Functional expression of a novel Kunitz type protease inhibitor from the human blood fluke Schistosoma mansoni

• Published in: Parasites & vectors Vol. 8; no. 1; p. 408
• Main Authors: Ranasinghe, Shiwanthi L., Fischer, Katja, Gobert, Geoffrey N., McManus, Donald P.
• Format: Journal Article
• Language: English
• Published: London BioMed Central 04.08.2015; BioMed Central Ltd; BMC
• Subjects: adults; Amino Acid Sequence; Animals; Anti-coagulant; Antibodies; anticoagulants; Biomedical and Life Sciences; Biomedicine; Blood; blood coagulation; Blood coagulation factors; blood flukes; cathepsins; chymotrypsin; Cloning, Molecular; coagulation; defense mechanisms; DNA, Helminth; eggs; elastase; Entomology; Enzymes; Escherichia coli; Gene Expression Regulation - physiology; Genetic aspects; Helminth Proteins - genetics; Helminth Proteins - metabolism; hosts; humans; Hydrolases; Infectious Diseases; inhibitory concentration 50; Kunitz type protease inhibitor; Medical examination; mice; Molecular Sequence Data; neutrophils; nucleotide sequences; Parasitology; Phylogeny; Physiological aspects; plasma kallikrein; Protease inhibitors; Protease Inhibitors - metabolism; protein secretion; proteinase inhibitors; Proteins; prothrombin; Schistosoma mansoni; Schistosoma mansoni - metabolism; schistosomiasis; SmKI-1; thromboplastin; Tropical Medicine; trypsin; vaccines; Veterinary Medicine/Veterinary Science; Viral antibodies; Virology; Western blotting; Australia; Germany; Kunitz type protease inhibitor; SmKI-1; Anti-coagulant
• ISSN: 1756-3305; 1756-3305
• DOI: 10.1186/s13071-015-1022-z

Background Schistosomes are able to survive for prolonged periods in the blood system, despite continuous contact with coagulatory factors and mediators of the host immune system. Protease inhibitors likely play a critical role in host immune modulation thereby promoting parasite survival in this extremely hostile environment. Even though Kunitz type serine protease inhibitors have been shown to play important physiological functions in a range of organisms these proteins are less well characterised in parasitic helminths. Methods We have cloned one gene sequence from S. mansoni , Smp_147730 ( SmKI-1 ) which is coded for single domain Kunitz type protease inhibitor, E. coli -expressed and purified. Immunolocalisation and western blotting was carried out using affinity purified polyclonal anti-SmKI-1 murine antibodies to determine SmKI-1 expression in the parasite. Protease inhibitor assays and coagulation assays were performed to evaluate the functional roles of SmKI-1. Results SmKI-1 is localised in the tegument of adult worms and the sub-shell region of eggs. Furthermore, this Kunitz protein is secreted into the host in the ES products of the adult worm. Recombinant SmKI-1 inhibited mammalian trypsin, chymotrypsin, neutrophil elastase, FXa and plasma kallikrein with IC 50 values of 35 nM, 61 nM, 56 nM, 142 nM and 112 nM, respectively. However, no inhibition was detected for pancreatic elastase or cathepsin G. SmKI-1 (4 μM) delayed blood clot formation, reflected in an approximately three fold increase in activated partial thromboplastin time and prothrombin time. Conclusions We have functionally characterised the first Kunitz type protease inhibitor (SmKI-1) from S. mansoni and show that it has anti-inflammatory and anti-coagulant properties. SmKI-1 is one of a number of putative Kunitz proteins in schistosomes that have presumably evolved as an adaptation to protect these parasites from the defence mechanisms of their mammalian hosts. As such they may represent novel vaccine candidates and/or drug targets for schistosomiasis control.