Candida albicans produces large amounts of the pentitol D-arabitol in culture and in infected mammalian hosts, but the functional and pathogenic significance of D-arabitol in C. albicans is not known. In this study, we sought to elucidate the pathway by which C. albicans synthesizes D-arabitol and to identify and characterize key enzymes in this pathway. C. albicans B311 produced D-[14C-l_arabitol from [14C-21glucose; Acyclic polyols are produced in large amounts by many fungi, and several functions have been ascribed to these compounds. For example, it has been proposed that fungi utilize polyols as storage or transport carbohydrates, as intracellular osmoregulatory solutes, and as metabolic reducing equivalents. Also, it has been suggested that fungal polyol metabolic pathways participate in the regulation of intracellular NAD(P)/NAD(P)H ratios and pH levels (14, 16). In 1979, Kiehn et al. (19) showed that cultures of the human pathogen Candida albicans produced large amounts of the acyclic pentitol D-arabitol and that people with invasive candidiasis had higher concentrations of arabitol in the blood than did normal or superficially colonized controls. Subsequent studies established that tissue and body fluid arabitol concentrations rose directly in proportion to fungal load in animals with C. albicans infection (42), that the excess arabitol in infected animals and humans was D-arabitol (the enantiomer produced by Candida spp.) (2, 43), that most humans with invasive candidiasis had high serum D-arabitol levels (11,44), and that these levels declined with effective therapy (40). Thus, D-arabitol is a quantitative diagnostic marker for invasive Candida infections.Several groups have investigated the clinical diagnostic implications of D-arabitol metabolism by C. albicans (9, 31, 36), but more basic aspects of D-arabitol metabolism have not been studied. Consequently, the metabolic pathway by which C. albicans synthesizes D-arabitol is not known, nor have the functional and pathogenetic significances of D-arabitol metabolism in C. albicans been examined. The goals of this study were to elucidate the D-arabitol biosynthetic pathway in C. albicans and to identify and characterize key * Corresponding author. enzymes in this pathway. This report describes (i) metabolic labeling studies that suggest that the pentose pathway intermediate D-ribulose-5-PO4 is the major metabolic precursor of D-arabitol in C. albicans, (ii) the presence in C. albicans lysates of a previously unknown NAD-dependent D-arabitol dehydrogenase (ArDH), and (iii) molecular cloning and analysis of the gene that encodes ArDH. Saccharomyces cerevisiae BWG 1-7A (MA4Ta his4-119 leu2-112 ura3-52 adel-100; from J. Loper, University of Cincinnati) was cultured in YPD or in 0.5% yeast extract-1% peptone-1% D-arabitol (YP-D-arab). Uracil prototrophs were selected on synthetic complete medium lacking uracil (12). MATERIALS AND METHODS StrainsEscherichia coli JM109 (45) and DH5-a (GIBCO BRL, Gaithersburg, Md.) were grown in LB medium (31) supplemented...

We have further characterized the functionality of the Saccharomyces cerevisiae gene SLT2(MPK1), coding for a MAP-kinase homolog essential for cell integrity, which is involved in the Pkc1p signalling pathway. This gene was isolated on the basis of its capacity to complement the thermosensitive-autolytic, osmotic-remediable phenotype of lyt2 mutants. Both slt2delta and lyt2 mutants displayed a caffeine-sensitive phenotype consisting of cell lysis that was not dependent on temperature. Caffeine concentrations affecting the growth of these mutant strains were dependent on the genetic background, the SSD1 allele being very significant in this regard. The SLT2 allele of several lyt2 strains was both rescued and amplified by PCR. The recovered allele was shown to be non-functional as it could not complement the lytic phenotype of both deletion (slt2delta) and lyt2 strains. After nucleotide sequencing of the recovered allele, we found that the defect of lyt2 mutants consists in a substitution of an aspartic acid for a glycine at position 35 of the amino-acid sequence of Slt2p. Gly35 is the third glycine of a glycine cluster (Gly-X-Gly-X-X-Gly), a conserved region in protein kinases and other nucleotide-binding proteins. Keywords Yeast middle dot SLT2 middle dot MAP-kinase middle dot Caffeine

No abstract

We have further characterized the functionality of the Saccharomyces cerevisiae gene SLT2(MPK1), coding for a MAP-kinase homolog essential for cell integrity, which is involved in the Pkc1p signalling pathway. This gene was isolated on the basis of its capacity to complement the thermosensitive-autolytic, osmotic-remediable phenotype of lyt2 mutants. Both slt2delta and lyt2 mutants displayed a caffeine-sensitive phenotype consisting of cell lysis that was not dependent on temperature. Caffeine concentrations affecting the growth of these mutant strains were dependent on the genetic background, the SSD1 allele being very significant in this regard. The SLT2 allele of several lyt2 strains was both rescued and amplified by PCR. The recovered allele was shown to be non-functional as it could not complement the lytic phenotype of both deletion (slt2delta) and lyt2 strains. After nucleotide sequencing of the recovered allele, we found that the defect of lyt2 mutants consists in a substitution of an aspartic acid for a glycine at position 35 of the amino-acid sequence of Slt2p. Gly35 is the third glycine of a glycine cluster (Gly-X-Gly-X-X-Gly), a conserved region in protein kinases and other nucleotide-binding proteins. Keywords Yeast middle dot SLT2 middle dot MAP-kinase middle dot Caffeine

No abstract