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Defining Substrate Specificities for Lipase and Phospholipase Candidates
Published on: November 23, 2016
Evidence of multiple extracellular phospholipase activities of Aspergillus fumigatus
Abstract:
Extracellular phospholipase activity has been implicated in the pathogenesis of several bacterial infections. Recently, extracellular phospholipase activity has been proposed as a virulence factor in the opportunistic yeast Candida albicans. Aspergillus fumigatus is the most pathogenic member of its genus, responsible for > 90% of infections. Previously, no specific virulence factors have been determined. We investigated the ability of A. fumigatus to produce extracellular phospholipases at 37 degrees C. Fast atom bombardment was used to compare lipid-containing media before and at 5-h intervals during shaking culture of A. fumigatus. Lipids were extracted and analyzed. Many anions corresponding to phospholipid breakdown products were identified. Specific anion species identified indicated phospholipase A, B, C (PLC), and D activities. PLC activity was further investigated by using the synthetic substrate p-nitrophenylphosphorylcholine. PLC activity was initially observed after 30 h of growth and accumulated in broth cultures up to 50 h. At 55 h, there was a sharp increase in PLC activity which coincided with cultures reaching the stationary phase. Activity of the PLC was measured at different temperatures, with greater activity occurring at 37 degrees C than at lower temperatures. Phospholipases could represent a virulence determinant in A. fumigatus.
Insights
Aspergillus fumigatus produces extracellular phospholipases, including phospholipase C, at 37°C, suggesting these enzymes may be virulence factors in fungal infections.
Area of Science:
- Mycology
- Medical Microbiology
- Biochemistry
Background:
- Extracellular phospholipase activity is a known virulence factor in bacterial infections and Candida albicans.
- Aspergillus fumigatus is a highly pathogenic fungus, but its specific virulence factors remain largely undetermined.
- Understanding fungal virulence mechanisms is crucial for developing effective antifungal therapies.
Purpose of the Study:
- To investigate the production of extracellular phospholipases by Aspergillus fumigatus at 37°C.
- To identify the types of phospholipase activities present in A. fumigatus cultures.
- To assess the correlation between phospholipase activity and fungal growth phases.
Main Methods:
- Lipid analysis of culture media using fast atom bombardment mass spectrometry at 5-hour intervals.
- Identification of phospholipid breakdown products to determine phospholipase activities (A, B, C, and D).
- Quantification of phospholipase C (PLC) activity using the synthetic substrate p-nitrophenylphosphorylcholine at varying temperatures.
Main Results:
- Analysis revealed anion species indicative of phospholipase A, B, C, and D activities.
- PLC activity was detected after 30 hours, accumulating up to 50 hours, with a sharp increase at 55 hours (stationary phase).
- Optimal PLC activity was observed at 37°C, higher than at lower temperatures.
Conclusions:
- Aspergillus fumigatus produces multiple extracellular phospholipases, including significant PLC activity.
- Phospholipase production, particularly PLC, is linked to the stationary phase of fungal growth.
- These extracellular phospholipases are potential virulence determinants in Aspergillus fumigatus infections.
