Fungal Pathogen Coccidioides Immitis Research Articles

Proteinase production by the parasitic cycle of the pathogenic fungus Coccidioides immitis.

Coccidioides immitis is the causative agent of coccidioidomycosis (valley fever), a potentially disseminated fungal disease. We hypothesized that proteinases are expressed by the parasitic life cycle of C. immitis and that they might play an important role in the pathogenesis of coccidioidomycosis by facilitating spherule rupture, endospore dissemination, and tissue invasion and destruction. Filtrate from cultures of the parasitic life cycle of C. immitis was therefore assayed for proteolytic activity at neutral pH. The filtrate degraded 68% of a radiolabeled model of an elastin-rich extracellular matrix. The principal activity was against elastin and glycoprotein in the matrix. Degradation of purified elastin by filtrate was 222 micrograms/h per mg of filtrate protein at 37 degrees C. Denatured type I collagen (Azocoll) degradation was 13.5 mg/h per mg of filtrate protein at 37 degrees C. Proteinase activity peaked at 60 h of culture, correlating with release of endospores from mature spherules in the in vitro culture system. Elastase activity was attributed to a serine proteinase which exhibited an active-site preference for phenylalanine at the P1 site. The subunit molecular mass of the elastase determined by [3H]diisopropylfluorophosphate labeling was approximately 25 kilodaltons. Inhibition of the azocollytic activity of crude filtrate by 2 mM 1,10-phenanthroline and 10 mM EDTA, and stimulation by 2 mM CaCl2, suggested that a metalloproteinase was also present. Gelatin substrate gel electrophoresis with and without inhibitors confirmed that two proteinases were expressed, and they were separated by fast protein liquid chromatography.

Isocitrate lyase and malate synthase activities of Coccidioides immitis

The saprophytic phase of the fungal pathogen Coccidioides immitis contains both of the enzymes unique to the glyoxylate cycle (isocitrate lyase, EC 4.1.3.1, and malate synthase, EC 4.1.3.2) when grown in a variety of media regardless of whether or not glucose is present in those media. Significantly higher levels of each enzyme are found in cells grown in defined media. Enzyme activity varied as a function of growth, being higher during the germination phase than the exponential phase of growth. C. immitis arthroconidia do not germinate in medium containing glucose as the sole carbon source, but will germinate in this same medium with acetate as the carbon source. Glucose alone does not support vegetative growth and acetate is utilized preferentially over glucose when both are present in defined minimal medium. An inhibitor of isocitrate lyase, itaconic acid, inhibits germination and vegetative growth of C. immitis in all growth supporting media tested. Itaconic acid acts synergistically with amphotericin B to inhibit arthroconidia germination and growth.