was identified, encoding a predicted protein of 624 amino acids and a molecular mass of 70 5 kDa. The identity between the deduced amino acid sequences of CaFET3 and the Saccharomyces cerevisiae FET3 gene is 55 %. CaFET3 was localized on chromosome 6. A null mutant (fet3∆/fet3∆) was constructed by sequential gene disruption. Unlike the C. albicans SC5314 wildtype strain the fet3∆ mutant was unable to grow in low-iron medium. The lack of growth of a S. cerevisiae fet3∆ mutant in iron-limited medium was compensated by transformation with CaFET3. The null mutant strain showed no change in pathogenicity compared with the wild-type strain in the mouse model of systemic candidiasis.

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“…The methods were carried out by procedures described previously (Boeke et al, 1984 ;Swoboda et al, 1995 ;Eck et al, 1997).…”

Section: Transformation Of C Albicans Isolation Of Heterozygous Andmentioning

confidence: 99%

A multicopper oxidase gene from Candida albicans: cloning, characterization and disruption b bThe EMBL accession number for the sequence reported in this paper is Y09329.

et al. 1999

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“…lactis and C. albicans and trehalases from these species are known to be regulated by reversible phosphorylation [22,23]. Hence, one or several of these eight sites are likely to be involved in the control of trehalase by reversible phosphorylation.…”

Section: Figure 3 Trehalase Activity Is Negatively Correlated With Hementioning

confidence: 99%

“…Trehalases from the latter organisms are also known to be regulated by PKA-mediated phosphorylation [22,23]. Eight potential PKA phosphorylation sites appeared to be conserved in trehalases from all three organisms.…”

Section: Introductionmentioning

confidence: 96%

“…Recently, we developed an EXCEL-based method that allows the prediction of potential Ser\Thr phosphorylation sites in proteins [21]. We used this method in combination with a sequence comparison of Nth1 with trehalases from Kluy ero- Figure 1 Sequence alignment of neutral trehalases from S. cerevisiae [4,8], K. lactis [22] and C. albicans [23] The eight conserved putative phosphorylation sites and Thr260 are indicated in black. Regions that are identical between two or three proteins are shaded grey.…”

Section: Introductionmentioning

confidence: 99%

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Opposite roles of trehalase activity in heat-shock recovery and heat-shock survival in Saccharomyces cerevisiae

Wera

Schrijver

Geyskens

et al. 1999

Biochemical Journal

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A variety of results has been obtained consistent with activation of neutral trehalase in Saccharomyces cerevisiae through direct phosphorylation by cAMP-dependent protein kinase (PKA). A series of neutral trehalase mutant alleles, in which all evolutionarily conserved putative phosphorylation sites were changed into alanine, was tested for activation in vitro (by PKA) and in vivo (by glucose addition). None of the mutations alone affected the activation ratio, whereas all mutations combined resulted in an inactive enzyme. All mutant alleles were expressed to similar levels, as shown by Western blotting. Several of the point mutations significantly lowered the specific activity. Using this series of mutants with different activity levels we show an inverse relationship between trehalase activity and heat-shock survival during glucose-induced trehalose mobilization. This is consistent with a stress-protective function of trehalose. On the other hand, reduction of trehalase activity below a certain threshold level impaired recovery from a sublethal heat shock. This suggests that trehalose breakdown is required for efficient recovery from heat shock, and that the presence of trehalase protein alone is not sufficient for efficient heat-stress recovery.

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“…The methods used for transformation of C. albicans and selection of Ura Ϫ auxotrophs were the methods described previously (3,16,43).…”

Section: Methodsmentioning

confidence: 99%

Defective Hyphal Induction of a Candida albicans Phosphatidylinositol 3-Phosphate 5-Kinase Null Mutant on Solid Media Does Not Lead to Decreased Virulence

Augsten¹,

Hübner²,

Nguyen³

et al. 2002

Infect Immun

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A phosphatidylinositol 3-phosphate [PI(3)P] 5-kinase gene (CaFAB1) of the most important human pathogenic yeast, Candida albicans, was cloned and sequenced. An open reading frame was detected which encodes a 2,369-amino-acid protein with a calculated molecular mass of 268 kDa and a relative isoelectric point of 6.76. This protein exhibits 38% overall amino acid sequence identity with Saccharomyces cerevisiae Fab1p. We localized the CaFAB1 gene on chromosome R. To determine the influence of the PI(3)P 5-kinase CaFab1p on processes involved in C. albicans morphogenesis and pathogenicity, we sequentially disrupted both copies of the gene. Homozygous deletion of C. albicans CaFAB1 resulted in a mutant strain which exhibited defects in morphogenesis. A Cafab1 null mutant had enlarged vacuoles, an acidification defect, and increased generation times and was unable to form hyphae on different solid media. The sensitivities to hyperosmotic and hightemperature stresses, adherence, and virulence compared to those of wild-type strain SC5314 were not affected.Candida albicans is the major fungal pathogen in humans, and its medical significance is increasing (34). This dimorphic yeast is capable of causing life-threatening infections in immunocompromised patients, as well as a variety of mucosal infections in healthy individuals. The present evidence suggests that several factors contribute to the virulence of C. albicans; these factors include the ability to switch between different morphogenetic forms, host epithelial and endothelial cell recognition and adhesion, and secretion of proteinases and phospholipases (12,20,24,29,35). While a number of virulence factors of C. albicans have been characterized, the mechanism which enables this opportunistic fungus to become pathogenic has not been unraveled yet.In eukaryotic cells many biochemical processes are based on the interactions between cytosolic proteins and intracellular membranes. Diverse cellular processes, such as vesicular transport of proteins, signal transduction, cell adhesion, mitogenesis, growth factor downregulation, cytoskeleton rearrangement, and the osmotic stress response, are processes that demand regulated and reversible docking of cytosolic proteins on specific membranes (13,15,44). The proteins bind to cytosolic domains of transmembrane proteins or to the GTP-associated forms of membrane-associated small GTPases. However, it seems that reversible membrane binding of proteins through interactions with specific lipid head groups is an additional mechanism whose importance has increased. In this process phosphatidylinositol (PI) and derivatives of PI that are phosphorylated at positions D-3, D-4, and D-5 play an important role. The different phosphorylated inositols are recognized by different groups of cytosolic proteins, most of which are unknown. However, target proteins of phosphatidylinositol 3-phosphate [PI(3)P] were identified in the yeast Saccharomyces cerevisiae and mammalian cells as proteins that contain a PI(3)P-binding FYVE (Fab1p, YOTB, Vps27...

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A neutral trehalase gene from Candida albicans: molecular cloning, characterization and disruption

Cited by 44 publications

References 44 publications

A multicopper oxidase gene from Candida albicans: cloning, characterization and disruption b bThe EMBL accession number for the sequence reported in this paper is Y09329.

A multicopper oxidase gene from Candida albicans: cloning, characterization and disruption b bThe EMBL accession number for the sequence reported in this paper is Y09329.

Opposite roles of trehalase activity in heat-shock recovery and heat-shock survival in Saccharomyces cerevisiae

Defective Hyphal Induction of a Candida albicans Phosphatidylinositol 3-Phosphate 5-Kinase Null Mutant on Solid Media Does Not Lead to Decreased Virulence

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