Discussion

Proteases are multi-functional, proteolytic enzymes that have complex roles in human health and disease, and therefore, detecting the activities of these enzymes can yield important insights into communicable and non-communicable diseases. Thus, the development of novel detection strategies for proteases may be beneficial to a host of global health applications. Using a synthetic biology approach, systematically, we have developed Advanced proteoLytic detector PolyHydroxyAlkanoates beads (AL-PHA) – a library of low-cost, biodegradable, bioplastic-based protease biosensors. These biosensors utilise PhaC-superfolder GFP (sfGFP) reporter fusion proteins that are bound to microbially manufactured polyhydroxyalkanoate (PHA) bioplastic beads. The design of the PhaC-sfGFP reporter fusions also incorporate modular, specific protease cleavage sites, thereby enabling the detection of specific proteases of choice. In the presence of a specific protease, sfGFP reporter proteins are cleaved off of the AL-PHA beads - resulting in a loss of bead fluorescence. These AL-PHA biosensors were initially optimised using a commercially available Tobacco Etch Virus (TEV) protease. Our third generation TEV biosensor (PhaC-112L-T-G) detected 0.5-1 U of AcTEV activity and 10 units of AcTEV protease activity resulted in a visually noticeable loss in AL-PHA bead fluorescence.

It is known that parasites employ proteases to support pathogenesis. In the case of Schistosomiasis (Bilharzia), a disease that affects over 200 million people, the invasive Schistosoma cercariae release a cocktail of proteases, including elastase, that help the parasite to invade into a host through the skin. We have therefore, designed AL-PHA beads that contain a protease cleavage site specific for Schistosoma mansoni cercarial elastase. These S. mansoni cercarial elastase specific AL-PHA beads were validated using S. mansoni cercarial transformation fluid (SmCTF) samples, and were able to detect elastase. We envision that AL-PHA beads could be adapted into a low-cost and high-throughput protease detection assay for global health applications.