The proposed research seeks to elucidate biochemical and ultrastructural aspects of the cell envelopes of pathogenic yeasts. It focuses on the enzymology of Sporothrix schenckii, the causative agent of sporotrichosis. Through a combination of methods and approaches this proposal is an investigation in depth of the acid phosphatases of this dimorphic fungus. S. schenckii grows as mycelium in the saprophytic state (in soil and on plants) and exhibits a yeast-like phase as a pathogen in man. Preliminary findings are presented to indicate five electrophoretically distinct acid phosphatases (""""""""isoenzymes"""""""") in yeast cells and only one in mycelium. Furthermore, cytochemistry at the electron microscope level indicates that acid phosphatase activity occurs in the cell envelopes (cell wall surface, periplasmic space, and possibly the plasma membrane) and also in cytoplasmic vacuoles. Through enzyme separation and characterization from cell-free extracts, by sub-cellular localizational studies, and complementary electron microscopy, the properties and the distribution of the isoenzymes within yeast and mycelial cells will be established. Enzyme purification procedures will include classical fractionation methods as well as affinity chromatography, and an application of the plant lectin concanavalin A. Characterization will include pH-activity relationships, responses to selected inhibitors, and substrate specificity. The latter will include an exploration of phosphoprotein dephosphorylation and the role of one or more of these isoenzymes of acid phosphatase in the regulation of other enzymes via that mechanism. Key experimental findings with laboratory strains will be repeated with more recently isolated strains of S. schenckii from human infections to test the generality of our results and to explore a possible role of acid phosphatase in the pathogenic phase. Strains of Ceratocystis stenoceras and other Ceratocystis species have also been assembled to test a long-standing, but unresolved, phylogenetic relationship among S. schenckii and Ceratocystis species from the point of view of the number and mobility of their acid phosphatases.
| Arnold, W N; Garrison, R G; Mann, L C et al. (1988) The acid phosphatases of Thermoascus crustaceus, a thermophilic fungus. Microbios 54:101-12 |
| Arnold, W N; Mann, L C; Garrison, R G (1987) Media-induced departures from the usual, temperature-dependent cell shapes of Sporothrix schenckii and concomitant changes in the acid phosphatase isoenzyme patterns. Microbios 52:161-71 |
| Arnold, W N; Sakai, K H; Mann, L C (1987) Selective inactivation of an extra-cytoplasmic acid phosphatase of yeast-like cells of Sporothrix schenckii by sodium fluoride. J Gen Microbiol 133:1503-9 |
| Arnold, W N; Mann, L C; Sakai, K H et al. (1986) Acid phosphatases of Sporothrix schenckii. J Gen Microbiol 132:3421-32 |
