Calcium dependence and binding in cultures of Histoplasma capsulatum

Histoplasma capsulatum is a pathogenic fungus with two distinct morphologies and lifestyles. The saprophytic form of this organism, a mold, thrives in soil and is especially abundant in the Ohio and Mississippi River valleys. Its parasitic counterpart, a yeast, colonizes phagolysosomes of mammalian macrophages. We have observed a major difference in the calcium requirements of the two forms of Histoplasma, potentially implicating the phagolysosome as a calcium-limiting compartment. Deprivation of calcium by the addition of EGTA to culture media inhibited the growth of mycelial H. capsulatum but had no effect on yeast growth in vitro. In addition, yeasts released a calcium-binding protein (CBP) detectable by a 45CaCl2 blotting technique. CBP was a major component of yeast culture supernatant and was also detectable by ruthenium red staining, another assay for calcium-binding activity. Conversely, mycelial H. capsulatum did not produce CBP, a finding that correlates with the dependence of mycelia on calcium for growth. We also describe here the purification of CBP from yeast culture supernatant by reversed-phase high-pressure liquid chromatography.