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Candida albicans, when cultivated in a medium containing insoluble bovine achilles tendon as a nitrogen source, was able to produce a collagen degrading proteinase. The degradation of achilles tendon collagen by the proteinase was verified by morphological change and the release of hydroxyproline. The proteinase activity was inhibited by pepstatin.Candida albicans, a dimorphic yeast, is an important opportunistic pathogen. It causes cutaneous or serious systemic candidiasis in a variety of clinical forms. However, the factors responsible for the pathogenicity of C. albicans have yet to be established. C. albicans proteinase has been proposed as one of the important virulence factors by Remold et al. [9], Chattaway et al. [1], MacDonald & Odds [6], Rfichel [10] and Odds [7]. Recently, Hattori et aL [4] described the relationship between proteolytic enzyme and candidiasis on the stratum corneum. Previously, we have reported on the dentinal collagen (type 1) degrading proteinase produced by C. albicans and we have suggested that this proteinase might be related to the progress of dentinal caries [3,5]. The present experiments provide evidence which shows that the C. albicans proteinase degrades native bovine achilles tendon collagen.The proteinase was prepared by the method described previously [5] except that dentinal collagen was replaced by bovine achilles tendon collagen. Briefly, C. albicans ATCC 1002 was cultured at 37°C in Yeast Carbon Base (Difco) containing 2% (w:v) achilles tendon collagen (type 1, insoluble, Sigma Chemical Co., St. Louis, Mo) as a nitrogen source. After 1 week of incubation, the culture supernatant was concentrated and the active proteinase fractions were separated by DEAE Sephacel columnchromatography at pH 6.7. The optimum pH for proteinase activity was 3.5-4.0. When collagen was used as a nitrogen source in culture media used to obtain the proteinase, it was added to the media after sterilization with ethylene oxide (1.0kgcm -2 at 50°C) for 6h.

It has been previously shown that the induction of germination in Candida albicans occurs following its cessation of growth as a yeast. Similarly, mammalian cells undergo a differentiation process that is preceded by a growth cessation associated with a hypophosphorylation of proteins of the retinoblastoma gene family. It is postulated that a similar type of mechanism may be operative in C. albicans and protein phosphorylation inhibitors: forskolin (stimulates cyclic adenosine monophosphate production), okadaic acid (phosphatase inhibitor) and D-erythro-sphingosine (retinoblastoma protein phosphorylation inhibitor) have been used to further strengthen this hypothesis. Okadaic acid (1-1000 nM) and D-erythro-sphingosine (100 microM) significantly inhibited the growth of yeast cells of C. albicans. D-Erythro-sphingosine at 1000 microM was candidicidal. Forskolin did not significantly affect growth. Exponentially grown C. albicans pretreated with forskolin (10 microM), okadaic acid (1000 nM) or D-erythro-sphingosine (100 microM) readily germinated. In comparison, when these inhibitors were incorporated in the same medium, germination of exponentially grown cells did not occur. These results suggest that protein dephosphorylation may be necessary at an early stage of the yeast-hyphae transition in C. albicans.