Fasciola hepatica: the biological, biochemical and mechanical characterisation of a novel serpin, FhSrp-6

Fasciola hepatica, known as liver fluke, is a globally distributed parasitic helminth (worm) capable of infecting a wide range of mammal hosts, including humans. It is an economically important pathogen of livestock (particularly sheep and cattle), responsible for extensive losses of around 2.5 billion euro worldwide, and approximately 90 million euro to the Irish livestock industry annually. Drug resistant parasites, especially to the frontline flukicide triclabendazole, are emerging in all continents leaving farmers devoid of means to control the parasite on their farms. It is imperative that we develop new means of parasite control and, at the same time, move away from the use of environmentally damaging chemicals. Therefore, the future of parasite control depends on the development of efficacious vaccines. Serine protease inhibitors (serpins) regulate proteolytic events within diverse biological processes, including digestion, coagulation, inflammation and immune responses. The presence of serpins in juvenile and mature F. hepatica excretory-secretory products indicates that the parasite uses these inhibitors to alter protease activity encountered during its development within the mammalian host. Interrogation of the F. hepatica genome identified a multi-gene serpin family of seven members that has expanded by gene duplication and divergence to create an array of inhibitors with distinct inhibitory profiles. Here, we investigated the molecular properties and functions of F. hepatica serpin-6 (FhSrp-6), which is expressed throughout the lifecycle of the parasite. The recombinant version of FhSrp-6 produced exhibited a unique inhibitory profile for serine proteases compared to other F. hepatica serpins, namely FhSrp-1 and FhSrp-2, previously characterised in our laboratory. This inhibitory profile revealed that rFhSrp-6 is potent against a range of proteases involved in digestion and the inflammatory immune response. Furthermore, we show that the unique inhibitory profile of this serpin was consistent with differences in amino acids presents in the reactive centre loop (RCL) of the inhibitor. rFhSrp-6 efficiently inhibited chymotrypsin (Ki = 1.38 nM), chymase (Ki = 9.65 nM) and cathepsin G (Ki = 2.25 nM). The potency at which rFhSrp-6 inhibits the activity of these proteases suggests a high level of complexity of F. hepatica and its ability to expand the serpin family to allow it to survive within the definitive host. The vulnerability of the parasite shows why such potent inhibitors are needed to deter any harmful proteolytic effects of host proteases. Future investigation is needed into this superfamily of serine protease inhibitors to understand the complexity of the host-parasite interaction. However, given the proposed importance of serpins in fluke biology and host-parasite interactions we suggest that this and other members of the serpin family are potential candidates at which new anti-fluke vaccines could be directed.

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University of Galway

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Attribution-NonCommercial-NoDerivatives 4.0 International

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University of Galway Theses (Research Masters Theses)