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Robinson, M.W., Buchtmann, K., Jenkins, C., Tacchi, J.L., Raymond, B.B., To, J., Bhattacharya, P., 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, p. 130017.
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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.
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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., 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, p. e1002042.
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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.

Robinson, M.W., Corvo, I., Jones, P.M., George, A.M., Padula, M.P., 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, p. e1012.
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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.

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.
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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.R., 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.L. 2010, 'Aminopeptidases of malaria parasites: New targets for chemotherapy.', Infectious Disorders Drug Targets, vol. 10, no. 3, pp. 217-225.
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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.F., 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.
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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.

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.
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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.