The Zoonotic Helminth Parasite Fasciola hepatica: Virulence-Associated Cathepsin B and Cathepsin L Cysteine Peptidases Secreted by Infective Newly Excysted Juveniles (NEJ)

SIMPLE SUMMARY: Fasciolosis, caused by the worm parasite Fasciola hepatica (liver fluke), is a global disease of farm animals and a neglected disease of humans. Infection arises from the ingestion of resistant metacercariae that contaminate vegetation. Within the intestine, the parasite excysts as an active larvae, the newly excysted juvenile (NEJ), that borrows through the intestinal wall to infect the host and migrates to the liver. NEJ release, tissue penetration and migration are facilitated by enzymes secreted by the parasite, namely, cathepsin B1 (FhCB1), cathepsin B2 (FhCB2), cathepsin B3 (FhCB3) and cathepsin L3 (FhCL3). While our knowledge of these enzymes is growing, we have yet to understand why the parasites require all four of them to invade the host. In this study, we produced functional recombinant forms of these enzymes and demonstrated that they vary greatly in terms of activity, optimal pH and substrate specificity, suggesting that, combined, these enzymes provide the parasite with an efficient digestion system for different host tissues and molecules. We also identified several compounds that inhibited the activity of these enzymes, but did not affect the ability of the larvae to excyst or survive. However, this does not exclude these enzymes as targets for development of drugs or vaccines. ABSTRACT: Fasciolosis caused by Fasciola hepatica is a major global disease of livestock and an important neglected helminthiasis of humans. Infection arises when encysted metacercariae are ingested by the mammalian host. Within the intestine, the parasite excysts as a newly excysted juvenile (NEJ) that penetrates the intestinal wall and migrates to the liver. NEJ excystment and tissue penetration are facilitated by the secretion of cysteine peptidases, namely, cathepsin B1 (FhCB1), cathepsin B2 (FhCB2), cathepsin B3 (FhCB3) and cathepsin L3 (FhCL3). While our knowledge of these peptidases is growing, we have yet to understand why multiple enzymes are required for parasite invasion. Here, we produced functional recombinant forms of these four peptidases and compared their physio-biochemical characteristics. Our studies show great variation of their pH optima for activity, substrate specificity and inhibitory profile. Carboxy-dipeptidase activity was exhibited exclusively by FhCB1. Our studies suggest that, combined, these peptidases create a powerful hydrolytic cocktail capable of digesting the various host tissues, cells and macromolecules. Although we found several inhibitors of these enzymes, they did not show potent inhibition of metacercarial excystment or NEJ viability in vitro. However, this does not exclude these peptidases as targets for future drug or vaccine development.