Evidence for the presence of complex carbohydrates in Candida albicans cell wall glycoproteins.

In order to test the hypothesis that cell wall glycoproteins of Candida albicans contained non-mannan oligosaccharides, the sugar composition of cell wall extracts and fractions of cell wall extracts was examined by means of fluorophore-assisted carbohydrate electrophoresis (FACE). In addition to the expected mannose, glucose, and N-acetyl-glucosamine, this analysis showed the presence of galactose, N-acetyl-galactosamine, fucose, and sialic acid and two unknown sugars. These sugars are also associated with complex oligosaccharides of mammalian glycoproteins. Presence of fucosylated cell wall components was further demonstrated by lectin-blotting analysis of cell wall extracts. Besides their structural role, complex carbohydrate structures on the surface of C. albicans may represent additional motifs through which interactions of this fungus with host cells and tissues could be established.

Evidence for presence in the cell wall of Candida albicans of a protein related to the hsp70 family.

We have previously reported the isolation of several clones from a cDNA expression library from Candida albicans, one of which was associated with a constitutively expressed 70-kDa protein. The moiety was present in the beta-mercaptoethanol extracts of cell walls from both blastoconidia and germ tubes. The surface expression of this moiety was revealed by an indirect immunofluorescence assay using affinity-purified antibody to the fusion protein produced by the clone. The 0.68-kb cDNA insert was sequenced. A database search revealed extensive homology with the 70-kDa family of stress or heat shock proteins (hsps). The 77% homology with another C. albicans HSP70 sequence suggested that this fragment represented a second member of the HSP70 family in this organism. Homology ranging from 65 to 76% was observed with members of four subfamilies (SSA, SSB, SSC, and SSD) of the Saccharomyces cerevisiae HSP70 gene family. The nucleic acid sequence and the deduced amino acid sequence of the open reading frame showed greatest homology with SSA1 and SSA2 sequences, and the gene corresponding to the cDNA clone was designated C. albicans SSA2. The relationship with the SSA family was supported by reactivity of the 70-kDa component with antibody recognizing the Ssa proteins of S. cerevisiae. The presence of an hsp70 in the cell wall was confirmed by two additional methods. Cell wall proteins were biotinylated with a non-membrane-permeable derivative to distinguish extracellular from cytosolic proteins. Biotinylated hsp70 was detected by Western blotting (immunoblotting) among the biotinylated components affinity purified by chromatography on streptavidin, thereby establishing its presence in the cell wall. Immunoelectron microscopy showed that the 70-kDa component was present at the cell surface as well as the outer surface of the plasma membrane and extended through the cell wall, occasionally appearing to reach the cell surface through channels. Northern (RNA) blot analysis showed that the gene was expressed in yeast cells growing in yeast extract-peptone medium at both 25 and 37 degrees C and in Lee medium at 25 degrees C and during formation of germ tubes in Lee medium 37 degrees C. No obvious increase in the expression level was detected after the temperature shift. Members of the hsp70 family have been reported to be immunoreactive. The fusion protein produced by the cDNA clone was recognized by serum from healthy individuals and patients with candidiasis. Since members of the hsp70 family of eucaryotic proteins are associated with chaperone and translocation functions, in addition to being immunogenic, this protein may play a role in the assembly and function of other cell wall proteins.

Dynamic expression of cell wall proteins of Candida albicans revealed by probes from cDNA clones.

Five cDNA clones were selected from the positive clones detected by screening a germ tube expression library constructed in lambda gt11 with rabbit antisera raised against cell wall extracts of Candida albicans. The selected clones were amplified and used to obtain affinity purified antibodies by eluting from the expressed proteins that had been previously transferred onto nitrocellulose discs. The antibodies obtained were used as probes in immunoblots of the cell wall extracts separated by denaturing polyacrylamide electrophoresis. A single protein band was detected for each clone. Detection of products of the cloned sequences varied according to the extraction procedure and/or cell morphology. These products included bands exhibiting apparent molecular weights of 40, 58, 68 and 70 kDa present in beta-mercaptoethanol (beta ME) extracts from both yeast and germ tubes, and a 30 kDa beta ME extracted protein specific for germ tubes. The expression of these products at the cell surface was confirmed by indirect immunofluorescence. Expression of the mRNAs of the different cDNA clones varied according to growth- and morphology-related factors and showed no direct correlation between expression and presence in the cell wall. These observations suggest that complex mechanisms are involved in the regulation and expression of cell surface components of C. albicans.

Characterisation of a partially purified uracil phosphoribosyltransferase from the opportunistic pathogen Candida albicans.

This paper describes for the first time the partial purification and properties of uracil phosphoribosyltransferase (UPRTase) from the yeast Candida albicans. UPRTase was purified 38 fold by acid precipitation, DEAE-Sephacel chromatography and ultrafiltration. Further purification of UPRTase was unsuccessful due to the labile nature of the enzyme and the failure in obtaining satisfactory stabilizing conditions. SDS-PAGE suggested that the enzyme exists as a dimer of two dissimilar subunits with molecular masses of 47 and 38 kDa. The pH optimum for phosphoribosylation was about 7.5 and the optimal Mg++ concentration was 2 mM. The kinetics of the enzymes for its substrates, uracil and 5-phosphoribosyl-1-pyrophosphate (PRPP) were determined by measuring initial enzyme velocities over a wide range of concentrations of either substrate at different fixed concentrations of the second substrate. Graphic analysis of the data by Hanes-Woolf plots indicated that the reaction is indistinguishable from a double displacement reaction. 'Ping pong' mechanism has been previously reported for other phosphoribosyltransferases. The enzyme has a low affinity for its substrates (Km = 70.5 and 186 microM for uracil and PRPP, respectively) as compared with those of E. coli and baker's yeast. Inhibition studies indicate that 5-fluorouracil acts as an alternative substrate for UPRTase with 1.6 times higher specific activity.