Fasciola hepatica is refractory to complement killing by preventing attachment of mannose binding lectin (MBL) and inhibiting MBL-associated serine proteases (MASPs) with serpins

The complement system is a first-line innate host immune defence against invading pathogens. It is activated via three pathways, termed Classical, Lectin and Alternative, which are mediated by antibodies, carbohydrate arrays or microbial liposaccharides, respectively. The three complement pathways converge in the formation of C3-convertase followed by the assembly of a lethal pore-like structure, the membrane attack complex (MAC), on the pathogen surface. We found that the infectious stage of the helminth parasite Fasciola hepatica, the newly excysted juvenile (NEJ), is resistant to the damaging effects of complement. Despite being coated with mannosylated proteins, the main initiator of the Lectin pathway, the mannose binding lectin (MBL), does not bind to the surface of live NEJ. In addition, we found that recombinantly expressed serine protease inhibitors secreted by NEJ (rFhSrp1 and rFhSrp2) selectively prevent activation of the complement via the Lectin pathway. Our experiments demonstrate that rFhSrp1 and rFhSrp2 inhibit native and recombinant MBL-associated serine proteases (MASPs), impairing the primary step that mediates C3b and C4b deposition on the NEJ surface. Indeed, immunofluorescence studies show that MBL, C3b, C4b or MAC are not deposited on the surface of NEJ incubated in normal human serum. Taken together, our findings uncover new means by which a helminth parasite prevents the activation of the Lectin complement pathway to become refractory to killing via this host response, in spite of presenting an assortment of glycans on their surface.</jats:p

Production of a functionally active recombinant SARS-CoV-2 (COVID-19) 3C-Like protease and a soluble inactive 3C-like protease-RBD chimeric in a prokaryotic expression system

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for the establishment of the viral replication and transcription complex (RTC), crucial for viral replication. Hence, these proteases are considered prime targets against which anti-coronavirus disease 2019 (COVID-19) drugs could be developed. Here, we describe the expression of a highly soluble and functionally active recombinant 3CL-pro using Escherichia coli BL21 cells. We show that the enzyme functions in a dimeric form and exhibits an unexpected inhibitory profile because its activity is potently blocked by serine rather than cysteine protease inhibitors. In addition, we assessed the ability of our 3CL-pro to function as a carrier for the receptor binding domain (RBD) of the Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro activity, but its enhanced solubility made purification easier and improved RBD antigenicity when tested against serum from vaccinated individuals in ELISAs. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines

Two Distinct Superoxidase Dismutases (SOD) Secreted by the Helminth Parasite Fasciola hepatica Play Roles in Defence against Metabolic and Host Immune Cell-Derived Reactive Oxygen Species (ROS) during Growth and Development

The antioxidant superoxide dismutase (SOD) catalyses the dismutation of superoxide, a dangerous oxygen free radical, into hydrogen peroxide and molecular oxygen. Superoxide generation during the oxidative burst of the innate immune system is considered a key component of the host defence against invading pathogens. We demonstrate the presence and differential expression of two SODs in Fasciola hepatica, a leaderless cytosolic (FhSOD1) and an extracellular (FhSOD3) form containing a secretory signal peptide, suggesting that the parasites exploit these enzymes in distinct ways to counteract reactive oxygen species (ROS) produced by cellular metabolism and immune defences. Both enzymes are highly expressed by the infective newly excysted juvenile (NEJ) stages and are found in abundance in their excretory&ndash;secretory products (ES), but only FhSOD1 is present in adult ES, suggesting that the antioxidants have different functions and pathways of secretion, and are under separate temporal expression control during the migration, growth, and development of the parasite. Functionally, the recombinant FhSOD1 and FhSOD3 exhibit similar activity against superoxide to their mammalian counterparts. Confocal immuno-localisation studies demonstrated the presence of FhSOD1 and FhSOD3 on the NEJ tegument and parenchyma, supporting our suggestion that these enzymes are secreted during host invasion to protect the parasites from the harmful oxidative bursts produced by the activated innate immune response. By producing superoxide enzymatically in vitro, we were able to demonstrate robust killing of F. hepatica NEJ within 24 h post-excystment, and that the lethal effect of ROS was nullified with the addition of SOD and catalase (the antioxidant enzyme responsible for the dismutation of hydrogen peroxide, a by-product of the SOD reaction). This study further elucidates the mechanism by which F. hepatica protects against ROS derived from cellular metabolism and how the parasite could mitigate damage caused by the host&rsquo;s immune response to benefit its survival

Regulation of the Fasciola hepatica newly excysted juvenile cathepsin L3 (FhCL3) by its propeptide: a proposed 'clamp-like' mechanism of binding and inhibition

AbstractBackgroundThe zoonotic worm parasiteFasciola hepaticasecretes an abundance of cathepsin L peptidases that are associated with virulence, invasiveness, feeding and migration. The peptidases are produced as inactive zymogens that activate at low pH by autocatalytic removal of their N-terminal pro-domain or propeptide. Propeptides bind to their cognate enzyme with high specificity. Little is known, however, about the mechanism by which the propeptide of FhCL3, a cathepsin L peptidase secreted by the infective newly excysted juveniles (NEJs), regulates the inhibition and activation of the mature enzyme before it is secreted into host tissues.ResultsImmunolocalisation/immunoblotting studies show that the FhCL3 zymogen is produced and secreted by gastrodermal cells of the NEJs gut. A recombinant propeptide of FhCL3 (ppFhCL3) was shown to be a highly potent and selective inhibitor of native and recombinantF. hepaticaFhCL3 peptidase, and other members of the cathepsin L family; inhibition constant (Ki) values obtained for FhCL1, FhCL2 and FhCL3 were 0.04 nM, 0.004 nM and &lt; 0.002 nM, respectively. These values are at least 1000-fold lower than thoseKiobtained for human cathepsin L (HsCL) and human cathepsin K (HsCK) demonstrating the selectivity of the ppFhCL3 for parasite cathepsins L. By exploiting 3-D structural data we identified key molecular interactions in the specific binding between the ppFhCL3 and FhCL3 mature domain. Using recombinant variants of ppFhCL3 we demonstrated the critical importance of a pair of propeptide residues (Tyr46Lys47) for the interaction with the propeptide binding loop (PBL) of the mature enzyme and other residues (Leu66and Glu68) that allow the propeptide to block the active site.ConclusionsThe FhCL3 peptidase involved in host invasion byF. hepaticais produced as a zymogen in the NEJs gut. Regulation of its activation involves specific binding sites within the propeptide that are interdependent and act as a “clamp-like” mechanism of inhibition. These interactions are disrupted by the low pH of the NEJs gut to initiate autocatalytic activation. Our enzyme kinetics data demonstrates high potency and selectivity of the ppFhCL3 for its cognate FhCL3 enzyme, information that could be utilised to design inhibitors of parasite cathepsin L peptidases.</jats:sec

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

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.This study was funded by the Science Foundation Ireland (SFI, Republic of Ireland)-Department of Education and Learning (DEL, Northern Ireland) grant 14/IA/2304 and Science Foundation Ireland (SFI, Republic of Ireland) grant 17/RP/5368. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.peer-reviewe