Catalytically active recombinant cysteine proteases of Haemonchus contortus: their ability to degrade host blood proteins and modulate coagulation

Authors: KARUNAKARAN, ATHIRA - Orise Fellow; BAKSHI, MARIAM - University Of Maryland; RAJENDRAKUMAR, ARUNRAJ - University Of Maryland; Wilson-Welder, Jennifer; AROIAN, RAFFI - University Of Massachusetts; SCHWARZ, ERICH - Cornell University; HOMAN, E. JANE - Iogenetics, Llc; OSTROFF, GARY - University Of Massachusetts; Beshah, Ethiopia; Miramontes, Eliseo; Papadopoulos, Marianne Dia; BOWDRIDGE, SCOTT - West Virginia University; ZARLENGA, DANTE - Former ARS Employee; ZHU, XIAOPING - University Of Maryland; Tuo, Wenbin

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/26/2025
Publication Date: 12/16/2025
Citation: Karunakaran, A.C., Bakshi, M., Rajendrakumar, A.M., Wilson-Welder, J.H., Aroian, R.V., Schwarz, E.M., Homan, E., Ostroff, G.R., Beshah, E., Miramontes, E.N., Papadopoulos, M., Bowdridge, S.A., Zarlenga, D.S., Zhu, X., Tuo, W. 2025. Catalytically active recombinant cysteine proteases of Haemonchus contortus: their ability to degrade host blood proteins and modulate coagulation. International Journal of Molecular Sciences. 26(24). Article e12077. https://doi.org/10.3390/ijms262412077.
DOI: https://doi.org/10.3390/ijms262412077

Interpretive Summary: The infection of small ruminants by the parasitic stomach worm, Haemonchus contortus, is one of the top causes of production losses. Rapid emergence of chemical drug resistance in the parasite leads to failure to control the infection in livestock. This highlights the need to find vaccine targets to control H. contortus. Our studies focused on a group of proteins, called proteases, which the parasite makes and that are essential for its feeding and survival. The parasite proteases produced in mammalian cells maintained in the laboratory retain normal biological function, which is to break down the major host blood proteins such as immunoglobulins, albumin, hemoglobin, and blood clot proteins. Those blood derived proteins could provide sufficient nutrients necessary for the parasite's survival. Therefore, creating a vaccine that targets these proteins could hamper the ability of the parasite to survive and infect the animals. The results from this research will be useful to veterinarians and ruminant producers.

Technical Abstract: Haemonchus contortus is a blood-feeding gastrointestinal nematode that significantly impacts the health and productivity of small ruminants. The burden of parasitism and the escalating incidence of anthelmintic resistance necessitate alternative control methods. Here, we characterize the enzymatic activities of five mammalian cell-expressed recombinant H. contortus cysteine proteases (HcCPs), which include two cathepsin B-like proteins (HcCBP1 and HcCBP2) and three cysteine protease 1 proteins (HcCP1a, HcCP1b, and HcCP1c). We hypothesize that these enzymes degrade host blood proteins, thereby facilitating the parasite’s nutrient acquisition and survival. Using synthetic cathepsin (cat) substrates, we show that HcCBP2 was the only protein that exhibited high catB/L but low catB or catK activity, which was inhibited by the cysteine protease inhibitor E-64. All mHcCPs degraded fibrinogen (Fg), which led to delayed plasma clotting, reduced clot density, and lysed plasma clots. All HcCPs partially degraded hemoglobin (Hb), except for mHcCBP2, which degraded Hb and bovine serum albumin completely and bovine IgG partially in the presence of a reducing agent. In conclusion, by sustaining blood feeding and facilitating immune evasion and nutrient acquisition, the HcCPs may play an essential role in the parasite’s survival. Thus, vaccines or cysteine protease inhibitors targeting these parasitic enzymes may mitigate or prevent infections.