Measuring A. alternata Protease Activity and Their Effects on Asthma

Abstract

Alternaria alternata, is a key factor in childhood allergic asthma in semi-arid climates. We have shown that filtrates from A. alternata can both cause asthma symptoms in a mouse model as well as directly activate human bronchial epithelial cells, which provide the first point of contact for microbes in the airway lumen. These interactions are dependent on A. alternata protease activity within the host acting on protease activated receptor-2 (PAR₂) (1). Proteolysis of PAR₂ results in an exposed “tethered ligand” that initiates host intracellular signaling pathways and subsequent physiological response (1). In this report, we measured the protease activity of various A. alternata filtrate samples using a standard, casein-based fluorescent protease detection assay and a novel fluorescent probe we designed to include the specific proteolytic sequence of PAR₂. Both assays showed protease activity of A. alternata filtrates that matched their respective signaling responses in host epithelial cells. Conditions that limited protease activity effectively blocked protease detections in the assays and cell signaling responses. We conclude that A. alternata filtrates induce airway epithelial cell signaling and asthmatic response partly via protease activity on PAR₂. By utilizing our novel proteolytic assay we may better predict potency of various PAR₂-dependent asthmagens in vitro.