Sequential action of cell wall hydrolases in the germination and outgrowth of Microsporum gypseum macroconidia.
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Biomedical subjects
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A normally asporogenous pleomorphic strain of Microsporum gypseum was induced to sporulate by controlled aeration and dehydration. Aeration of the pleomorphic strain under optimal cultivation conditions caused the initiation of a sporulation cycle with equivalent growth parameters and percentage intracellular water loss as the wild-type strain. Initiation of sporulation was not due to alteration of the medium's nutrient concentration or consistency, concentration of fungal growth by-products, or removal of volatile "staling factors." Macroconidia formed by the pleomorphic colonies were of characteristic wildtype morphology, but germinated to form typical pleomorphic colonies, indicating that the induced sporulation was strictly phenotypic and reversible. Other asporogenous pleomorphic strains from different dermatophyte genera also were induced to form macroconidia by aeration, suggesting a similarity in sporulation induction in Microsporum sp., Epidermophyton floccosum, and Trichophyton violaceum. Initiation of sporulation by aeration further suggested that the pleomorphic mutation was one which affected the sensitivity of the pleomorphic aerial hyphae to natural sporulation inducers (i.e., decreased humidity) and did not represent a loss in the ability to form fertile macroconidia.
Three types of lysosomes containing either acid protease, alkaline protease, or phosphodiesterase were isolated from a Microsporum gypseum macroconidial homogenate on Ficoll gradients. The acid protease was contained in an assimilative lysosome since its activity was affected by the complexity of the exogenous nitrogen source. Ultracentrifugation and electron microscopy revealed that the alkaline protease-containing vesicles were associated with the spore coat material prior to macroconidial germination. During macroconidial germination, zones of spore coat hydrolysis were seen surrounding these vesicles. Other larger vesicles, believed to contain the phosphodiesterase, were also observed in the spore coat during macroconidial germination.
Germination of Microsporum gypseum macroconidia was accompanied by the release of alkaline protease, calcium ions, and inorganic phosphate into the germination fluid. The rate of germination was greatest during the first 2 hr, decreasing thereafter. This decrease in rate was accompanied by a decrease in protease activity, which was caused by an interaction of the enzyme with the inorganic phosphate released from the spores and accumulated in the germination medium after 2 hr. Germination of high spore densities was regulated by the ratio of released phosphate to protease protein, resulting in a constant percentage of germination at both high and low spore densities. A germination-defective mutant strain failed to germinate normally and released excessively high concentrations of phosphate into the germination medium during the initial 2 hr of incubation. Addition of calcium ions to germination mutant macroconidia stabilized spore morphology, prevented protease inactivation, and allowed normal germ-tube outgrowth. The germination of macroconidia appears to be regulated by the release of phosphate ions, which then inhibit the alkaline protease.