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An atypical and functionally diverse family of Kunitz-type cysteine/serine proteinase inhibitors secreted by the helminth parasite Fasciola hepatica.

ABSTRACT: Fasciola hepatica is a global parasite of humans and their livestock. Regulation of parasite-secreted cathepsin L-like cysteine proteases associated with virulence is important to fine-tune parasite-host interaction. We uncovered a family of seven Kunitz-type (FhKT) inhibitors dispersed into five phylogenetic groups. The most highly expressed FhKT genes (group FhKT1) are secreted by the newly excysted juveniles (NEJs), the stage responsible for host infection. The FhKT1 inhibitors do not inhibit serine proteases but are potent inhibitors of parasite cathepsins L and host lysosomal cathepsin L, S and K cysteine proteases (inhibition constants?15 in the reactive site loop P1 position that sits at the water-exposed interface of the S1 and S1' subsites of the cathepsin protease, and (b) Arg¹⁹ which forms cation-? interactions with Trp²⁹¹ of the S1' subsite and electrostatic interactions with Asp¹²⁵ of the S2' subsite. FhKT1.3 is exceptional, however, as it also inhibits the serine protease trypsin due to replacement of the P1 Leu¹⁵ in the reactive loop with Arg¹⁵. The atypical Kunitz-type inhibitor family likely regulate parasite cathepsin L proteases and/or impairs host immune cell activation by blocking lysosomal cathepsin proteases involved in antigen processing and presentation.

SUBMITTER: Smith D

PROVIDER: S-EPMC7692546 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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An atypical and functionally diverse family of Kunitz-type cysteine/serine proteinase inhibitors secreted by the helminth parasite Fasciola hepatica.

Smith David D Cwiklinski Krystyna K Jewhurst Heather H Tikhonova Irina G IG Dalton John P JP

Scientific reports 20201126 1

Fasciola hepatica is a global parasite of humans and their livestock. Regulation of parasite-secreted cathepsin L-like cysteine proteases associated with virulence is important to fine-tune parasite-host interaction. We uncovered a family of seven Kunitz-type (FhKT) inhibitors dispersed into five phylogenetic groups. The most highly expressed FhKT genes (group FhKT1) are secreted by the newly excysted juveniles (NEJs), the stage responsible for host infection. The FhKT1 inhibitors do not inhibit ...[more]

PMID: 33244035

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Project description:Helminth parasites secrete extracellular vesicles (EVs) that can be internalised by host immune cells resulting in modulation of host immunity. While the molecular cargo of EVs have been characterised in many parasites, little is known about the surface-exposed molecules that participate in ligand-receptor interactions with the host cell surface to initiate vesicle docking and subsequent internalisation. Using a membrane-impermeable biotin reagent to capture proteins displayed on the outer membrane surface of two EV sub-populations (termed 15k and 120k EVs) released by adult F. hepatica, we describe 380 surface proteins including an array of virulence factors, membrane transport proteins and molecules involved in EV biogenesis/trafficking. Proteomics and immunohistochemical analysis show that the 120k EVs have an endosomal origin and may be released from the parasite via the protonephridial (excretory) system whilst the larger 15k EVs are released from the gastrodermal epithelial cells that line the fluke gut. A parallel lectin microarray strategy was used to profile the topology of major surface oligosaccharides of intact fluorogenically-labelled EVs as they would be displayed to the host. Lectin profiles corresponding to glycoconjugates exposed on the surface of the 15 K and 120K EV sub-populations are practically identical but are distinct from those of the parasite surface tegument, although all are predominated by high mannose sugars. We found that while the F. hepatica EVs were resistant to exo- and endo-glycosidases, the glyco-amidase PNGase F drastically remodelled the surface oligosaccharides and blocked the uptake of EVs by host macrophages. In contrast, pre-treatment with antibodies obtained from infected hosts, or purified antibodies raised against the extracellular domains of specific EV surface proteins (DM9-containing protein, CD63 receptor and myoferlin), significantly enhanced their cellular internalisation. This work highlights the diversity of EV biogenesis and trafficking pathways used by F. hepatica and sheds light on the molecular interaction between parasite EVs and host cells.

Project description: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.

Dataset Information

An atypical and functionally diverse family of Kunitz-type cysteine/serine proteinase inhibitors secreted by the helminth parasite Fasciola hepatica.

Publications

An atypical and functionally diverse family of Kunitz-type cysteine/serine proteinase inhibitors secreted by the helminth parasite Fasciola hepatica.

Similar Datasets

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