Hydrolase and transferase activities of the beta-1,3-exoglucanase of Candida albicans.

The exo-beta-1,3-glucanase of Candida albicans (Exg) has a marked specificity for beta-1,3-glucosidic linkages as judged by the kinetic constants for p-nitophenyl beta-glucoside, beta-linked disaccharides of glucose (laminaribiose, gentiobiose, and cellobiose), oligosaccharides of the laminari series, laminarin and pustulan. The kcat/Km ratios for a series of laminari oligosaccharides from -biose to -heptaose showed that Exg has an extended substrate-binding site which contains at least five binding sites for sugar residues. Binding at position +2 (the third sugar residue) increases the kcat twofold while positions +3 and +4 lower the Km value further and thereby increase the catalytic efficiency. Exg catalyses an efficient transglucosylation reaction with high concentrations of laminari-oligosaccharides which specifically form beta-1,3 linkages and with yields up to 50%. The rate of the transglucosylation is concentration-dependent and can be more than 10 times faster than the hydrolytic reaction with excess donor substrates such as laminaritriose and laminarihexaose. The kinetics of Exg and the predicted substrate-binding site for up to five sugar residues are consistent with a recent structural analysis of the enzyme-binding site.

The cell wall of the oleaginous yeast Trichosporon cutaneum.

The cell wall of Trichosporon cutaneum consists of 11% protein, 63% neutral carbohydrate, 9% glucosamine and 13% glucuronic acid. The sugars include glucose (32%), mannose (6%) and traces of xylose and galactose. The cell wall was fractionated with alkali to yield a mixture of alkali-soluble matrix components, and an alkali-insoluble glucan associated with chitin. The alkali-insoluble glucan contained a mixture of (1-3) and (1-6) glycosidic linkages. It was only partly susceptible to digestion by the beta(1-3) glucanase, Zymolyase. The alkali-soluble fraction contained glucan, mannan and acidic polymers. The glucan was (1-3)-linked with no (1-6) linkages and only trace amounts of (1-3-6)-linked glucose. It was resistant to digestion by Zymolyase. Extensive hydrolysis of this fraction with trifluoroacetic acid released a high-molecular-mass glucuronan which had 1H- and 13C-NMR profiles matching those of the beta(1-4) glucuronan, mucoric acid. Xylomannan was purified from isolated cell walls and from whole cells. It contained glucose, mannose, xylose, and D-glucuronic acid. It was very similar in composition and structure to the capsular polysaccharides of Cryptococcus neoformans, and to an extracellular polysaccharide produced by another yeast described as T. cutaneum. Electron microscopy showed that the cell wall of T. cutaneum has a lamellar structure characteristic of a basidiomycetous yeast rather than the electron-dense 'fuzzy coat' seen in Candida albicans.

An exo-beta-(1,3)-glucanase of Candida albicans: purification of the enzyme and molecular cloning of the gene.

A nucleotide sequence encoding an exo-beta-(1,3)-glucanase was cloned from a library of genomic DNA of Candida albicans ATCC 10261. The sequenced gene encodes a protein of 438 amino acid residues. The amino terminal and an internal peptide sequence of the enzyme matched with deduced sequences within the cloned gene. Analysis of the sequence indicated that the nascent protein is processed during secretion by the signal peptidase and a Kex2-like proteinase, yielding a predicted mature enzyme of 400 residues. There is 58% identity and 85% similarity between the amino acid sequences of this exoglucanase and the homologous enzyme of Saccharomyces cerevisiae. An antiserum to the purified exoglucanase cross-reacted with the S. cerevisiae exoglucanase and a similar protein secreted by other C. albicans strains and Candida species. There are no sites for N-linked glycosylation in the sequence and this is consistent with the carbohydrate content of the secreted enzyme. Putative upstream promoter elements are associated with the gene. Southern analysis of the gene indicated that it was present at one copy per genome and that the diploid genome of C. albicans ATCC 10261 is heterozygous at this locus for a BglII RFLP. A 2.5 kb mRNA transcript was detected by Northern analysis and gene expression, as monitored by Northern and Western blots, reflected the growth rates of the cultures.

A second gene for a secreted aspartate proteinase in Candida albicans.

The gene (PRA11) encoding a secreted aspartate proteinase of Candida albicans has been cloned and sequenced. The nucleotide and deduced amino acid sequences of PRA11 are 77 and 73% identical, respectively, with the reported sequences of PRA10 also cloned from C. albicans. Southern analyses indicated that the genome of each strain examined (ATCC 10231 and ATCC 10261) contains PRA10 and PRA11. Northern (RNA) analyses showed that PRA11 was expressed at a much higher level than was PRA10 when secretion of the proteinase by strain ATCC 10261 was induced with albumin.

A secreted beta-glucan-branching enzyme from Candida albicans.

A Mr 34,000 wall protein was isolated as a by-product of the purification of an endo-(1-3)-beta-glucanase from the culture filtrate of Candida albicans. The purified fraction contained no exo- or endo-beta-glucanase activity, and analysis by SDS poly-acrylamide gel electrophoresis (SDS-PAGE) showed one protein band at Mr 34,000. Analysis by gel filtration high performance liquid chromatography (HPLC) of reaction products from incubations of the protein fraction with laminarioligosaccharides of five glucosyl units or greater revealed a unique glucanosyl transferase activity. The enzyme specifically cleaved laminaribiaose (G2) from the reducing-end of a linear beta-(1-3)-glucan and transferred the remainder to another laminarioligosaccharide. The reaction with laminaripentaose (G5) produced G2 and a product eluting at the position of G8. Analysis of the latter transferase product by 13C- and 1H-nuclear magnetic resonance (NMR) spectroscopy shows it to be a branched molecule containing a beta-(1-3)-beta-(1-6)-branchpoint. It is suggested that the Mr 34,000 wall protein is a glucan branching enzyme, perhaps the key enzyme responsible for the transformation of the initial linear beta-(1-3)-glucan into the branched beta-(1-3)-beta-1-6)-glucan as found in the cell wall of C. albicans.

Purification and properties of two lactose hydrolases from Trichosporon cutaneum.

Two enzymes that hydrolysed lactose were purified essentially to homogeneity from cell extracts of the oleaginous yeast Trichosporon cutaneum. One enzyme of Mr 120,000 had properties typical of a beta-galactosidase (EC 3.2.1.23). It hydrolysed lactose, lactulose and nitrophenyl-beta-D-galactosides. The enzyme required K+ or Rb+ for activity, and other monovalent cations tested were not effective. Enzyme activity was abolished by EDTA and stimulated by Mg2+, Mn2+ and Ca2+. The beta-galactosidase was induced by lactose, galactose, lactulose and lactobionic acid. The other enzyme, a beta-glycosidase (EC 3.2.1.21) of Mr 52,000 showed no ionic requirements and it hydrolysed lactose, nitrophenyl-beta-D-galactosides, 4-nitrophenyl-beta-D-glucoside, cellobiose, laminaribiose, laminaritriose and sophorose, but not gentiobiose, 4-nitrophenyl-beta-D-mannoside or sucrose. This enzyme was induced by lactose, galactose and lactulose, and also by cellobiose.

Evidence for a role for secreted aspartate proteinase of Candida albicans in vulvovaginal candidiasis.

The presence of the secretory aspartate (acid) proteinase in the vaginal fluid of candidal vaginitis patients and controls was studied by ELISA and immunoblot (Western blot). In addition, a proteinase-deficient mutant strain of Candida albicans (IR24) was compared with the wild-type parent strain (10261) for ability to infect the vagina of pseudoestrus rats under estradiol treatment. Among the 67 women examined, proteinase was detected only in 22 harboring C. albicans (range, 42-233 ng/ml of vaginal fluid), at concentrations significantly higher in the 14 vaginitis patients than in the 8 asymptomatic fungal carriers. Western blots confirmed the presence of only one protein band of approximately 43 kDa, corresponding to that of the purified proteinase, in the ELISA-positive vaginal fluids. Experimental vaginal infection was significantly more extensive and persistent in rats infected with the proteinase-producer strain than in those challenged with the proteinase-deficient mutant, and the enzyme was detected in the vaginas of the former but not of the latter animals. Both strains 10261 and IR24 developed hyphal forms to a roughly similar extent during infection, and both showed a comparable adherence in vitro to vaginal and buccal epithelial cells. The clinical and experimental evidence support a role for secretory proteinase as a virulence factor in the pathogenesis of candidal vaginitis.

The secreted aspartate proteinase of Candida albicans: physiology of secretion and virulence of a proteinase-deficient mutant.

It was established that Candida albicans grew rapidly in a simple medium containing yeast extract (0.2%, w/v) plus glucose (2%, w/v). These cultures were in or near to a state of nitrogen limitation and the concentration of secreted aspartate proteinase increased rapidly (within 3-4 h) on addition of BSA. Synthesis and secretion were apparently controlled both positively (induction by albumin or, more probably, the peptides produced from it) and negatively (repression by NH4Cl). A small intracellular pool of the enzyme was detected during production of the enzyme and this pool decreased with the cessation of synthesis and secretion. A stable mutant, IR24, was isolated which secreted less than 0.3% of the amount of the proteinase exported by the parent strain ATCC 10261. The LD50 values for mutant IR24 and the parent strain administered intravenously to mice were greater than 1.0 x 10(9) and 1.6 x 10(6) c.f.u. kg-1 respectively.