The Effects of pH and Temperature on Cysteine Protease (Cathepsin B) Activity in Miracidia and Eggs of Fasciola hepatica
Background: Fascioliasis is a worldwide zoonotic disease caused by the trematodes Fasciola hepatica in humans and animals. Proteases are essential for the survival of parasites and have important activities such as penetration, tissue migration, and egg hatching. This study was conducted to analyze cysteine protease of the miracidia and eggs of F. hepatica, and to assess the effects of pH and temperature on the proteases activity and stability.
Methods: Adults F. hepatica were isolated from infected livers and were morphologically identified in 2018. Eggs collected from the adults and incubated in distilled water at 28 °C for 16 d to produce miracidia. The extract was collected from miracidia and eggs. A substrate for cathepsin B (Z-Arg-Arg-Pna) was used to assess the enzyme activity at different (2-12) pH levels. After homogenization, protein level was measured with Bradford method. Estimation of optimum temperature and pH was performed in the temperature range of 10-90 ° C and pH values from 2-12.
Results: The highest activity of the miracidia and eggs enzyme extracts for Z-Arg-Arg-pNA was at pH 4. The miracidia extract was most stable at neutral pH and the eggs extract was most stable in acidic pH. The optimum temperature activity for both stages was 40 °C. These proteases were stable up to 40 °C.
Conclusion: Upon the importance of pH and temperature in the life cycle of F. hepatica, the current findings can be used for induction of some modifications in pH and preventing the activity of the enzymes for decrement of the efficacy of miracidia penetration into the intermediate snails and egg hatching of this zoonotic parasite.
2. WHO. Prevention and control of schistosomiasis and soil-transmitted helminthiasis. World Health Organization, 2002; Geneva.
3. Mas-Coma S. Epidemiology of fascioliasis in hu-man endemic areas. J Helminthol. 2005; 79(3):207-16.
4. Rawlings ND, Barrett AJ, Finn R. Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 2016; 44(D1):D343-50.
5. Dalton JP, Caffrey CR, Sajid M, et al. Proteases in trematode biology. In: Maule AG, Marks NJ edi-tors. Parasitic Flatworms: Molecular Biology, Bio-chemistry, Immunology and Physiology. Cam-bridge: CAB International; 2006. p. 348-368.
6. Tallima H, Dvorak J, Kareem S, et al. Protective immune responses against Schistosoma mansoni infec-tion by immunization with functionally active gut-derived cysteine peptidases alone and in combina-tion with glyceraldehyde 3-phosphate dehydrogen-ase. PLOS Negl Trop dis. 2017; 11(3):440-3.
7. Smooker PM, Jayaraj R, Pike RN, et al. Cathepsin B proteases of flukes: the key to facilitating parasite control? Trends Parasitol. 2010; 26(10):506-14.
8. Cancela M, Acosta D, Rinaldi G, et al. A distinctive repertoire of cathepsins is expressed by juvenile in-vasive Fasciola hepatica. Biochimie. 2008; 90(10):1461-75.
9. Abdulla MH, Lim KC, Sajid M, et al. Schistosoma mansoni: novel chemotherapy using a cysteine prote-ase inhibitor. PLOS Med. 2007; 4(1):10-4.
10. Engel JC, Doyle PS, Hsieh I,et al. Cysteine protease inhibitors cure an experimental Trypanosoma cruzi in-fection. J Exp Med. 1998; 188(4):725-34.
11. Beckham SA, Piedrafita D, Phillips CI, et al. A ma-jor cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a poten-tial role in the digestive tract of newly excysted ju-venile parasites. Int J Biochem Cell Biol. 2009; 41(7):1601-12.
12. McGonigle L, Mousley A, Marks NJ, et al. The silencing of cysteine proteases in Fasciola hepatica newly excysted juveniles using RNA interference reduces gut penetration. Int J Parasitol. 2008; 38(2):149-55.
13. Law RH, Smooker PM, Irving JA,et al. Cloning and expression of the major secreted cathepsin B-like protein from juvenile Fasciola hepatica and analysis of immunogenicity following liver fluke infection. In-fect Immun. 2003; 71(12):6921-32.
14. Dalton JP, McGonigle S, Rolph TP, et al. Induction of protective immunity in cattle against infection with Fasciola hepatica by vaccination with cathepsin L proteinases and with hemoglobin. Infect Immun. 1996; 64(12):5066-74.
15. Jayaraj R, Piedrafita D, Dynon K, et al. Vaccination against Fascioliasis by a multivalent vaccine of stage-specific antigens. Vet Parasitol. 2009; 160(3-4):230-6.
16. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ana-lytic Biochem. 1976; 72:248-54.
17. Wijffels GL, Panaccio M, Salvatore L, et al. The secreted cathepsin L-like proteinases of the trema-tode, Fasciola hepatica, contain 3-hydroxyproline resi-dues. Biochem J. 1994; 299 (3):781-90.
18. Dowd AJ, Dooley M, Fagain C, et al. Stability stud-ies on the cathepsin L proteinase of the helminth parasite, Fasciola hepatica. Enzyme Microb Technol. 2000; 27(8):599-604.
19. Siklos M, BenAissa M, Thatcher GR. Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathep-sin inhibitors. Acta Pharmaceutica Sinica B. 2015; 5(6):506-19.
20. Harmsen MM, Cornelissen JB, Buijs HE,et al. Identification of a novel Fasciola hepatica cathepsin L protease containing protective epitopes within the propeptide. Int J Parasitol. 2004; 34(6):675-82.
21. Corvo I, Cancela M, Cappetta M, et al. The major cathepsin L secreted by the invasive juvenile Fasciola hepatica prefers proline in the S2 subsite and can cleave collagen. Molecular Biochem Parasitol. 2009; 167(1):41-7.
22. Smith AM, Dowd AJ, Heffernan M, et al. Fasciola hepatica: a secreted cathepsin L-like proteinase cleaves host immunoglobulin. Int J Parasitol. 1993; 23(8):977-83.
23. Johnston KA, Lee MJ, Brough C, et al. Protease activities in the larval midgut of Heliothis virescens: evi-dence for trypsin and chymotrypsin-like enzymes. Insect Biochem Molecular Biol.1995; 25(3): 375–83.
24. Carmona C, Dowd AJ, Smith AM, et al. Cathepsin L proteinase secreted by Fasciola hepatica in vitro pre-vents antibody-mediated eosinophil attachment to newly excysted juveniles. Mol Biochem Parasitol. 1993; 62(1):9-17.
25. Fagbemi BO, Hillyer GV. The purification and characterization of a cysteine protease of Fasciola gi-gantica adult worms. Vet Parasitol. 1992; 43(3-4):223-32.
26. Mohamed SA, Fahmy AS, Mohamed TM, et al. Proteases in egg, miracidia and adult of Fasciola gigan-tica. Characterization of serine and cysteine proteas-es from adult. Comparative Biochem Physiol Part B, Biochem Mol Biol. 2005; 142(2):192-200.
27. Bromme D, Bonneau PR, Lachance P, et al. Func-tional expression of human cathepsin S in Saccharo-myces cerevisiae. Purification and characterization of the recombinant enzyme. J Biol Chem. 1993; 268(7):4832-38.
28. Turk V, Stoka V, Vasiljeva O, et al. Cysteine ca-thepsins: from structure, function and regulation to new frontiers. Biochimica et Biophysica Acta. 2012; 1824(1):68-88.
29. Da Silva-Lopez RE, Giovanni-De-Simone S. Leishmania (Leishmania) amazonensis: purification and characterization of a promastigote serine protease. Exp Parasitol. 2004; 107(3-4):173-82.
30. Michalski WP, Crooks JK, Prowse SJ. Purification and characterization of a serine-type protease from Eimeria tenella oocytes. Int J Parasitol.1994; 24(2):189–95.
31. Kong HH, Kim TH, Chung DI. Purification and characterization of a secretory serine proteinase of Acanthamoeba healyi isolated from GAE. J Parasitol. 2000; 86(1):12-17.
32. Li S, Chung YB, Chung BS,et al. The involvement of the cysteine proteases of Clonorchis sinensis meta-cercariae in excystment. Parasitol Res. 2004; 93(1):36-40.
33. Hurtrez-Bousses S, Meunier C, Durand P, et al. Dynamics of host-parasite interactions: the example of population biology of the liver fluke (Fasciola hepat-ica). Microbes Infect. 2001; 3(10):841-849.
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