UBC Theses and Dissertations
Characterization of the physiological role of the Microsporum gypseum alkaline protease during macroconidium germination and outgrowth Page, William James
The results of this investigation suggested that Microsporum gypseum macroconidium germination was initiated by an alkaline protease and that this enzyme's activity, and hence the number of spores initiated to germinate, was regulated by inorganic phosphate. Inorganic phosphate altered the germination protease pH optimum from pH 9 to pH 7 and decreased its molecular weight from 33,000 to 16,000. Phosphate acted as a hyperbolic competitive inhibitor of germination protease activity, resulting in decreased activity towards ethyl-ester and spore coat substrates, and increased activity towards keratin substrates. The alkaline protease was inserted into the maturing spore coat as lysosomal vesicles, which upon spore rehydration, released their lytic contents into the cell wall. Owing to the complex construction of the fungal cell wall, the sequential action of other hydrolases was necessary to ensure sufficient spore coat lysis for germling outgrowth. Germination initiation also involved the action of β1,3 glucanase, followed by chitinase, phosphodiesterase, and ethyl-esterase activities. The phosphodiesterase, which also was lysosomal, released spore coat phosphates for subsequent protease inactivation. Spore coat glycoproteins, extractable with ethylene diamine, were examined as possible germination protease substrates. A water-insoluble glycoprotein, which accounted for 10% of the spore coat dry weight and was modified in its total protein and amino acid content during sporulation, possibly served as the germination protease substrate in vivo. The alkaline protease also functioned in the turnover of a water-soluble glycoprotein, which was not involved with sporulation, in terminal stages of mycelial starvation. Spore coat phosphates were determined to be located internally as phosphodiesters, and externally as exposed phosphate groups. These phosphate groups were deemed the possible sites of phosphodiesterase action, as the phosphate content of both sources decreased during spore germination. The degree of alkaline protease inactivation by inorganic phosphate was found to be dependent on the ratio of phosphate to protease protein. For example, in a high density of spores the ratio of phosphate to protease protein increased two-fold and the activity of the alkaline protease decreased stoichiometrically. By this mechanism, a constant number of spores were germinated in any given population. Calcium ions also were released prior to germination initiation. These ions possibly precipitated phosphates washed from the spores, thus preventing premature germination protease inactivation. Immediately after release from the fungal spore, the germination protease was active against the outer keratinized protein of hair (β keratin). After phosphate-treatment, the protease also hydrolysed the internal fibrillar keratin of hair (α keratin). Germination protease activity towards keratin also was enhanced by disulfide reducing agents and by keratin degradation products. The results suggested that the germination protease was converted into a functional keratinase, immediately after germ tube emergence from the spore, thus ensuring the parasitic survival of this fungus on keratinized tissues. M. gypseum sporulation was initiated by the hyphal tip after its emergence from a submerged to aerial environment. Reduction of aerial environment humidity was implicated as the sporulation inducing factor. Forced dehydration by increased aeration caused a normally asporogenous pleomorphic strain of M. gypseum to sporulate. The induced wild-type strain characteristics, however, were strictly phenotypic and reversible. Other pleomorphic strains of different dermatophyte species similarly were induced to sporulate by increased aeration, suggesting that the pleomorphic variant resulted from a common alteration.
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