Exploring the utility of circulating miRNAs as diagnostic biomarkers of fasciolosis.

Effective management and control of parasitic infections on farms depends on their early detection. Traditional serological diagnostic methods for Fasciola hepatica infection in livestock are specific and sensitive, but currently the earliest detection of the parasite only occurs at approximately three weeks post-infection. At this timepoint, parasites have already entered the liver and caused the tissue damage and immunopathology that results in reduced body weight and loss in productivity. Here, we investigated whether the differential abundance of micro(mi)miRNAs in sera of F. hepatica-infected sheep has potential as a tool for the early diagnosis of infection. Using miRNA sequencing analysis, we discovered specific profiles of sheep miRNAs at both the pre-hepatic and hepatic infection phases in comparison to non-infected sheep. In addition, six F. hepatica-derived miRNAs were specifically identified in sera from infected sheep. Thus, a panel of differentially expressed miRNAs comprising four sheep (miR-3231-3p; miR133-5p; 3957-5p; 1197-3p) and two parasite miRNAs (miR-124-3p; miR-Novel-11-5p) were selected as potential biomarkers. The expression of these candidates in sera samples from longitudinal sheep infection studies collected between 7 days and 23 weeks was quantified using RT-qPCR and compared to samples from age-matched non-infected sheep. We identified oar-miR-133-5p and oar-miR-3957-5p as promising biomarkers of fasciolosis, detecting infection as early as 7 days. The differential expression of the other selected miRNAs was not sufficient to diagnose infection; however, our analysis found that the most abundant forms of fhe-miR-124-3p in sera were sequence variants (IsomiRs) of the canonical miRNA, highlighting the critical importance of primer design for accurate diagnostic RT-qPCR. Accordingly, this investigative study suggests that certain miRNAs are biomarkers of F. hepatica infection and validates miRNA-based diagnostics for the detection of fasciolosis in sheep.

Glycan Complexity and Heterogeneity of Glycoproteins in Somatic Extracts and Secretome of the Infective Stage of the Helminth Fasciola hepatica.

Fasciola hepatica is a global helminth parasite of humans and their livestock. The invasive stage of the parasite, the newly excysted juvenile (NEJs), relies on glycosylated excreted-secreted (ES) products and surface/somatic molecules to interact with host cells and tissues and to evade the host's immune responses, such as disarming complement and shedding bound antibody. While -omics technologies have generated extensive databases of NEJs' proteins and their expression, detailed knowledge of the glycosylation of proteins is still lacking. Here, we employed glycan, glycopeptide, and proteomic analyses to determine the glycan profile of proteins within the NEJs' somatic (Som) and ES extracts. These analyses characterized 123 NEJ glycoproteins, 71 of which are secreted proteins, and allowed us to map 356 glycopeptides and their associated 1690 N-glycan and 37 O-glycan forms to their respective proteins. We discovered abundant micro-heterogeneity in the glycosylation of individual glycosites and between different sites of multi-glycosylated proteins. The global heterogeneity across NEJs' glycoproteome was refined to 53 N-glycan and 16 O-glycan structures, ranging from highly truncated paucimannosidic structures to complex glycans carrying multiple phosphorylcholine (PC) residues, and included various unassigned structures due to unique linkages, particularly in pentosylated O-glycans. Such exclusive glycans decorate some well-known secreted molecules involved in host invasion, including cathepsin B and L peptidases, and a variety of membrane-bound glycoproteins, suggesting that they participate in host interactions. Our findings show that F. hepatica NEJs generate exceptional protein variability via glycosylation, suggesting that their molecular portfolio that communicates with the host is far more complex than previously anticipated by transcriptomic and proteomic analyses. This study opens many avenues to understand the glycan biology of F. hepatica throughout its life-stages, as well as other helminth parasites, and allows us to probe the glycosylation of individual NEJs proteins in the search for innovative diagnostics and vaccines against fascioliasis.

A new multiplex SARS-CoV-2 antigen microarray showed correlation of IgG, IgA, and IgM antibodies from patients with COVID-19 disease severity and maintenance of relative IgA and IgM antigen binding over time.

Zoonotic spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to humans in December 2019 caused the coronavirus disease 2019 (COVID-19) pandemic. Serological monitoring is critical for detailed understanding of individual immune responses to infection and protection to guide clinical therapeutic and vaccine strategies. We developed a high throughput multiplexed SARS-CoV-2 antigen microarray incorporating spike (S) and nucleocapsid protein (NP) and fragments expressed in various hosts which allowed simultaneous assessment of serum IgG, IgA, and IgM responses. Antigen glycosylation influenced antibody binding, with S glycosylation generally increasing and NP glycosylation decreasing binding. Purified antibody isotypes demonstrated a binding pattern and intensity different from the same isotype in whole serum, probably due to competition from the other isotypes present. Using purified antibody isotypes from naïve Irish COVID-19 patients, we correlated antibody isotype binding to different panels of antigens with disease severity, with binding to the S region S1 expressed in insect cells (S1 Sf21) significant for IgG, IgA, and IgM. Assessing longitudinal response for constant concentrations of purified antibody isotypes for a patient subset demonstrated that the relative proportion of antigen-specific IgGs decreased over time for severe disease, but the relative proportion of antigen-specific IgA binding remained at the same magnitude at 5 and 9 months post-first symptom onset. Further, the relative proportion of IgM binding decreased for S antigens but remained the same for NP antigens. This may support antigen-specific serum IgA and IgM playing a role in maintaining longer-term protection, important for developing and assessing vaccine strategies. Overall, these data demonstrate the multiplexed platform as a sensitive and useful platform for expanded humoral immunity studies, allowing detailed elucidation of antibody isotypes response against multiple antigens. This approach will be useful for monoclonal antibody therapeutic studies and screening of donor polyclonal antibodies for patient infusions.

In vitro evaluation of the potential use of snake-derived peptides in the treatment of respiratory infections using inhalation therapy: A proof of concept study.

Inhalation therapy using nebulisers is an attractive non-invasive route for drug delivery, particularly for the treatment of lung infections with anti-inflammatory and anti-microbial compounds. This study evaluated the suitability of three snake-derived peptides (termed Sn1b, SnE1 and SnE1-F), which we have recently shown have potent anti-inflammatory and bacteriostatic activities, for nebulisation using a vibrating mesh nebuliser (VMN). The effect of nebulisation on peptide concentration, stability and function were assessed, prior to progression to aerodynamic particle size distribution, and in vitro drug delivery in simulated adult spontaneous breathing and mechanical ventilated patient models. When nebulised, all three peptides exhibited similar functions to their non-nebulised counterparts and were found to be respirable during simulated mechanical ventilation. Based on the assessment of the droplet distributions of nebulised peptides using a Next Generation Impactor (NGI) demonstrated that if administered in vivo each peptide would likely be delivered to the lower airways. These data suggest that nebulisation using a VMN is a viable means of anti-microbial / anti-inflammatory peptide delivery targeting microbial respiratory infections, and possibly even systemic infections.

Omics tools enabling vaccine discovery against fasciolosis.

In the past decade significant advances in our understanding of liver fluke biology have been made through in-depth interrogation and analysis of evolving Fasciola hepatica and Fasciola gigantica omics datasets. This information is crucial for developing novel control strategies, particularly vaccines necessitated by the global spread of anthelmintic resistance. Distilling them down to a manageable number of testable vaccines requires combined rational, empirical, and collaborative approaches. Despite a lack of clear outstanding vaccine candidate(s), we must continue to identify salient parasite-host interacting molecules, likely in the secretory products, tegument, or extracellular vesicles, and perform robust trials especially in livestock, using present and emerging vaccinology technologies to discover that elusive liver fluke vaccine. Omics tools are bringing this prospect ever closer.

Stage-specific miRNAs regulate gene expression associated with growth, development and parasite-host interaction during the intra-mammalian migration of the zoonotic helminth parasite Fasciola hepatica.

BACKGROUND: MiRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression in organisms ranging from viruses to mammals. There is great relevance in understanding how miRNAs regulate genes involved in the growth, development, and maturation of the many parasitic worms (helminths) that together afflict more than 2 billion people. RESULTS: Here, we describe the miRNAs expressed by each of the predominant intra-mammalian development stages of Fasciola hepatica, a foodborne flatworm that infects a wide range of mammals worldwide, most importantly humans and their livestock. A total of 124 miRNAs were profiled, 72 of which had been previously reported and three of which were conserved miRNA sequences described here for the first time. The remaining 49 miRNAs were novel sequences of which, 31 were conserved with F. gigantica and the remaining 18 were specific to F. hepatica. The newly excysted juveniles express 22 unique miRNAs while the immature liver and mature bile duct stages each express 16 unique miRNAs. We discovered several sequence variant miRNAs (IsomiRs) as well as miRNA clusters that exhibit strict temporal expression paralleling parasite development. Target analysis revealed the close association between miRNA expression and stage-specific changes in the transcriptome; for example, we identified specific miRNAs that target parasite proteases known to be essential for intestinal wall penetration (cathepsin L3). Moreover, we demonstrate that miRNAs fine-tune the expression of genes involved in the metabolic pathways that allow the parasites to move from an aerobic external environment to the anerobic environment of the host. CONCLUSIONS: These results provide novel insight into the regulation of helminth parasite development and identifies new genes and miRNAs for therapeutic development to limit the virulence and pathogenesis caused by F. hepatica.

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.

Tuaimenal A, a Meroterpene from the Irish Deep-Sea Soft Coral Duva florida, Displays Inhibition of the SARS-CoV-2 3CLpro Enzyme.

Cold water benthic environments are a prolific source of structurally diverse molecules with a range of bioactivities against human disease. Specimens of a previously chemically unexplored soft coral, Duva florida, were collected during a deep-sea cruise that sampled marine invertebrates along the Irish continental margin in 2018. Tuaimenal A (1), a cyclized merosesquiterpenoid representing a new carbon scaffold with a highly substituted chromene core, was discovered through exploration of the soft coral secondary metabolome via NMR-guided fractionation. The absolute configuration was determined through vibrational circular dichroism. Functional biochemical assays and in silico docking experiments found tuaimenal A selectively inhibits the viral main protease (3CLpro) of SARS-CoV-2.

Targeting Secreted Protease/Anti-Protease Balance as a Vaccine Strategy against the Helminth Fasciola hepatica.

The liver fluke Fasciola hepatica is an economically important global pathogen of humans and their livestock. To facilitate host invasion and migration, F. hepatica secretes an abundance of cathepsin peptidases but prevents excessive damage to both parasite and host tissues by co-secreting regulatory peptidase inhibitors, cystatins/stefins and Kunitz-type inhibitors. Here, we report a vaccine strategy aimed at disrupting the parasite's protease/anti-protease balance by targeting these key inhibitors. Our vaccine cocktail containing three recombinant stefins (rFhStf-1, rFhStf-2, rFhStf-3) and a Kunitz-type inhibitor (rFhKT1) formulated in adjuvant Montanide 61VG was assessed in two independent sheep trials. While fluke burden was not reduced in either trial, in Trial 1 the vaccinated animals showed significantly greater weight gain (p < 0.05) relative to the non-vaccinated control group. In both trials we observed a significant reduction in egg viability (36-42%). Multivariate regression analyses showed vaccination and increased levels of IgG2 antibodies specific for the F. hepatica peptidase inhibitors were positive indicators for increased weight gain and levels of haemoglobin within the normal range at 16 weeks post-infection (wpi; p < 0.05). These studies point to the potential of targeting peptidase inhibitors as vaccine cocktails for fasciolosis control in sheep.

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.

The Impact of Lung Proteases on Snake-Derived Antimicrobial Peptides.

Respiratory infections are a leading cause of global morbidity and mortality and are of significant concern for individuals with chronic inflammatory lung diseases. There is an urgent need for novel antimicrobials. Antimicrobial peptides (AMPs) are naturally occurring innate immune response peptides with therapeutic potential. However, therapeutic development has been hindered by issues with stability and cytotoxicity. Availing of direct drug delivery to the affected site, for example the lung, can reduce unwanted systemic side effects and lower the required dose. As cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) lungs typically exhibit elevated protease levels, the aim of this study was to assess their impact on snake-derived AMPs. Peptide cleavage was determined using SDS-PAGE and antimicrobial and anti-inflammatory activities of neutrophil elastase (NE)-incubated peptides were assessed using a radial diffusion assay (RDA) and an in vitro LPS-induced inflammation model, respectively. Although the snake-derived AMPs were found to be susceptible to cleavage by lung proteases including NE, several retained their function following NE-incubation. This facilitated the design of novel truncated derivatives that retained functionality following NE incubation. Snake-derived AMPs are tractable candidate treatments for use in environments that feature elevated NE levels, such as the CF airways.

Recognition Pattern of the Fasciola hepatica Excretome/Secretome during the Course of an Experimental Infection in Sheep by 2D Immunoproteomics.

Excretory/secretory products released by helminth parasites have been widely studied for their diagnostic utility, immunomodulatory properties, as well as for their use as vaccines. Due to their location at the host/parasite interface, the characterization of parasite secretions is important to unravel the molecular interactions governing the relationships between helminth parasites and their hosts. In this study, the excretory/secretory products from adult worms of the trematode Fasciola hepatica (FhES) were employed in a combination of two-dimensional electrophoresis, immunoblot and mass spectrometry, to analyze the immune response elicited in sheep during the course of an experimental infection. Ten different immunogenic proteins from FhES recognized by serum samples from infected sheep at 4, 8, and/or 12 weeks post-infection were identified. Among these, different isoforms of cathepsin L and B, peroxiredoxin, calmodulin, or glutathione S-transferase were recognized from the beginning to the end of the experimental infection, suggesting their potential role as immunomodulatory antigens. Furthermore, four FhES proteins (C2H2-type domain-containing protein, ferritin, superoxide dismutase, and globin-3) were identified for the first time as non-immunogenic proteins. These results may help to further understand host/parasite relationships in fasciolosis, and to identify potential diagnostic molecules and drug target candidates of F. hepatica.

Antigen-specific response of CD4+ T cells and hepatic lymph node cells to Fasciola hepatica-derived molecules at the early and late stage of the infection in sheep.

The immunomodulatory capacity of F. hepatica antigens is probably one of the main reasons for the development of a driven non-protective Th2 immune response. In this study, we analysed the cellular response of hepatic lymph node cells and CD4+ T cells in terms of proliferative response, efficiency of antigen presentation and cytokine production, to F. hepatica-derived molecules, at early and late stages of the infection. Thirty-one sheep were allocated into five groups and were slaughtered at 16 dpi and 23 wpi. In order to analyse antigen-specific response, the following F. hepatica recombinant molecules were used: rFhCL1, rFhCL2, rFhCL3, rFhCB1, rFhCB2, rFhCB3, rFhStf-1, rFhStf-2, rFhStf-3 and rFhKT1. A cell proliferation assay using hepatic lymph node cells and an antigen presentation cell assay using CD4+ T cells were performed. At 16 dpi, all molecules but rFhStf-2 and rFhKT1 elicited a significant cell proliferative response on hepatic lymph node cells of infected animals. At both early and late stage of the infection, antigen presentation of rFhCB3 and rFhCL2 resulted in higher stimulation index of CD4+ T cells which was IL-2 mediated, although no statistically significant when compared to uninfected animals. Significant cytokine production (IL-4, IL-10 and IFN-γ) was conditioned by the antigen-specific cell stimulation. No CD4+ T cell exhaustion was detected in infected sheep at the chronic stage of the infection. This study addressed antigen-specific response to F. hepatica-derived molecules that are involved in key aspects of the parasite survival within the host.

Improved diagnosis of SARS-CoV-2 by using nucleoprotein and spike protein fragment 2 in quantitative dual ELISA tests.

The novel coronavirus, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), is the causative agent of the 2020 worldwide coronavirus pandemic. Antibody testing is useful for diagnosing historic infections of a disease in a population. These tests are also a helpful epidemiological tool for predicting how the virus spreads in a community, relating antibody levels to immunity and for assessing herd immunity. In the present study, SARS-CoV-2 viral proteins were recombinantly produced and used to analyse serum from individuals previously exposed, or not, to SARS-CoV-2. The nucleocapsid (Npro) and spike subunit 2 (S2Frag) proteins were identified as highly immunogenic, although responses to the former were generally greater. These two proteins were used to develop two quantitative enzyme-linked immunosorbent assays (ELISAs) that when used in combination resulted in a highly reliable diagnostic test. Npro and S2Frag-ELISAs could detect at least 10% more true positive coronavirus disease-2019 (COVID-19) cases than the commercially available ARCHITECT test (Abbott). Moreover, our quantitative ELISAs also show that specific antibodies to SARS-CoV-2 proteins tend to wane rapidly even in patients who had developed severe disease. As antibody tests complement COVID-19 diagnosis and determine population-level surveillance during this pandemic, the alternative diagnostic we present in this study could play a role in controlling the spread of the virus.

Autonomous Non Antioxidant Roles for Fasciola hepatica Secreted Thioredoxin-1 and Peroxiredoxin-1.

Trematode parasites of the genus Fasciola are the cause of liver fluke disease (fasciolosis) in humans and their livestock. Infection of the host involves invasion through the intestinal wall followed by migration in the liver that results in extensive damage, before the parasite settles as a mature egg-laying adult in the bile ducts. Genomic and transcriptomic studies revealed that increased metabolic stress during the rapid growth and development of F. hepatica is balanced with the up-regulation of the thiol-independent antioxidant system. In this cascade system thioredoxin/glutathione reductase (TGR) reduces thioredoxin (Trx), which then reduces and activates peroxiredoxin (Prx), whose major function is to protect cells against the damaging hydrogen peroxide free radicals. F. hepatica expresses a single TGR, three Trx and three Prx genes; however, the transcriptional expression of Trx1 and Prx1 far out-weighs (>50-fold) other members of their family, and both are major components of the parasite secretome. While Prx1 possesses a leader signal peptide that directs its secretion through the classical pathway and explains why this enzyme is found freely soluble in the secretome, Trx1 lacks a leader peptide and is secreted via an alternative pathway that packages the majority of this enzyme into extracellular vesicles (EVs). Here we propose that F. hepatica Prx1 and Trx1 do not function as part of the parasite's stress-inducible thiol-dependant cascade, but play autonomous roles in defence against the general anti-pathogen oxidative burst by innate immune cells, in the modulation of host immune responses and regulation of inflammation.

Eudiplozoon nipponicum (Monogenea, Diplozoidae) and its adaptation to haematophagy as revealed by transcriptome and secretome profiling.

BACKGROUND: Ectoparasites from the family Diplozoidae (Platyhelminthes, Monogenea) belong to obligate haematophagous helminths of cyprinid fish. Current knowledge of these worms is for the most part limited to their morphological, phylogenetic, and population features. Information concerning the biochemical and molecular nature of physiological processes involved in host-parasite interaction, such as evasion of the immune system and its regulation, digestion of macromolecules, suppression of blood coagulation and inflammation, and effect on host tissue and physiology, is lacking. In this study, we report for the first time a comprehensive transcriptomic/secretome description of expressed genes and proteins secreted by the adult stage of Eudiplozoon nipponicum (Goto, 1891) Khotenovsky, 1985, an obligate sanguivorous monogenean which parasitises the gills of the common carp (Cyprinus carpio). RESULTS: RNA-seq raw reads (324,941 Roche 454 and 149,697,864 Illumina) were generated, de novo assembled, and filtered into 37,062 protein-coding transcripts. For 19,644 (53.0%) of them, we determined their sequential homologues. In silico functional analysis of E. nipponicum RNA-seq data revealed numerous transcripts, pathways, and GO terms responsible for immunomodulation (inhibitors of proteolytic enzymes, CD59-like proteins, fatty acid binding proteins), feeding (proteolytic enzymes cathepsins B, D, L1, and L3), and development (fructose 1,6-bisphosphatase, ferritin, and annexin). LC-MS/MS spectrometry analysis identified 721 proteins secreted by E. nipponicum with predominantly immunomodulatory and anti-inflammatory functions (peptidyl-prolyl cis-trans isomerase, homolog to SmKK7, tetraspanin) and ability to digest host macromolecules (cathepsins B, D, L1). CONCLUSIONS: In this study, we integrated two high-throughput sequencing techniques, mass spectrometry analysis, and comprehensive bioinformatics approach in order to arrive at the first comprehensive description of monogenean transcriptome and secretome. Exploration of E. nipponicum transcriptome-related nucleotide sequences and translated and secreted proteins offer a better understanding of molecular biology and biochemistry of these, often neglected, organisms. It enabled us to report the essential physiological pathways and protein molecules involved in their interactions with the fish hosts.

Fasciola hepatica hijacks host macrophage miRNA machinery to modulate early innate immune responses.

Fasciola hepatica, a global worm parasite of humans and their livestock, regulates host innate immune responses within hours of infection. Host macrophages, essential to the first-line defence mechanisms, are quickly restricted in their ability to initiate a classic protective pro-inflammatory immune response. We found that macrophages from infected animals are enriched with parasite-derived micro(mi)RNAs. The most abundant of these miRNAs, fhe-miR-125b, is released by the parasite via exosomes and is homologous to a mammalian miRNA, hsa-miR-125b, that is known to regulate the activation of pro-inflammatory M1 macrophages. We show that the parasite fhe-miR-125b loads onto the mammalian Argonaut protein (Ago-2) within macrophages during infection and, therefore, propose that it mimics host miR-125b to negatively regulate the production of inflammatory cytokines. The hijacking of the miRNA machinery controlling innate cell function could be a fundamental mechanism by which worm parasites disarm the early immune responses of their host to ensure successful infection.

Biochemical and cellular characterisation of the Plasmodium falciparum M1 alanyl aminopeptidase (PfM1AAP) and M17 leucyl aminopeptidase (PfM17LAP).

The Plasmodium falciparum M1 alanyl aminopeptidase and M17 leucyl aminopeptidase, PfM1AAP and PfM17LAP, are potential targets for novel anti-malarial drug development. Inhibitors of these aminopeptidases have been shown to kill malaria parasites in culture and reduce parasite growth in murine models. The two enzymes may function in the terminal stages of haemoglobin digestion, providing free amino acids for protein synthesis by the rapidly growing intra-erythrocytic parasites. Here we have performed a comparative cellular and biochemical characterisation of the two enzymes. Cell fractionation and immunolocalisation studies reveal that both enzymes are associated with the soluble cytosolic fraction of the parasite, with no evidence that they are present within other compartments, such as the digestive vacuole (DV). Enzyme kinetic studies show that the optimal pH of both enzymes is in the neutral range (pH 7.0-8.0), although PfM1AAP also possesses some activity (< 20%) at the lower pH range of 5.0-5.5. The data supports the proposal that PfM1AAP and PfM17LAP function in the cytoplasm of the parasite, likely in the degradation of haemoglobin-derived peptides generated in the DV and transported to the cytosol.

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

BACKGROUND: The major pathogenesis associated with Fasciola hepatica infection results from the extensive tissue damage caused by the tunnelling and feeding activity of immature flukes during their migration, growth and development in the liver. This is compounded by the pathology caused by host innate and adaptive immune responses that struggle to simultaneously counter infection and repair tissue damage. RESULTS: Complementary transcriptomic and proteomic approaches defined the F. hepatica factors associated with their migration in the liver, and the resulting immune-pathogenesis. Immature liver-stage flukes express ~ 8000 transcripts that are enriched for transcription and translation processes reflective of intensive protein production and signal transduction pathways. Key pathways that regulate neoblast/pluripotent cells, including the PI3K-Akt signalling pathway, are particularly dominant and emphasise the importance of neoblast-like cells for the parasite's rapid development. The liver-stage parasites display different secretome profiles, reflecting their distinct niche within the host, and supports the view that cathepsin peptidases, cathepsin peptidase inhibitors, saposins and leucine aminopeptidases play a central role in the parasite's destructive migration, and digestion of host tissue and blood. Immature flukes are also primed for countering immune attack by secreting immunomodulating fatty acid binding proteins (FABP) and helminth defence molecules (FhHDM). Combined with published host microarray data, our results suggest that considerable immune cell infiltration and subsequent fibrosis of the liver tissue exacerbates oxidative stress within parenchyma that compels the expression of a range of antioxidant molecules within both host and parasite. CONCLUSIONS: The migration of immature F. hepatica parasites within the liver is associated with an increase in protein production, expression of signalling pathways and neoblast proliferation that drive their rapid growth and development. The secretion of a defined set of molecules, particularly cathepsin L peptidases, peptidase-inhibitors, saponins, immune-regulators and antioxidants allow the parasite to negotiate the liver micro-environment, immune attack and increasing levels of oxidative stress. This data contributes to the growing F. hepatica -omics information that can be exploited to understand parasite development more fully and for the design of novel control strategies to prevent host liver tissue destruction and pathology.

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

BACKGROUND: The zoonotic worm parasite Fasciola hepatica secretes 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. RESULTS: Immunolocalisation/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 recombinant F. hepatica FhCL3 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 < 0.002 nM, respectively. These values are at least 1000-fold lower than those Ki obtained 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 (Leu66 and Glu68) that allow the propeptide to block the active site. CONCLUSIONS: The FhCL3 peptidase involved in host invasion by F. hepatica is 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.