Wong, S.L., To, J., Santos, J., Allam, V.S.R.R., Dalton, J.P., Djordjevic, S.P., Donnelly, S., Padula, M.P. & Sukkar, M.B. 2018, 'Proteomic Analysis of Extracellular HMGB1 Identifies Binding Partners and Exposes Its Potential Role in Airway Epithelial Cell Homeostasis.', Journal of proteome research, vol. 17, no. 1, pp. 33-45.View/Download from: UTS OPUS or Publisher's site
The release of damage-associated molecular patterns (DAMPs) by airway epithelial cells is believed to play a crucial role in the initiation and development of chronic airway conditions such as asthma and chronic obstructive pulmonary disease (COPD). Intriguingly, the classic DAMP high-mobility group box-1 (HMGB1) is detected in the culture supernatant of airway epithelial cells under basal conditions, indicating a role for HMGB1 in the regulation of epithelial cellular and immune homeostasis. To gain contextual insight into the potential role of HMGB1 in airway epithelial cell homeostasis, we used the orthogonal and complementary methods of high-resolution clear native electrophoresis, immunoprecipitation, and pull-downs coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) to profile HMGB1 and its binding partners in the culture supernatant of unstimulated airway epithelial cells. We found that HMGB1 presents exclusively as a protein complex under basal conditions. Moreover, protein network analysis performed on 185 binding proteins revealed 14 that directly associate with HMGB1: amyloid precursor protein, F-actin-capping protein subunit alpha-1 (CAPZA1), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), ubiquitin, several members of the heat shock protein family (HSPA8, HSP90B1, HSP90AA1), XRCC5 and XRCC6, high mobility group A1 (HMGA1), histone 3 (H3F3B), the FACT (facilitates chromatin transcription) complex constituents SUPT1H and SSRP1, and heterogeneous ribonucleoprotein K (HNRNPK). These studies provide a new understanding of the extracellular functions of HMGB1 in cellular and immune homeostasis at the airway mucosal surface and could have implications for therapeutic targeting.
Tanaka, A., To, J., O'Brien, B., Donnelly, S. & Lund, M. 2017, 'Selection of reliable reference genes for the normalisation of gene expression levels following time course LPS stimulation of murine bone marrow derived macrophages.', BMC immunology, vol. 18, no. 1, p. 43.View/Download from: UTS OPUS or Publisher's site
Macrophages are key players in the initiation, perpetuation and regulation of both innate and adaptive immune responses. They largely perform these roles through modulation of the expression of genes, especially those encoding cytokines. Murine bone marrow derived macrophages (BMDMs) are commonly used as a model macrophage population for the study of immune responses to pro-inflammatory stimuli, notably lipopolysaccharide (LPS), which may be pertinent to the human situation. Evaluation of the temporal responses of LPS stimulated macrophages is widely conducted via the measurement of gene expression levels by RT-qPCR. While providing a robust and sensitive measure of gene expression levels, RT-qPCR relies on the normalisation of gene expression data to a stably expressed reference gene. Generally, a normalisation gene(s) is selected from a list of "traditional" reference genes without validation of expression stability under the specific experimental conditions of the study. In the absence of such validation, and given that many studies use only a single reference gene, the reliability of data is questionable.The stability of expression levels of eight commonly used reference genes was assessed during the peak (6 h) and resolution (24 h) phases of the BMDM response to LPS. Further, this study identified two additional genes, which have not previously been described as reference genes, and the stability of their expression levels during the same phases of the inflammatory response were validated. Importantly, this study demonstrates that certain "traditional" reference genes are in fact regulated by LPS exposure, and, therefore, are not reliable candidates as their inclusion may compromise the accuracy of data interpretation. Testament to this, this study shows that the normalisation of gene expression data using an unstable reference gene greatly affects the experimental data obtained, and, therefore, the ultimate biological conclusions drawn.This study reaffirms ...
Alvarado, R., To, J., Lund, M.E., Pinar, A., Mansell, A., Robinson, M.W., O'Brien, B.A., Dalton, J.P. & Donnelly, S. 2017, 'The immune modulatory peptide FhHDM-1 secreted by the helminth Fasciola hepatica prevents NLRP3 inflammasome activation by inhibiting endolysosomal acidification in macrophages.', FASEB journal : official publication of the Federation of American Societies for Experimental Biology, vol. 31, no. 1, pp. 85-95.View/Download from: UTS OPUS or Publisher's site
The NLRP3 inflammasome is a multimeric protein complex that controls the production of IL-1, a cytokine that influences the development of both innate and adaptive immune responses. Helminth parasites secrete molecules that interact with innate immune cells, modulating their activity to ultimately determine the phenotype of differentiated T cells, thus creating an immune environment that is conducive to sustaining chronic infection. We show that one of these molecules, FhHDM-1, a cathelicidin-like peptide secreted by the helminth parasite, Fasciola hepatica, inhibits the activation of the NLRP3 inflammasome resulting in reduced secretion of IL-1 by macrophages. FhHDM-1 had no effect on the synthesis of pro-IL-1. Rather, the inhibitory effect was associated with the capacity of the peptide to prevent acidification of the endolysosome. The activation of cathepsin B protease by lysosomal destabilization was prevented in FhHDM-1-treated macrophages. By contrast, peptide derivatives of FhHDM-1 that did not alter the lysosomal pH did not inhibit secretion of IL-1. We propose a novel immune modulatory strategy used by F. hepatica, whereby secretion of the FhHDM-1 peptide impairs the activation of NLRP3 by lysosomal cathepsin B protease, which prevents the downstream production of IL-1 and the development of protective T helper 1 type immune responses that are detrimental to parasite survival.-Alvarado, R., To, J., Lund, M. E., Pinar, A., Mansell, A., Robinson, M. W., O'Brien, B. A., Dalton, J. P., Donnelly, S. The immune modulatory peptide FhHDM-1 secreted by the helminth Fasciola hepatica prevents NLRP3 inflammasome activation by inhibiting endolysosomal acidification in macrophages.
Lund, M.E., To, J., O'Brien, B.A. & Donnelly, S. 2016, 'The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus', JOURNAL OF IMMUNOLOGICAL METHODS, vol. 430, pp. 64-70.View/Download from: UTS OPUS or Publisher's site
Lund, M.E., Greer, J., Dixit, A., Alvarado, R., McCauley-Winter, P., To, J., Tanaka, A., Hutchinson, A.T., Robinson, M.W., Simpson, A.M., O'Brien, B.A., Dalton, J.P. & Donnelly, S. 2016, 'A parasite-derived 68-mer peptide ameliorates autoimmune disease in murine models of Type 1 diabetes and multiple sclerosis.', Scientific Reports, vol. 6, pp. 1-11.View/Download from: UTS OPUS or Publisher's site
Helminth parasites secrete molecules that potently modulate the immune responses of their hosts and, therefore, have potential for the treatment of immune-mediated human diseases. FhHDM-1, a 68-mer peptide secreted by the helminth parasite Fasciola hepatica, ameliorated disease in two different murine models of autoimmunity, type 1 diabetes and relapsing-remitting immune-mediated demyelination. Unexpectedly, FhHDM-1 treatment did not affect the proliferation of auto-antigen specific T cells or their production of cytokines. However, in both conditions, the reduction in clinical symptoms was associated with the absence of immune cell infiltrates in the target organ (islets and the brain tissue). Furthermore, after parenteral administration, the FhHDM-1 peptide interacted with macrophages and reduced their capacity to secrete pro-inflammatory cytokines, such as TNF and IL-6. We propose this inhibition of innate pro-inflammatory immune responses, which are central to the initiation of autoimmunity in both diseases, prevented the trafficking of autoreactive lymphocytes from the periphery to the site of autoimmunity (as opposed to directly modulating their function per se), and thus prevented tissue destruction. The ability of FhHDM-1 to modulate macrophage function, combined with its efficacy in disease prevention in multiple models, suggests that FhHDM-1 has considerable potential as a treatment for autoimmune diseases.
Spicer, T., Fernandez-Vega, V., Chase, P., Scampavia, L., To, J., Dalton, J.P., Da Silva, F.L., Skinner-Adams, T.S., Gardiner, D.L., Trenholme, K.R., Brown, C.L., Ghosh, P., Porubsky, P., Wang, J.L., Whipple, D.A., Schoenen, F.J. & Hodder, P. 2014, 'Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High-Throughput Screening.', Journal of biomolecular screening, vol. 19, no. 7, pp. 1107-1115.View/Download from: UTS OPUS or Publisher's site
The target of this study, the PfM18 aspartyl aminopeptidase (PfM18AAP), is the only AAP present in the genome of the malaria parasite Plasmodium falciparum. PfM18AAP is a metallo-exopeptidase that exclusively cleaves N-terminal acidic amino acids glutamate and aspartate. It is expressed in parasite cytoplasm and may function in concert with other aminopeptidases in protein degradation, of, for example, hemoglobin. Previous antisense knockdown experiments identified a lethal phenotype associated with PfM18AAP, suggesting that it is a valid target for new antimalaria therapies. To identify inhibitors of PfM18AAP function, a fluorescence enzymatic assay was developed using recombinant PfM18AAP enzyme and a fluorogenic peptide substrate (H-Glu-NHMec). This was screened against the Molecular Libraries Probe Production Centers Network collection of ~292,000 compounds (the Molecular Libraries Small Molecule Repository). A cathepsin L1 (CTSL1) enzyme-based assay was developed and used as a counter screen to identify compounds with nonspecific activity. Enzymology and phenotypic assays were used to determine mechanism of action and efficacy of selective and potent compounds identified from high-throughput screening. Two structurally related compounds, CID 6852389 and CID 23724194, yielded micromolar potency and were inactive in CTSL1 titration experiments (IC50>59.6 µM). As measured by the K(i) assay, both compounds demonstrated micromolar noncompetitive inhibition in the PfM18AAP enzyme assay. Both CID 6852389 and CID 23724194 demonstrated potency in malaria growth assays (IC504 µM and 1.3 µM, respectively).
Thivierge, K., Cotton, S., Schaefer, D., Riggs, M., To, J.H., Lund, M.E., Robinson, M.W., Dalton, J.P. & Donnelly, S.M. 2013, 'Cathelicidin-like Helminth Defence Molecules (HDMs): Absence of Cytotoxic, Anti-microbial and Anti-protozoan Activities Imply a Specific Adaptation to Immune Modulation', Plos Neglected Tropical Diseases, vol. 7, no. 7, pp. 1-14.View/Download from: UTS OPUS or Publisher's site
Host defence peptides (HDPs) are expressed throughout the animal and plant kingdoms. They have multifunctional roles in the defence against infectious agents of mammals, possessing both bactericidal and immune-modulatory activities. We have identified a
Hutchinson, A.T., Malik, A., Berkahn, M.B., Agostino, M., To, J., Tacchi, J.L., Djordjevic, S.P., Turnbull, L., Whitchurch, C.B., Edmundson, A.B., Jones, P.M., Raison, R.L. & Ramsland, P.A. 2013, 'Formation of Assemblies on Cell Membranes by Secreted Proteins: Molecular Studies of Free Lambda Light Chain Aggregates Found on the Surface of Myeloma Cells.', Biochemical Journal, vol. 454, no. 3, pp. 479-489.View/Download from: UTS OPUS or Publisher's site
We have described the presence of cell membrane-associated ? free immunoglobulin light chains (FLC) on the surface of myeloma cells. Notably, the anti-?FLC mAb, MDX-1097, is being assessed in clinical trials as a therapy for ? light chain isotype multiple myeloma. Despite the clinical potential of anti-FLC mAbs, there have been limited studies on characterizing membrane-associated FLCs at a molecular level. Furthermore, it is not known if ?FLCs can associate with cell membranes of myeloma cells. In this study, we describe the presence of ?FLCs on the surface of myeloma cells. We found that cell surface-associated ?FLC are bound directly to the membrane and in an aggregated form. Subsequently, membrane interaction studies revealed that ?FLCs interact with saturated zwitterionic lipids such as phosphatidylcholine and phosphatidylethanolamine, and using automated docking, we characterize a potential recognition site for these lipids. Atomic force microscopy confirmed that membrane-associated ?FLCs are aggregated. Given our findings, we propose a model whereby individual FLCs show modest affinity for zwitterionic lipids, with aggregation stabilizing the interaction due to multivalency. Notably, this is the first study to image FLCs bound to phospholipids and provides important insights into the possible mechanisms of membrane association by this unique myeloma surface antigen.
Robinson, M.W., Buchtmann, K.A., Jenkins, C., Tacchi, J.L., Raymond, B.B.A., To, J., Chowdhury, P.R., Woolley, L.K., Labbate, M., Turnbull, L., Whitchurch, C.B., Padula, M.P. & Djordjevic, S.P. 2013, 'MHJ_0125 is an M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of Mycoplasma hyopneumoniae', OPEN BIOLOGY, vol. 3.View/Download from: UTS OPUS or Publisher's site
Poreba, M., McGowan, S., Skinner-Adams, T.S., Trenholme, K.R., Gardiner, D.L., Whisstock, J.C., To, J., Salvesen, G.S., Dalton, J.P. & Drag, M. 2012, 'Fingerprinting the substrate specificity of M1 and M17 aminopeptidases of human malaria, Plasmodium falciparum.', PloS one, vol. 7, no. 2, p. e31938.View/Download from: UTS OPUS or Publisher's site
Plasmodium falciparum, the causative agent of human malaria, expresses two aminopeptidases, PfM1AAP and PfM17LAP, critical to generating a free amino acid pool used by the intraerythrocytic stage of the parasite for proteins synthesis, growth and development. These exopeptidases are potential targets for the development of a new class of anti-malaria drugs.To define the substrate specificity of recombinant forms of these two malaria aminopeptidases we used a new library consisting of 61 fluorogenic substrates derived both from natural and unnatural amino acids. We obtained a detailed substrate fingerprint for recombinant forms of the enzymes revealing that PfM1AAP exhibits a very broad substrate tolerance, capable of efficiently hydrolyzing neutral and basic amino acids, while PfM17LAP has narrower substrate specificity and preferentially cleaves bulky, hydrophobic amino acids. The substrate library was also exploited to profile the activity of the native aminopeptidases in soluble cell lysates of P. falciparum malaria.This data showed that PfM1AAP and PfM17LAP are responsible for majority of the aminopeptidase activity in these extracts. These studies provide specific substrate and mechanistic information important for understanding the function of these aminopeptidases and could be exploited in the design of new inhibitors to specifically target these for anti-malaria treatment.
Robinson, M.W., Alvarado, R., To, J., Hutchinson, A.T., Dowdell, S.N., Lund, M.E., Turnbull, L., Whitchurch, C.B., O'Brien, B., Dalton, J.P. & Donnelly, S.M. 2012, 'A helminth cathelicidin-like protein suppresses antigen processing and presentation in macrophages via inhibition of lysosomal vATPase', Faseb Journal, vol. 26, no. 11, pp. 4614-4627.View/Download from: UTS OPUS or Publisher's site
We previously reported the identification of a novel family of immunomodulatory proteins, termed helminth defense molecules (HDMs), that are secreted by medically important trematode parasites. Since HDMs share biochemical, structural, and functional characteristics with mammalian cathelicidin-like host defense peptides (HDPs), we proposed that HDMs modulate the immune response via molecular mimicry of host molecules. In the present study, we report the mechanism by which HDMs influence the function of macrophages. We show that the HDM secreted by Fasciola hepatica (FhHDM-1) binds to macrophage plasma membrane lipid rafts via selective interaction with phospholipids and/or cholesterol before being internalized by endocytosis. Following internalization, FhHDM-1 is rapidly processed by lysosomal cathepsin L to release a short C-terminal peptide (containing a conserved amphipathic helix that is a key to HDM function), which then prevents the acidification of the endolysosomal compartments by inhibiting vacuolar ATPase activity. The resulting endolysosomal alkalization impedes macrophage antigen processing and prevents the transport of peptides to the cell surface in conjunction with MHC class II for presentation to CD4(+) T cells. Thus, we have elucidated a novel mechanism by which helminth pathogens alter innate immune cell function to assist their survival in the host.-Robinson, M. W., Alvarado, R., To, J., Hutchinson, A. T., Dowdell, S. N., Lund, M., Turnbull, L., Whitchurch, C. B., O'Brien, B. A., Dalton, J. P., Donnelly, S. A helminth cathelicidin-like protein suppresses antigen processing and presentation in macrophages via inhibition of lysosomal vATPase
Robinson, M.W., Corvo, I., Jones, P.M., George, A.M., Padula, M., To, J., Cancela, M., Rinaldi, G., Tort, J.F., Roche, L. & Dalton, J.P. 2011, 'Collagenolytic Activities of the Major Secreted Cathepsin L Peptidases Involved in the Virulence of the Helminth Pathogen, Fasciola hepatica', Plos Neglected Tropical Diseases, vol. 5, no. 4, pp. 1-13.View/Download from: UTS OPUS or Publisher's site
Background: The temporal expression and secretion of distinct members of a family of virulence-associated cathepsin L cysteine peptidases (FhCL) correlates with the entry and migration of the helminth pathogen Fasciola hepatica in the host. Thus, infective larvae traversing the gut wall secrete cathepsin L3 (FhCL3), liver migrating juvenile parasites secrete both FhCL1 and FhCL2 while the mature bile duct parasites, which are obligate blood feeders, secrete predominantly FhCL1 but also FhCL2. Methodology/Principal Findings: Here we show that FhCL1, FhCL2 and FhCL3 exhibit differences in their kinetic parameters towards a range of peptide substrates. Uniquely, FhCL2 and FhCL3 readily cleave substrates with Pro in the P2 position and peptide substrates mimicking the repeating Gly-Pro-Xaa motifs that occur within the primary sequence of collagen. FhCL1, FhCL2 and FhCL3 hydrolysed native type I and II collagen at neutral pH but while FhCL1 cleaved only non-collagenous (NC, non-Gly-X-Y) domains FhCL2 and FhCL3 exhibited collagenase activity by cleaving at multiple sites within the alpha 1 and alpha 2 triple helix regions (Col domains). Molecular simulations created for FhCL1, FhCL2 and FhCL3 complexed to various seven-residue peptides supports the idea that Trp67 and Tyr67 in the S2 subsite of the active sites of FhCL3 and FhCL2, respectively, are critical to conferring the unique collagenase-like activity to these enzymes by accommodating either Gly or Pro residues at P2 in the substrate. The data also suggests that FhCL3 accommodates hydroxyproline (Hyp)-Gly at P3-P2 better than FhCL2 explaining the observed greater ability of FhCL3 to digest type I and II collagens compared to FhCL2 and why these enzymes cleave at different positions within the Col domains. Conclusions/Significance: These studies further our understanding of how this helminth parasite regulates peptidase expression to ensure infection, migration and establishment in host tissues.
Robinson, M.W., Donnelly, S.M., Hutchinson, A.T., To, J., Taylor, N.L., Norton, R.S., Perugini, M. & Dalton, J.P. 2011, 'A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides', Plos Pathogens, vol. 7, no. 5, pp. 1-15.View/Download from: UTS OPUS or Publisher's site
Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly alpha-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation.
Sripa, J., Laha, T., To, J., Brindley, P.J., Sripa, B., Kaewkes, S., Dalton, J.P. & Robinson, M.W. 2010, 'Secreted cysteine proteases of the carcinogenic liver fluke, Opisthorchis viverrini: regulation of cathepsin F activation by autocatalysis and trans-processing by cathepsin B.', Cellular Microbiology, vol. 12, no. 6, pp. 781-795.View/Download from: UTS OPUS or Publisher's site
Opisthorchis viverrini is an important helminth pathogen of humans that is endemic in Thailand and Laos. Adult flukes reside within host bile ducts and feed on epithelial tissue and blood cells. Chronic opisthorchiasis is associated with severe hepatobiliary diseases such as cholangiocarcinoma. Here we report that adult O. viverrini secrete two major cysteine proteases: cathepsin F (Ov-CF-1) and cathepsin B1 (Ov-CB-1). Ov-CF-1 is secreted as an inactive zymogen that autocatalytically processes and activates to a mature enzyme at pH 4.5 via an intermolecular cleavage at the prosegment-mature domain junction. Ov-CB-1 is also secreted as a zymogen but, in contrast to Ov-CF-1, is fully active against peptide and macromolecular substrates despite retaining the N-terminal prosegment. The active Ov-CB-1 zymogen was capable of trans-activating Ov-CF-1 by proteolytic removal of its prosegment at pH 5.5, a pH at which the Ov-CF-1 zymogen cannot autocatalytically activate. Both cathepsins hydrolyse human haemoglobin but their combined action more efficiently degrades haemoglobin to smaller peptides than each enzyme alone. Ov-CF-1 degraded extracellular matrix proteins more effectively than Ov-CB-1 at physiological pH. We propose that Ov-CB-1 regulates Ov-CF-1 activity and that both enzymes work together to degrade host tissue contributing to the development of liver fluke-associated cholangiocarcinoma.
Trenholme, K., Brown, C.L., Skinner-Adams, T.S., Stack, C.M., Lowther, J., To, J., Robinson, M.W., Donnelly, S.M., Dalton, J.P. & Gardiner, D. 2010, 'Aminopeptidases of malaria parasites: New targets for chemotherapy.', Infectious Disorders Drug Targets, vol. 10, no. 3, pp. 217-225.View/Download from: UTS OPUS or Publisher's site
Novel targets for new drug development are urgently required to combat malaria, a disease that puts half of the world's population at risk. One group of enzymes identified within the genome of the most lethal of the causative agents of malaria, Plasmodium falciparum, that may have the potential to become new targets for antimalarial drug development are the aminopeptidases. These enzymes catalyse the cleavage of the N-terminal amino acids from proteins and peptides. P. falciparum appears to encode for at least nine aminopeptidases, two neutral aminopeptidases, one aspartyl aminopeptidase, one aminopeptidase P, one prolyl aminopeptidase and four methionine aminopeptidases. Recent advances in our understanding of these genes and their protein products are outlined in this review, including their potential for antimalarial drug development
Villavedra Sierra, M., To, J., Lemke, S.J., Birch, D., Crosbie, P., Adams, M.A., Broady, K.W., Nowak, B., Raison, R.L. & Wallach, M. 2010, 'Characterisation Of An Immunodominant, High Molecular Weight Glycoprotein On The Surface Of Infectious Neoparamoeba Spp., Causative Agent Of Amoebic Gill Disease (Agd) In Atlantic Salmon', Fish and Shellfish Immunology, vol. 29, no. 6, pp. 946-955.View/Download from: UTS OPUS or Publisher's site
Amoebic gill disease can be experimentally induced by the exposure of salmonids to Neoparamoeba spp. freshly isolated from infected fish, while cultured amoebae are non-infective.
Pinlaor, P., Kaewpitoon, N., Laha, T., Sripa, B., Kaewkes, S., Morales, M.E., Mann, V.H., Parriott, S.K., Suttiprapa, S., Robinson, M.W., To, J., Dalton, J.P., Loukas, A. & Brindley, P.J. 2009, 'Cathepsin F cysteine protease of the human liver fluke, Opisthorchis viverrini.', PLoS neglected tropical diseases, vol. 3, no. 3, p. e398.View/Download from: Publisher's site
The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities.Here, we describe the cDNA, gene organization, phylogenetic relationships, immunolocalization, and functional characterization of the cathepsin F cysteine protease gene, here termed Ov-cf-1, from O. viverrini. The full length mRNA of 1020 nucleotides (nt) encoded a 326 amino acid zymogen consisting of a predicted signal peptide (18 amino acids, aa), prosegment (95 aa), and mature protease (213 aa). BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes, including Clonorchis sinensis (81%), Paragonimus westermani (58%), Schistosoma mansoni and S. japonicum (52%), and with vertebrate cathepsin F (51%). Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host. The Ov-cf-1 gene, of approximately 3 kb in length, included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length, the introns from 43 to 1,060 bp. The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene, although the gene structure of human cathepsin F is more complex. Unlike Ov-CF-1, human cathepsin F zymogen includes a cystatin domain in the prosegment region. Phylogenetic analysis revealed that the fluke, human, and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases. A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris. Although the recombinant protease did not autocatalytically process and activate to a m...
Porntip, P., Kaewpitoon, N., Laha, T., Sripa, B., Kaewkes, S., Morales, M.E., Mann, V.H., Parriott, S.K., Suttiprapa, S., Robinson, M.W., To, J., Dalton, J.P., Loukas, A.C. & Brindley, P.J. 2009, 'Cathepsin F cysteine protease of the human liver fluke, Opisthorchis viverrini', Plos Neglected Tropical Diseases, vol. 3, no. 3, pp. 1-15.View/Download from: UTS OPUS or Publisher's site
The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities.
Villavedra Sierra, M., Lemke, S.J., To, J.H., Broady, K.W., Wallach, M. & Raison, R.L. 2007, 'Carbohydrate epitopes are immunodominant at the surface of infectious Neoparamoeba spp', Journal Of Fish Diseases, vol. 30, no. 4, pp. 191-199.View/Download from: UTS OPUS or Publisher's site
Amoebic gill disease, the main disease of concern to the salmon industry is Tasmania is caused by the amoeba Neoparamoeba spp. Experimental infection can onlybe induced by exposure to wild-type (WT) oarasites isolated from the gills of infected fish, as cultured amoeba are non-infective. To characterise the surface antigens of WT parasites, we produced monoclonal antibodies (mAbs) using subtractive immunization. Mice inoculated with non-infective parasites were treated with cyclophosphamide, to deplete reactive lumphocytes, and then immunized with different antigen preparations from infective parasites. When whole parasites were used for boosting the percentage of WT unique mAbs was very high (86%) as was the percentage of mAbs specific for carbohydrate pritopes (89%). When degloycosylated membranes were used, the numbers of mAbs spefic for non-carbohydrate spitopes id not increase, but the total number of WT unique mAbs was reduced (86-40%). Using an untreated membrane preparation, the total number of mAbs to surface molecules was very high, but all recognized carbohydrate epitopes. The total number of MAbs recognising carbohydrate epitopes on the surface of the WT parasites was 97%, suggesting that the dominant epitopes on the surface molecules unique to WT parasites are carbohydrate in nature.
Villavedra Sierra, M., McCarthy, K., To, J.H., Morrison, R., Crosbie, P., Broady, K.W. & Raison, R.L. 2005, 'Changes in antigenic profile during culture of Neoparamoeba sp., causative agent of amoebic gill disease in Atlantic salmon', International Journal for Parasitology, vol. 35, pp. 1417-1423.View/Download from: UTS OPUS or Publisher's site
Amoebic gill diseas (AGD) the most serious infectious disease affecting farmed salmon in Tasmania, is caused by free-living marine amoeba Neoparamoeba sp. The parasites on the gills induce proliferation of epithelial cells initiating a hyperplastic response and reducing the surface area available for gaseous exchange. AGD casn be induced in salmon by exposure to frshly isolated Neoparamoeba from AGD infected fish, however cultured Neoparamoeba are non-infective. We describe here antigenic difference between freshly isolated and in vitro cultured parasites, and within individual isolates of the parasite cultured under different conditions.