Fasciola hepatica serine protease inhibitor family (serpins): Purposely crafted for regulating host proteases

Abstract

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 (K i = 40 nM) whilst rFhSrp2 was a highly potent inhibitor of chymotrypsin (K i = 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.

Complementary transcriptomic and proteomic analyses reveal the cellular and molecular processes that drive growth and development of Fasciola hepatica in the host liver

“…The 27 proteins found to be exclusively secreted by the immature flukes includes a range of uncharacterised proteins, a specific member of the cathepsin L3 peptidase group (FhCL3_2; BN1106_s8881B000009), FhKT1 group member FhKT1.1 (BN1106_s8826B000029), a saposin (BN1106_s4986B000028) and a ferritin protein (BN1106_s3950B000041). The NEJ-specific proteins also include a range of uncharacterised proteins, in addition to a serpin (FhSrp3, based on nomenclature of [ 57 ], a member of the cathepsin L3 peptidases (FhCL3_3; BN1106_s10139B000014) and thioredoxin glutathione reductase (TGR). Key proteins exclusively secreted in high amounts by the adults include a ferritin (BN1106_s709B000627) and a mu class GST (BN1106_s4370B000168).…”

“…The NEJ and adults secrete comparable levels of stefins/cystatins, which is comprised predominately of cathepsin L peptidase inhibitor, FhStefin-1. Similarly, both these stages also secrete members of the serpin family, which have distinct inhibitory profiles, specifically being potent inhibitors of host chymotrypsin and kallikrein [ 57 , 59 ]. FhSrp1 and FhSrp3 are secreted in greatest abundance by the NEJ compared with the FhSrp4 by the adults.…”

Proteomic Profiling of the Liver, Hepatic Lymph Nodes, and Spleen of Buffaloes Infected with Fasciola gigantica

“…Inside the buffaloes, the flukes feed on hepatic tissue and blood to support their own growth and development. During their migration and development within the host, F. gigantica flukes must overcome a variety of biological barriers and produce effector molecules, which alter the gene expression [5][6][7][8] and modulate the immune responses [9,10] of the affected animals.…”

Host-Parasite Relationships in Porcine Ascariosis: Anticoagulant Potential of the Third Larval Stage of Ascaris suum as a Possible Survival Mechanism

“…The subsequent analysis by LC-MS/MS of these complexes allowed us to identify three A. suum serpins as potential candidates to inhibit FXa. It is worth mentioning that, in addition to their role in disrupting coagulation cascade, helminth serpins have also been related to other important survival mechanisms such as the modulation of inflammation and the host immune system [ 55 , 56 , 57 ]. In regards to porcine ascariosis, complexes formed between A. suum serpins and host proteases have been postulated as a mechanism to mask the larvae surface in order to evade the host immune response during their migration [ 58 ].…”