Segregation of proteinase-negative mutants from heterozygous Candida albicans

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 degrees C, the sensitivity of proteinase detection was considerably greater than when assayed on BSA agar at 37 degrees 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 x 10(-3). Ultraviolet light increased the mitotic segregation of Prt- cells to a frequency of 1 x 10(-2). The Prt- phenotype showed a large inoculum effect, Prt- segregants reverted with a high frequency, and the revertants were unstable.