Centre for One Health and Ryan Institute, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
School of Pharmacy, Medical Biology Centre, Queen's University Belfast, Belfast, United Kingdom.
PLoS Negl Trop Dis. 2020 Aug 6;14(8):e0008510. doi: 10.1371/journal.pntd.0008510. eCollection 2020 Aug.
Serine protease inhibitors (serpins) regulate proteolytic events within diverse biological processes, including digestion, coagulation, inflammation and immune responses. The presence of serpins in Fasciola hepatica excretory-secretory products indicates that the parasite exploits these to regulate proteases encountered during its development within vertebrate hosts. 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 specificities. We investigated the molecular properties and functions of two representatives, FhSrp1 and FhSrp2, highly expressed in the invasive newly excysted juvenile (NEJ). Consistent with marked differences in the reactive centre loop (RCL) that executes inhibitor-protease complexing, the two recombinant F. hepatica serpins displayed distinct inhibitory profiles against an array of mammalian serine proteases. In particular, rFhSrp1 efficiently inhibited kallikrein (Ki = 40 nM) whilst rFhSrp2 was a highly potent inhibitor of chymotrypsin (Ki = 0.07 nM). FhSrp1 and FhSrp2 are both expressed on the NEJ surface, predominantly around the oral and ventral suckers, suggesting that these inhibitors protect the parasites from the harmful proteolytic effects of host proteases, such as chymotrypsin, during invasion. Furthermore, the unusual inhibition of kallikrein suggests that rFhSrp1 modulates host responses such as inflammation and vascular permeability by interfering with the kallikrein-kinin system. A vaccine combination of rFhSrp1 and rFhSrp2 formulated in the adjuvant Montanide ISA 206VG elicited modest but non-significant protection against a challenge infection in a rat model, but did induce some protection against liver pathogenesis when compared to a control group and a group vaccinated with two well-studied vaccine candidates, F. hepatica cathepsin L2 and L3. This work highlights the importance of F. hepatica serpins to regulate host responses that enables parasite survival during infection and, coupled with the vaccine data, encourages future vaccine trials in ruminants.
丝氨酸蛋白酶抑制剂（serpins）调节多种生物学过程中的蛋白水解事件，包括消化、凝血、炎症和免疫反应。在肝片形吸虫的排泄分泌产物中存在 serpins，表明寄生虫利用这些抑制剂来调节在脊椎动物宿主内发育过程中遇到的蛋白酶。对肝片形吸虫基因组的分析鉴定了一个多基因丝氨酸蛋白酶抑制剂家族，该家族通过基因复制和分化扩张，产生了具有不同特异性的抑制剂。我们研究了两个代表性成员，FhSrp1 和 FhSrp2 的分子特性和功能，它们在侵袭性新孵出的幼虫（NEJ）中高度表达。与执行抑制剂-蛋白酶复合物形成的反应中心环（RCL）的显著差异一致，两种重组肝片形吸虫丝氨酸蛋白酶抑制剂对一系列哺乳动物丝氨酸蛋白酶表现出不同的抑制谱。特别是，rFhSrp1 有效地抑制激肽释放酶（Ki = 40 nM），而 rFhSrp2 是糜蛋白酶的高效抑制剂（Ki = 0.07 nM）。FhSrp1 和 FhSrp2 都在 NEJ 表面表达，主要分布在口吸盘和腹吸盘周围，这表明这些抑制剂可以保护寄生虫免受宿主蛋白酶（如糜蛋白酶）在侵袭过程中的有害蛋白水解作用的影响。此外，激肽释放酶的异常抑制表明 rFhSrp1 通过干扰激肽释放酶-激肽系统来调节宿主反应，如炎症和血管通透性。rFhSrp1 和 rFhSrp2 与佐剂 Montanide ISA 206VG 联合制成的疫苗在大鼠模型中进行挑战感染时，仅产生适度但无统计学意义的保护，但与对照组和用两种经过充分研究的疫苗候选物（肝片形吸虫组织蛋白酶 L2 和 L3）进行疫苗接种的组相比，对肝发病机制有一定的保护作用。这项工作强调了肝片形吸虫丝氨酸蛋白酶抑制剂对调节宿主反应的重要性，这使得寄生虫能够在感染过程中存活下来，并且结合疫苗数据，鼓励在反刍动物中进行未来的疫苗试验。
