Exo-(l->3)- -glucanase, Autolysin and Trehalase Activities during Yeast Growth and Germ-tube Formation in Candida albicans

Abstract

Exo-(1----3)-beta-glucanase, beta-glucosidase, autolysin and trehalase were assayed in situ in Candida albicans during yeast growth, starvation and germ-tube formation. Cell viability, germ-tube formation, intracellular glucose-6-phosphate dehydrogenase and beta-glucosidase were unaffected in cells incubated in 0.1 M-HC1 for 15 min at 4 degrees C. However, in situ trehalase, (1----3)-beta-glucanase and autolysin activities in acid-treated cells decreased by 95, 50 and 35% respectively, indicating that these enzymes are, in part, associated with the cell envelope. Trehalase activity increased throughout yeast growth and remained elevated during the first hour of incubation for germ-tube formation. All of the in situ trehalase activity in starved yeast cells could be measured without the permeabilizing treatment. beta-Glucosidase activity declined throughout yeast growth and did not alter during germ-tube formation. Both the (1----3)-beta-glucanase and autolysin activities were optimal at pH 5 X 6, inhibited by gluconolactone and HgCl2, and maximal at 15-16 h during yeast growth. Although autolysin activity increased by 50-100% when starved yeast cells were incubated for germ-tube formation, the in situ (1----3)-beta-glucanase remained constant. When acid-treated starved yeast cells were similarly induced, in situ (1----3)-beta-glucanase increased 100% over 3 h of germ-tube formation. Yeast cells secreted (1----3)-beta-glucanase into the growth medium. This was highest in early exponential phase cultures (34% of the maximum in situ activity) and declined throughout growth. (1----3)-beta-Glucanase was also secreted into the medium during germ-tube formation and this represented 80-100% of the in situ activity in germ-tube forming cells. Both secretion of (1----3)-beta-glucanase and germ-tube formation were inhibited by 2-deoxyglucose, ethidium bromide, trichodermin and azaserine.

The Secretion of N-Acetylglucosaminidase during Germ-tube Formation in Candida albicans

“…Recently we found that exo-( 1 -, 3)-P-glucanase was secreted and exported in C. albicans during germ-tube formation but negligible enzyme was released into the yeast growth medium (Ram et al, 1984). It is suggested that the germ-tube wall is more porous than that of yeast cells and consequently export of enzymes is facilitated by the formation of hyphae.…”

“…The cellular distribution of N-acetylglucosaminidase was determined by measuring in siru enzyme activity before and after treatment of intact cells with 0-1 M-HCl (Ram et al, 1984). Washed cells were resuspended in 0.1 M-HCI at 4 "C, incubated for 15 min, neutralized with NaOH and washed three times with the potassium phosphate buffer.…”

The Cellular Location of Proteases in Candida albicans

“…Acid treatment (Ram et al, 1984) of C . albicans cells did not inactivate the in situ proteinase activity but a crude proteinase ycaB preparation was inactivated at pH 1.5 and 0 "C. The in situ proteinase activity was not increased by permeabilizing (Ram et al, 1984) cells. Intact cells are also permeable to Leu-Nna (Montes & Constantine, 1968).…”

“…Candida albicans (ATCC 10261) was grown as described previously (Farley et al, 1986) and germ-tube formation induced with starved yeast cells and N-acetylglucosamine at 37 "C (Ram et al, 1984). Starved cells incubated at 28 "C under these conditions retained the yeast morphology and are referred to as yeast control cells.…”

“…A similar result (105 % recovery in the 60000 g supernatant, specific activity 13 pmol min-I pg-l) was obtained for P-glucosidase in C. albicans. Since P-glucosidase is not periplasmic (Ram et al, 1984) it must be a cytoplasmic enzyme. The aminopeptidase activity in the 60000g supernatant exhibited optimum activity for Leu-Nna between pH 7.0 and 8.0, which is similar to the pH optima for the periplasmic and cytoplasmic aminopeptidases in S .…”