We isolated and sequenced a clone for Candida albicans enolase from a C. albicans cDNA library by using molecular genetic techniques. The 1.4-kbp cDNA encoded one long open reading frame of 440 amino acids which was 87 and 75% similar to predicted enolases of Saccharomyces cerevisiae and enolases from other organisms, respectively. The cDNA included the entire coding region and predicted a protein of molecular weight 47,178. The codon usage was highly biased and similar to that found for the highly expressed EF-1 alpha proteins of C. albicans. Northern (RNA) blot analysis showed that the enolase cDNA hybridized to an abundant C. albicans mRNA of 1.5 kb present in both yeast and hyphal growth forms. The polypeptide product of the cloned cDNA, which was purified as a recombinant protein fused to glutathione S-transferase, had enolase enzymatic activity and inhibited radioimmunoprecipitation of a single C. albicans protein of molecular weight 47,000. Analysis of the predicted C. albicans enolase showed strong conservation in regions of alpha helices, beta sheets, and beta turns, as determined by comparison with the crystal structure of apo-enolase A of S. cerevisiae. The lack of cysteine residues and a two-amino-acid insertion in the main domain differentiated C. albicans enolase from S. cerevisiae enolase. Immunofluorescence of whole C. albicans cells by using a mouse antiserum generated against the purified fusion protein showed that enolase is not located on the surface of C. albicans. Recombinant C. albicans enolase will be useful in understanding the pathogenesis and host immune response in disseminated candidiasis, since enolase is an immunodominant antigen which circulates during disseminated infections.

Immunoblot analysis showed that enolase is one of a subset of proteins found in cell supernatants of Candida albicans. Enzyme assays on whole cell extracts indicated that enolase is an abundant protein, comprising 0.7% and 2.0% of the total protein from yeast and hyphal forms of C. albicans, respectively. Comparison of enolase enzyme activities in whole cell extracts and cell culture supernatants showed the enzyme to be located primarily within cells. Extracellular glyceraldehyde-3-phosphate dehydrogenase activity was absent or lower than that of enolase, despite equivalent intracellular levels. The results suggest that enolase, released from fungi in the absence of host factors, may contribute to enolase found circulating in the blood of patients with hematogenously disseminated candidiasis. In addition, the release from cells of highly immunogenic fungal proteins, such as enolase, may be important in defining the selective stimulation of host antifungal responses during infection.

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Sexual fusion of haploid Chlamydomonas gametes produces a diploid zygote which undergoes sporulation (maturation). We have used a combination of genetic and cellular approaches to evaluate the role(s) of gametic cell and nuclear fusion in the progression of sporulation. A fusion‐arrested strain, zym‐26–3. was obtained following ultraviolet irradiation of vegetative haploid cells of the homothallic species Chlamydomonas monoica Strehlow. Using the DNA‐specific fluorochrome, DAPI, we determined that diploidy was rarely achieved although nuclear migration to the base of the cytoplasmic bridge connecting the gametes and attempted transit through the tubule could be easily documented. Unusual cytoplasmic‘buds’which developed adjacent to the cytoplasmic bridge in sporulating haploids were usually found to contain a migrant nucleus. Using transmission electron microscopy, we determined that ultrastructural changes typically associated with sporulation of a diploid zygote (e.g. spore wall formation; plastid dedifferentiation and associated lipid accumulation; nuclear migration and heterochromatization) could occur following arrested cell fusion despite the absence of nuclear fusion. Genetic analysis of the zym‐26–3 strain revealed two unlinked mutations: cf‐1 responsible for the failure to complete cell fusion; and ger‐8, a mutant allele not affecting cell fusion, but interfering with late stages of spore maturation and germination.‘Cytoplasmic budding’was observed in strains carrying each of these mutations singly and may be a common secondary consequence of disturbances in the relative timing of interrelated processes required for spare wall assembly.