Fasciola hepatica Kunitz-type inhibitors

• Main Author: Smith, David
• Published: Queen's University Belfast 2017
• Subjects: 636.089
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.728662

The aim of this thesis was to characterize Kunitz-type (KT) protease inhibitors expressed by the helminth parasite Fasciola hepatica during infection of the mammalian host. KT inhibitors have traditionally been considered serine protease inhibitors. A P1 residue, typically Lys or Arg, within a reactive loop, determines serine protease inhibition specificity. Recombinant KT inhibitor expression was carried out for the gene identified in F. hepatica (fhktl) that encodes an uncommon P1 Leu residue. Inhibition studies found that FhKT1 did not inhibit serine proteases, but did inhibit cysteine protease, particularly cathepsin L-like cysteine proteases. Molecular modelling predicted that residues P1 Leu15 to P4’ Arg19 of the KT inhibitor interact with the S2 and S2’ pockets of the cysteine protease. Interrogation of the F. hepatica draft genome identified seven KT inhibitors in this parasite, which were found to fall into 5 distinct groups. The fhktl group is made up of three highly similar KT genes, two with a P1 Leu residue (fh ktl.1 and fh k tl.2), but another with a P1 Arg (fh ktl.3). Recombinant FhKT1.3was found to inhibit cysteine proteases, as well as trypsin. Transcriptomic analysis revealed that the fhkt genes are temporally regulated across mammal-associated parasite life-cycle stages, with only the fhktl group expressed at all stages. The fhktl group was also found to be the most highly expressed, as well as the only FhKTs secreted by the parasite. Based on the inhibition specificity of FhKT1 inhibitors, their constituative expression, tissue-specific localization and their presence in parasite secretions, these inhibitors are proposed to be multi-functional, with a primary role in the regulation of F. hepatica and host cathepsin L- like cysteine proteases. Based on the ability of FhKT1.3 to inhibit trypsin, this inhibitor could also function in parasite defence. FhKT1 represents a target at which a novel drug or vaccine could be directed.