Segregation of Proteinase-negative Mutants from Heterozygous Candida albicans

SUMMARY: The extracellular acidic proteinase (EC 3.4.23.6) produced by Candida albicans has been reported to be a virulence factor. In studying the role of this proteinase in human disease, we determined the optimum conditions for stimulating proteinase production in order to isolate proteinase-negative (Prt-) mutants. We found that in liquid medium containing bovine serum albumin (BSA) as the sole nitrogen source, at pH 4 and 27°C, the sensitivity of proteinase detection was considerably greater than when assayed on BSA agar at 37°C. This observation is due, in part, to temperature sensitivity of proteinase induction. Nitrogen starvation did not induce proteinase. Proteinase production on agar was increased by adding 0.01% yeast extract (YE) to BSA medium. Using BSA + YE agar to isolate mutants, it was discovered that C. albicans ATCC 28366 was heterozygous for a Prt- mutation. Spontaneous Prt- mutants occurred at a frequency of 2 ×10−3 Ultraviolet light increased the mitotic segregation of Prt-cells to a frequency of 1 × −2 The Prt- phenotype showed a large inoculum effect, Prt-segregants reverted with a high frequency, and the revertants were unstable.