My interest has always been to understand host-pathogen interactions.
I qualified with a degree in Microbiology from Trinity College Dublin, Ireland and a PhD in Virology from the same university. My early postdoctoral work at the National University of Ireland Maynooth, focused primarily on the neuro-immunological response to vaccination. During this time, I developed and published the details of a murine model, which reported a definitive link between administration of whole cell vaccines and the induction of seizure behaviour.
In 2001, I moved to Dublin City University, Ireland to work on the isolation and characterization of immune modulating proteins secreted by helminth parasites, with the aim of identifying putative vaccine candidates for the prevention of ruminant worm infection. Research here led to the discovery of a protein secreted by helminth parasites that modulates cells of the innate immune system, specifically, activating macrophages towards an alternative phenotype which has downstream effects on the host T cell response. This was the first paper to describe the alternative activation of macrophages by an individual parasite-secreted protein independently of IL-4 and IL-13 signalling.
In 2004, I moved to UTS and since then have continued to investigate host-parasite interactions. This research has two streams: understanding the mechanisms by which helminth parasites successfully manipulate their host’s immune system with a view to developing novel anti-helminthic treatment strategies; and using this knowledge to develop novel therapeutics for the treatment of autoimmune disease.
- Member of Australian Society of Parasitology
- Member of the ARC/NHMRC Research Network for Parasitology
- Member of the Australasian Society for Immunology
- Host-Pathogen interactions
- Therapeutic potential of novel immunomodulators secreted by pathogens
- Role of macrophages in the regulation of immune responses
Biotechnology - subject coordinator
Immunology – Notre Dame Postgraduate Medical degree
Programme Director of the Undergraduate Biotechnology degrees at UTS
Shiels, J, Cwiklinski, K, Alvarado, R, Thivierge, K, Cotton, S, Gonzales Santana, B, To, J, Donnelly, S, Taggart, CC, Weldon, S & Dalton, JP 2020, 'Schistosoma mansoni immunomodulatory molecule Sm16/SPO-1/SmSLP is a member of the trematode-specific helminth defence molecules (HDMs).', PLoS neglected tropical diseases, vol. 14, no. 7, p. e0008470.View/Download from: Publisher's site
BACKGROUND:Sm16, also known as SPO-1 and SmSLP, is a low molecular weight protein (~16kDa) secreted by the digenean trematode parasite Schistosoma mansoni, one of the main causative agents of human schistosomiasis. The molecule is secreted from the acetabular gland of the cercariae during skin invasion and is believed to perform an immune-suppressive function to protect the invading parasite from innate immune cell attack. METHODOLOGY/PRINCIPAL FINDINGS:We show that Sm16 homologues of the Schistosomatoidea family are phylogenetically related to the helminth defence molecule (HDM) family of immunomodulatory peptides first described in Fasciola hepatica. Interrogation of 69 helminths genomes demonstrates that HDMs are exclusive to trematode species. Structural analyses of Sm16 shows that it consists predominantly of an amphipathic alpha-helix, much like other HDMs. In S. mansoni, Sm16 is highly expressed in the cercariae and eggs but not in adult worms, suggesting that the molecule is of importance not only during skin invasion but also in the pro-inflammatory response to eggs in the liver tissues. Recombinant Sm16 and a synthetic form, Sm16 (34-117), bind to macrophages and are internalised into the endosomal/lysosomal system. Sm16 (34-117) elicited a weak pro-inflammatory response in macrophages in vitro but also suppressed the production of bacterial lipopolysaccharide (LPS)-induced inflammatory cytokines. Evaluation of the transcriptome of human macrophages treated with a synthetic Sm16 (34-117) demonstrates that the peptide exerts significant immunomodulatory effects alone, as well as in the presence of LPS. Pathways most significantly influenced by Sm16 (34-117) were those involving transcription factors peroxisome proliferator-activated receptor (PPAR) and liver X receptors/retinoid X receptor (LXR/RXR) which are intricately involved in regulating the cellular metabolism of macrophages (fatty acid, cholesterol and glucose homeostasis) and are central to infl...
Cwiklinski, K, Donnelly, S, Drysdale, O, Jewhurst, H, Smith, D, De Marco Verissimo, C, Pritsch, IC, O'Neill, S, Dalton, JP & Robinson, MW 2019, 'The cathepsin-like cysteine peptidases of trematodes of the genus Fasciola.', vol. 104, pp. 113-164.View/Download from: Publisher's site
In this report, we review our present understanding of the cathepsin-like
cysteine peptidases of F. hepatica in the context of the parasite's relationship with its mammalian host. Wehave integrated information gathered frombasic molecular biology/biochemical studies and experimental laboratory techniques with the emerging –omics technologies (genomics, transcriptomics and proteomics) and immunological advances to clarify the structural make-up of the peptidase families and to help our continual re-assessment of the role each peptidase plays in the virulence, pathogenicity and, indeed, in the lengthy rapport between parasite and host.
Cwiklinski, K, Jewhurst, H, McVeigh, P, Barbour, T, Maule, AG, Tort, J, O'Neill, SM, Robinson, MW, Donnelly, S & Dalton, JP 2018, 'Infection by the Helminth Parasite Fasciola hepatica Requires Rapid Regulation of Metabolic, Virulence, and Invasive Factors to Adjust to Its Mammalian Host.', Molecular & cellular proteomics : MCP, vol. 17, no. 4, pp. 792-809.View/Download from: Publisher's site
The parasite Fasciola hepatica infects a broad range of mammals with impunity. Following ingestion of parasites (metacercariae) by the host, newly excysted juveniles (NEJ) emerge from their cysts, rapidly penetrate the duodenal wall and migrate to the liver. Successful infection takes just a few hours and involves negotiating hurdles presented by host macromolecules, tissues and micro-environments, as well as the immune system. Here, transcriptome and proteome analysis of ex vivo F. hepatica metacercariae and NEJ reveal the rapidity and multitude of metabolic and developmental alterations that take place in order for the parasite to establish infection. We found that metacercariae despite being encased in a cyst are metabolically active, and primed for infection. Following excystment, NEJ expend vital energy stores and rapidly adjust their metabolic pathways to cope with their new and increasingly anaerobic environment. Temperature increases induce neoblast proliferation and the remarkable up-regulation of genes associated with growth and development. Cysteine proteases synthesized by gastrodermal cells are secreted to facilitate invasion and tissue degradation, and tegumental transporters, such as aquaporins, are varied to deal with osmotic/salinity changes. Major proteins of the total NEJ secretome include proteases, protease inhibitors and anti-oxidants, and an array of immunomodulators that likely disarm host innate immune effector cells. Thus, the challenges of infection by F. hepatica parasites are met by rapid metabolic and physiological adjustments that expedite tissue invasion and immune evasion; these changes facilitate parasite growth, development and maturation. Our molecular analysis of the critical processes involved in host invasion has identified key targets for future drug and vaccine strategies directed at preventing parasite infection.
Tanaka, A, Allam, VSRR, Simpson, J, Tiberti, N, Shiels, J, To, J, Lund, M, Combes, V, Weldon, S, Taggart, C, Dalton, JP, Phipps, S, Sukkar, MB & Donnelly, S 2018, 'The parasitic 68-mer peptide FhHDM-1 inhibits mixed granulocytic inflammation and airway hyperreactivity in experimental asthma', JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, vol. 141, no. 6, pp. 2316-2319.View/Download from: Publisher's site
Wong, SL, To, J, Santos, J, Allam, VSRR, Dalton, JP, Djordjevic, SP, Donnelly, S, Padula, MP & Sukkar, MB 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: 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.
Dixit, A, Tanaka, A, Greer, JM & Donnelly, S 2017, 'Novel Therapeutics for Multiple Sclerosis Designed by Parasitic Worms.', International Journal of Molecular Sciences, vol. 18, no. 10, pp. 1-17.View/Download from: Publisher's site
The evolutionary response to endemic infections with parasitic worms (helminth) was the development of a distinct regulatory immune profile arising from the need to encapsulate the helminths while simultaneously repairing tissue damage. According to the old friend's hypothesis, the diminished exposure to these parasites in the developed world has resulted in a dysregulated immune response that contributes to the increased incidence of immune mediated diseases such as Multiple Sclerosis (MS). Indeed, the global distribution of MS shows an inverse correlation to the prevalence of helminth infection. On this basis, the possibility of treating MS with helminth infection has been explored in animal models and phase 1 and 2 human clinical trials. However, the possibility also exists that the individual immune modulatory molecules secreted by helminth parasites may offer a more defined therapeutic strategy.
Ruiz-Campillo, MT, Molina-Hernandez, V, Escamilla, A, Stevenson, M, Perez, J, Martinez-Moreno, A, Donnelly, S, Dalton, JP & Cwiklinski, K 2017, 'Immune signatures of pathogenesis in the peritoneal compartment during early infection of sheep with Fasciola hepatica', Scientific Reports, vol. 7, no. 1, pp. 1-14.View/Download from: Publisher's site
© The Author(s) 2017. Immune signatures of sheep acutely-infected with Fasciola hepatica, an important pathogen of livestock and humans were analysed within the peritoneal compartment to investigate early infection. Within the peritoneum, F. hepatica antibodies coincided with an intense innate and adaptive cellular immune response, with infiltrating leukocytes and a marked eosinophilia (49%). However, while cytokine qPCR analysis revealed IL-10, IL-12, IL-13, IL-23 and TGFβ were elevated, these were not statistically different at 18 days post-infection compared to uninfected animals indicating that the immune response is muted and not yet skewed to a Th2 type response that is associated with chronic disease. Proteomic analysis of the peritoneal fluid identified infection-related proteins, including several structural proteins derived from the liver extracellular matrix, connective tissue and epithelium, and proteins related to the immune system. Periostin and vascular cell adhesion protein 1 (VCAM-1), molecules that mediate leukocyte infiltration and are associated with inflammatory disorders involving marked eosinophilia (e.g. asthma), were particularly elevated in the peritoneum. Immuno-histochemical studies indicated that the source of periostin and VCAM-1 was the inflamed sheep liver tissue. This study has revealed previously unknown aspects of the immunology and pathogenesis associated with acute fascioliasis in the peritoneum and liver.
Alvarado, R, To, J, Lund, ME, Pinar, A, Mansell, A, Robinson, MW, O'Brien, BA, Dalton, JP & 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: Publisher's site
The NLRP3 inflammasome is a multimeric protein complex that controls the production of IL-1β, 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-1β by macrophages. FhHDM-1 had no effect on the synthesis of pro-IL-1β. 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-1β. 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-1β 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.
Donnelly, S, Huston, WM, Johnson, M, Tiberti, N, Saunders, B, O'Brien, B, Burke, C, Labbate, M & Combes, V 2017, 'Targeting the master regulator mTOR: a new approach to prevent the neurological of consequences of parasitic infections?', Parasites & Vectors, vol. 10, no. 1, pp. 1-6.View/Download from: Publisher's site
A systematic analysis of 240 causes of death in 2013 revealed that parasitic diseases were responsible for more than one million deaths. The vast majority of these fatalities resulted from protozoan infections presenting with neurological sequelae. In the absence of a vaccine, development of effective therapies is essential to improving global public health. In 2015, an intriguing strategy to prevent cerebral malaria was proposed by Gordon et al. 2015 mBio, 6:e00625. Their study suggested that inhibition of the mammalian target of rapamycin prevented experimental cerebral malaria by blocking the damage to the blood brain barrier and stopping the accumulation of parasitized red blood cells and T cells in the brain. Here, we hypothesize that the same therapeutic strategy could be adopted for other protozoan infections with a brain tropism, to prevent cerebral parasitosis by limiting pathogen replication and preventing immune mediated destruction of brain tissue.
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, pp. 1-12.View/Download from: 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 ...
Lund, ME, Greer, J, Dixit, A, Alvarado, R, McCauley-Winter, P, To, J, Tanaka, A, Hutchinson, AT, Robinson, MW, Simpson, AM, O'Brien, BA, Dalton, JP & 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: 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.
Lund, ME, To, J, O'Brien, BA & 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: Publisher's site
Molina-Hernandez, V, Mulcahy, G, Perez, J, Martinez-Moreno, A, Donnelly, S, O'Neill, SM, Dalton, JP & Cwiklinski, K 2015, 'Fasciola hepatica vaccine: We may not be there yet but we're on the right road', VETERINARY PARASITOLOGY, vol. 208, no. 1-2, pp. 101-111.View/Download from: Publisher's site
Alvarado, R, O'Brien, B, Tanaka, A, Dalton, JP & Donnelly, S 2015, 'A parasitic helminth-derived peptide that targets the macrophage lysosome is a novel therapeutic option for autoimmune disease', IMMUNOBIOLOGY, vol. 220, no. 2, pp. 262-269.View/Download from: Publisher's site
Adams, PN, Aldridge, A, Vukman, KV, Donnelly, S & O'Neill, SM 2014, 'Fasciola hepatica tegumental antigens indirectly induce an M2 macrophage-like phenotype in vivo.', Parasite immunology, vol. 36, no. 10, pp. 531-539.View/Download from: Publisher's site
The M2 subset of macrophages has a critical role to play in host tissue repair, tissue fibrosis and modulation of adaptive immunity during helminth infection. Infection with the helminth, Fasciola hepatica, is associated with M2 macrophages in its mammalian host, and this response is mimicked by its excretory-secretory products (FhES). The tegumental coat of F. hepatica (FhTeg) is another major source of immune-modulatory molecules; we have previously shown that FhTeg can modulate the activity of both dendritic cells and mast cells inhibiting their ability to prime a Th1 immune response. Here, we report that FhTeg does not induce Th2 immune responses but can induce M2-like phenotype in vivo that modulates cytokine production from CD4(+) cells in response to anti-CD3 stimulation. FhTeg induces a RELMα expressing macrophage population in vitro, while in vivo, the expression of Arg1 and Ym-1/2 but not RELMα in FhTeg-stimulated macrophages was STAT6 dependent. To support this finding, FhTeg induces RELMα expression in vivo prior to the induction of IL-13. FhTeg can induce IL-13-producing peritoneal macrophages following intraperitoneal injection This study highlights the important role of FhTeg as an immune-modulatory source during F. hepatica infection and sheds further light on helminth-macrophage interactions.
El Ridi, R, Tallima, H, Dalton, JP & Donnelly, S 2014, 'Induction of protective immune responses against schistosomiasis using functionally active cysteine peptidases', FRONTIERS IN GENETICS, vol. 5.View/Download from: Publisher's site
El Ridi, R, Tallima, H, Selim, S, Donnelly, S, Cotton, S, Santana, BG & Dalton, JP 2014, 'Cysteine Peptidases as Schistosomiasis Vaccines with Inbuilt Adjuvanticity', PLOS ONE, vol. 9, no. 1.View/Download from: Publisher's site
Lund, ME, O'Brien, B, Hutchinson, AT, Robinson, MW, Simpson, AM, Dalton, JP & Donnelly, SM 2014, 'Secreted proteins from the helminth Fasciola hepatica inhibit the initiation of autoreactive T cell responses and prevent diabetes in the NOD mouse', PLoS One, vol. 9, no. 1, p. e86289.View/Download from: Publisher's site
Infections with helminth parasites prevent/attenuate auto-inflammatory disease. Here we show that molecules secreted by a helminth parasite could prevent Type 1 Diabetes (T1D) in nonobese diabetic (NOD) mice. When delivered at 4 weeks of age (coincident with the initiation of autoimmunity), the excretory/secretory products of Fasciola hepatica (FhES) prevented the onset of T1D, with 84% of mice remaining normoglycaemic and insulitis-free at 30 weeks of age. Disease protection was associated with suppression of IFN-? secretion from autoreactive T cells and a switch to the production of a regulatory isotype (from IgG2a to IgG1) of autoantibody. Following FhES injection, peritoneal macrophages converted to a regulatory M2 phenotype, characterised by increased expression levels of Ym1, Arg-1, TGFß and PD-L1. Expression of these M2 genetic markers increased in the pancreatic lymph nodes and the pancreas of FhES-treated mice. In vitro, FhES-stimulated M2 macrophages induced the differentiation of Tregs from splenocytes isolated from naïve NOD mice. Collectively, our data shows that FhES contains immune-modulatory molecules that mediate protection from autoimmune diabetes via the induction and maintenance of a regulatory immune environment.
Dalton, JP, Robinson, MW, Mulcahy, G, O'neill, S & Donnelly, SM 2013, 'Immunomodulatory molecules of Fasciola hepatica:Candidates for both vaccine and immunotherapeuticdevelopment', Veterinary Parasitology, vol. 195, no. 3-4, pp. 272-285.View/Download from: Publisher's site
tThe liver fluke, Fasciola hepatica, causes fascioliasis in domestic animals (sheep, cattle),a global disease that is also an important infection of humans. As soon as the parasiteinvades the gut wall its interaction with various host immune cells (e.g. dendritic cells,macrophages and mast cells) is complex. The parasite secretes a myriad of molecules thatdirect the immune response towards a favourable non-protective Th2-mediate/regulatoryenvironment. These immunomodulatory molecules, such as cathepsin L peptidase (FhCL1),are under development as the first generation of fluke vaccines. However, this peptidase andother molecules, such as peroxiredoxin (FhPrx) and helminth defence molecule (FhHDM-1), exhibit various immunomodulatory properties that could be harnessed to help treatimmune-related conditions in humans and animals.
Thivierge, K, Cotton, S, Schaefer, D, Riggs, M, To, JH, Lund, ME, Robinson, MW, Dalton, JP & Donnelly, SM 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: 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
Robinson, MW, Dalton, JP, O'Brien, B & Donnelly, SM 2013, 'Fasciola hepatica: The therapeutic potential of a worm secretome', International Journal For Parasitology, vol. 43, no. 3-4, pp. 283-291.View/Download from: Publisher's site
The success of helminth parasites is partly related to their ability to modulate host immune responses towards an anti-inflammatory/regulatory phenotype. This ability resides with the molecules contained in the secretome of various helminths that have been shown to interact with host immune cells and influence their function. Consequently, there exists a unique opportunity to exploit these molecules for the prophylactic and therapeutic treatment of human pro- and auto-inflammatory disorders (for example septic shock, transplant rejection and autoimmune disease). In this review, we describe the mechanisms used by the trematode parasite, Fasciola hepatica, to modulate the immune responses of its host and discuss the potent immune-modulatory effects of three individual molecules within the secretome; namely cathepsin L1, peroxiredoxin and helminth defence molecule. With a focus on the requirements from industry, we discuss the strategies by which these molecules may be clinically developed to control human immune responses in a way that is conducive to the prevention of immune-mediated diseases.
Cotton, S, Donnelly, SM, Robinson, MW, Dalton, JP & Thivierge, K 2012, 'Defense peptides secreted by helminth pathogens: antimicrobial and/or immunomodulator molecules?', Frontiers in Immunology, vol. 3, pp. 1-7.View/Download from: Publisher's site
Host defense peptides (HDPs) are an evolutionarily conserved component of the innate immune response found in all living species. They possess antimicrobial activities against a broad range of organisms including bacteria, fungi, eukaryotic parasites, and viruses. HDPs also have the ability to enhance immune responses by acting as immunomodulators. We discovered a new family of HDPs derived from pathogenic helminth (worms) that cause enormous disease in animals and humans worldwide. The discovery of these peptides was based on their similar biochemical and functional characteristics to the human defense peptide LL-37. We propose that these new peptides modulate the immune response via molecular mimicry of mammalian HDPs thus providing a mechanism behind the anti-inflammatory properties of helminth infections.
Robinson, MW, Alvarado, R, To, J, Hutchinson, AT, Dowdell, SN, Lund, ME, Turnbull, L, Whitchurch, CB, O'Brien, B, Dalton, JP & Donnelly, SM 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: 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
Donnelly, SM, Dalton, JP & Robinson, MW 2011, 'How pathogen-derived cysteine proteases modulate host immune responses', Advances in Experimental Medicine and Biology, vol. 712, pp. 192-207.View/Download from: Publisher's site
In mammals, cysteine proteases are essential for the induction and development of both innate and adaptive immune responses. These proteases play a role in antigen-and pathogen-recognition and elimination, signal processing and cell homeostasis. Many pathogens also secrete cysteine proteases that often act on the same target proteins as the mammalian proteases and thereby can modulate host immunity from initial recognition to effector mechanisms. Pathogen-derived proteases range from nonspecific proteases that degrade multiple proteins involved in the immune response to enzymes that are very specific in their mode of action. Here, we overview current knowledge of pathogen-derived cysteine proteases that modulate immune responses by altering the normal function of key receptors or pathways in the mammalian immune system.
Robinson, MW, Donnelly, SM, Hutchinson, AT, To, J, Taylor, NL, Norton, RS, Perugini, M & Dalton, JP 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: 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.
Donnelly, SM, O'Neill, S, Stack, CM, Robinson, MW, Turnbull, L, Whitchurch, CB & Dalton, JP 2010, 'Helminth cysteine proteases inhibit TRIF - dependent activation of macrophages via degradation of TLR3.', Journal Of Biological Chemistry, vol. 285, no. 5, pp. 3383-3392.View/Download from: Publisher's site
Helminth pathogens prepare a Th2 type immunological environment in their hosts to ensure their longevity. They achieve this by secreting molecules that not only actively drive type 2 responses but also suppress type 1 responses. Here, we show that the major cysteine proteases secreted from the helminth pathogens Fasciola hepatica (FheCLl) and Schistosoma mansoni (SmCBI) protect mice from the lethal effects oflipopolysaccharide by preventing the release of inflammatory mediators, nitric oxide, interleukin-6, tumor necrosis factor a, and interleukin- 12, from macro phages. The proteases specifically blocl< the MyDSS-independent TRIF-dependent signaling pathway of Toll-like receptor (TLR)4 and TLR3. Microscopical and flow cytometric studies, however, show that alteration of macrophage function by cysteine protease is not mediated by cleavage of components of the TLR4 complex on the cell surface but occurs by degradation ofTLR3 within the endosome. This is the first study to describe a parasite molecule that degrades this receptor and pinpoints a novel mechanism by which helminth parasites modulate the innate immune responses of their hosts to suppress the development ofThl responses.
Dowling, D, Hamilton, C, Donnelly, SM, La Course, J, Brophy, P, Dalton, JP & O'neill, S 2010, 'Major Secretory Antigens Of The Helminth Fasciola Hepatica Activate A Suppressive Dendritic Cell Phenotype That Attenuates Th17 Cells But Fails To Activate Th2 Immune Responses', Infection And Immunity, vol. 78, no. 2, pp. 793-801.View/Download from: Publisher's site
Fasciola hepatica is a helminth pathogen that drives Th2/Treg immune responses in its mammalian host. The parasite releases a large number of molecules that are critical to inducing this type of immune response. Here we have selected recombinant forms of
Golden, O, Flynn, RY, Read, C, Sekiya, M, Donnelly, SM, Stack, CM, Dalton, JP & Mulcahy, G 2010, 'Protection Of Cattle Against A Natural Infection Of Fasciola Hepatica By Vaccination With Recombinant Cathepsin L1 (Rfhcl1)', Vaccine, vol. 28, no. 34, pp. 5551-5557.View/Download from: Publisher's site
The liver fluke, Fasciola hepatica causes liver fluke disease, or fasciolosis, in ruminants such as cattle and sheep. An effective vaccine against the helminth parasite is essential to reduce our reliance on anthelmintics, particularly in light of freque
Robinson, MW, Hutchinson, AT, Donnelly, SM & Dalton, JP 2010, 'Peroxiredoxin: a central player in immune modulation.', Parasite Immunology, vol. 32, no. 5, pp. 305-313.View/Download from: Publisher's site
Peroxiredoxins (Prx) are a family of anti-oxidants that protect cells from metabolically produced reactive oxygen species (ROS). The presence of these enzymes in the secretomes of many parasitic helminths suggests they provide protection against ROS released by host immune effector cells. However, we recently reported that helminth-secreted Prx also contribute to the development of Th2-responses via a mechanism involving the induction of alternatively activated macrophages. In this review, we discuss the role helminth Prx may play in modulating the immune responses of their hosts.
Damage-associated molecular pattern molecules (DAMPs) are endogenous leaderless molecules that are released into the extracellular environment in response to tissue damage. Most DAMPs have recognised intracellular housekeeping functions and are traditionally regarded as cytosolic or nuclear proteins (Table 1). They activate inflammatory responses by interacting with pattern recognition receptors (PRRs) expressed by immune cells and thus set off an `alarm system, the outcome of which depends on the collection of DAMPs released by the damaged or infected tissue. Although the collection of DAMPs vary considerably depending on the type of tissue involved, these often lead to a wound-healing scenario in response to tissue damage . Besides being released passively by necrotic or damaged cells, it is also known that many DAMPS exit the cell via non-classical secretory pathways
Trenholme, K, Brown, CL, Skinner-Adams, TS, Stack, CM, Lowther, J, To, J, Robinson, MW, Donnelly, SM, Dalton, JP & 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: 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
Miller, CM, Smith, NC, Ikin, RJ, Boulter, N, Dalton, JP & Donnelly, SM 2009, 'Immunological interactions between 2common pathogens, the Th1-inducing protozoan Toxoplasma gondii and the Th2-inducing helminth Fasciola hepatica', PLoS ONE, vol. 4, no. 5, pp. 1-10.
The nature of the immune response to infection is dependent on the type of infecting organism. Intracellular organisms such as Toxoplasma gondii stimulate a Th1-driven response associated with production of IL-12, IFN-?, nitric oxide and IgG2a antibodies and classical activation of macrophages. In contrast, extracellular helminths such as Fasciola hepatica induce Th2 responses characterised by the production of IL-4, IL-5, IL-10 and IgG1 antibodies and alternative activation of macrophages. As co-infections with these types of parasites commonly exist in the field it is relevant to examine how the various facets of the immune responses induced by each may influence or counter-regulate that of the other.
Miller, CMD, Smith, NC, Ikin, RJ, Boulter, NR, Dalton, JP & Donnelly, S 2009, 'Immunological interactions between 2 common pathogens, Th1-inducing protozoan Toxoplasma gondii and the Th2-inducing helminth Fasciola hepatica.', PloS one, vol. 4, no. 5, p. e5692.View/Download from: Publisher's site
BACKGROUND: The nature of the immune response to infection is dependent on the type of infecting organism. Intracellular organisms such as Toxoplasma gondii stimulate a Th1-driven response associated with production of IL-12, IFN-gamma, nitric oxide and IgG2a antibodies and classical activation of macrophages. In contrast, extracellular helminths such as Fasciola hepatica induce Th2 responses characterised by the production of IL-4, IL-5, IL-10 and IgG1 antibodies and alternative activation of macrophages. As co-infections with these types of parasites commonly exist in the field it is relevant to examine how the various facets of the immune responses induced by each may influence or counter-regulate that of the other. PRINCIPAL FINDINGS: Regardless, of whether F. hepatica infection preceded or succeeded T. gondii infection, there was little impact on the production of the Th1 cytokines IL-12, IFN-gamma or on the development of classically-activated macrophages induced by T. gondii. By contrast, the production of helminth-specific Th2 cytokines, such as IL-4 and IL-5, was suppressed by infection with T. gondii. Additionally, the recruitment and alternative activation of macrophages by F. hepatica was blocked or reversed by subsequent infection with T. gondii. The clinical symptoms of toxoplasmosis and the survival rate of infected mice were not significantly altered by the helminth. CONCLUSIONS: Despite previous studies showing that F. hepatica suppressed the classical activation of macrophages and the Th1-driven responses of mice to bystander microbial infection, as well as reduced their ability to reject these, here we found that the potent immune responses to T. gondii were capable of suppressing the responses to helminth infection. Clearly, the outcome of particular infections in polyparasitoses depends on the means and potency by which each pathogen controls the immune response.
Dalton, JP, Brindley, PJ, Donnelly, SM & Robinson, MW 2009, 'The enigmatic asparaginyl endopeptidase of helminth parasites', Trends In Parasitology, vol. 25, no. 2, pp. 59-61.View/Download from: Publisher's site
The clan CD cysteine protease of Schistosoma mansoni (SmAE, also known as Sm32 or schistosome legumain) is an asparaginyl endopeptidase that cleaves C-terminal to asparaginyl (Asn) residues. The enzyme was considered to function in the hydrolytic degradation of host haemoglobin within the parasite gut 1 B. Götz and M.Q. Klinkert, Expression and partial characterization of a cathepsin B-like enzyme (Sm31) and a proposed `haemoglobinase (Sm32) from Schistosoma mansoni, Biochem. J. 290 (1993), pp. 801806. View Record in Scopus | Cited By in Scopus (26). However, Dalton and Brindley  proposed that the primary role of the enzyme was the trans-processing and activation of other schistosome proteases after noting that zymogens of clan AA aspartic proteases (cathepsin D) and clan CA cysteine proteases (cathepsins L, F, B1 and C) each possessed an asparaginyl-endopeptidase-cleavage site at the juncture between the prosegment and mature enzyme domain. Prosegment removal by trans-processing exposes the active site of the mature enzyme to enable entry of haemoglobin substrate. Cytochemistry revealed that schistosome asparaginyl endopeptidase is expressed in the gastrodermis surrounding the gut lumen, the same locality as the haemoglobin-degrading enzymes. Sajid et al.  provided support for this trans-processing hypothesis by showing that a recombinant S. mansoni asparaginyl endopeptidase could convert the zymogen of schistosome cathepsin B1 to a mature enzyme in vitro. Delcroix et al.  also showed that 20% of cathepsin B activity was lost when the expression of asparaginyl endopeptidase of three-week-old male and female schistosomes was knocked down using RNA interference (RNAi).
Lowther, J, Robinson, MW, Donnelly, SM, Xu, W, Stack, CM, Matthews, J & Dalton, JP 2009, 'The Importance of pH in Regulating the Function of the Fasciola hepatica Cathepsin L1 Cysteine Protease', Plos Neglected Tropical Diseases, vol. 3, no. 1, pp. 1-11.View/Download from: Publisher's site
The helminth parasite Fasciola hepatica secretes cathepsin L cysteine proteases to invade its host, migrate through tissues and digest haemoglobin, its main source of amino acids. Here we investigated the importance of pH in regulating the activity and functions of the major cathepsin L protease FheCL1. The slightly acidic pH of the parasite gut facilitates the auto-catalytic activation of FheCL1 from its inactive proFheCL1 zymogen; this process was similar to 40-fold faster at pH 4.5 than at pH 7.0. Active mature FheCL1 is very stable at acidic and neutral conditions ( the enzyme retained similar to 45% activity when incubated at 37 degrees C and pH 4.5 for 10 days) and displayed a broad pH range for activity peptide substrates and the protein ovalbumin, peaking between pH 5.5 and pH 7.0. This pH profile likely reflects the need for FheCL1 to function both in the parasite gut and in the host tissues. FheCL1, however, could not cleave its natural substrate Hb in the pH range pH 5.5 and pH 7.0; digestion occurred only at pH <= 4.5, which coincided with pH-induced dissociation of the Hb tetramer. Our studies indicate that the acidic pH of the parasite relaxes the Hb structure, making it susceptible to proteolysis by FheCL1. This process is enhanced by glutathione (GSH), the main reducing agent contained in red blood cells. Using mass spectrometry, we show that FheCL1 can degrade Hb to small peptides, predominantly of 4-14 residues, but cannot release free amino acids. Therefore, we suggest that Hb degradation is not completed in the gut lumen but that the resulting peptides are absorbed by the gut epithelial cells for further processing by intracellular di- and amino-peptidases to free amino acids that are distributed through the parasite tissue for protein anabolism.
Maric, S, Donnelly, SM, Robinson, MW, Skinner-Adams, TS, Trenholme, K, Gardiner, D, Dalton, JP, Stack, CM & Lowther, J 2009, 'The M17 Leucine Aminopeptidase of the Malaria Parasite Plasmodium falciparum: Importance of Active Site Metal Ions in the Binding of Substrates and Inhibitors', Biochemistry, vol. 48, no. 23, pp. 5435-5439.View/Download from: Publisher's site
The M17 leucine aminopeptidase of the intraerythrocytic stages of the malaria parasite Plasmodium falciparum (PfLAP) plays it role in releasing amino acids from host hemoglobin that are used for parasite protein synthesis, growth, and development. This enzyme represents a target at which new antimalarials Could be designed since metalloaminopeptidase inhibitors prevent the growth of the parasites in vitro and in vivo. A study on the metal ion binding characteristics of recombinant P. falciparum M17 leucine aminopeptidase (rPfLAP) shows that the active site of this exopeptidase contains two metal-binding sites, a readily exchangeable site (site 1) and a tight binding site (site 2). The enzyme retains activity when the metal ion is removed from site 1, while removal of metal ions from both sites results in an inactive apoenzyme that cannot be reactivated by the addition of divalent metal cations. The metal Ion at site I is readily exchangeable with several divalent metal ions and displays a preference in the order of preference Zn2+ > Mn2+ > Co2+ > Mg2+. While it is likely that native PfLAP contains a Zn2+ in site 2, the metal ion located in site 1 may be dependent on the type and concentration of metal ions in the cytosolic compartment of the parasite. Importantly, the type of metal ion present at site 1 influences not only the catalytic efficiency of the enzyme for peptide Substrates but also the mode of binding by bestatin, a metal-chelating inhibitor of M17 aminopeptidases with antimalarial activity.
McGowan, S, Porter, CJ, Lowther, J, Stack, CM, Golding, SJ, Skinner-Adams, TS, Trenholme, K, Teuscher, F, Donnelly, SM, Grembecka, J, Mucha, A, Kafarski, P, DeGori, R, Buckle, AM, Gardiner, D, Whisstock, J & Dalton, JP 2009, 'Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase', Proceedings Of The National Academy Of Sciences Of The United States Of America, vol. 106, no. 8, pp. 2537-2542.View/Download from: Publisher's site
Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH2]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.
Robinson, MW, Menon, R, Donnelly, SM, Dalton, JP & Ranganathan, S 2009, 'An Integrated Transcriptomics And Proteomics Analysis Of The Secretome Of The Helminth Pathogen Fasciola Hepatica Proteins Associated With Invasion And Infection Of The Mammalian Host', Molecular & Cellular Proteomics, vol. 8, no. 8, pp. 1891-1907.View/Download from: Publisher's site
To infect their mammalian hosts, Fasciola hepatica larvae must penetrate and traverse the intestinal wall of the duodenum, move through the peritoneum, and penetrate the liver. After migrating through and feeding on the liver, causing extensive tissue damage, the parasites move to their final niche in the bile ducts where they mature and produce eggs. Here we integrated a transcriptomics and proteomics approach to profile Fasciola secretory proteins that are involved in host-pathogen interactions and to correlate changes in their expression with the migration of the parasite. Prediction of F. hepatica secretory proteins from 14,031 expressed sequence tags (ESTs) available from the Wellcome Trust Sanger Centre using the semiautomated EST2Secretome pipeline showed that the major components of adult parasite secretions are proteolytic enzymes including cathepsin L, cathepsin B, and asparaginyl endopeptidase cysteine proteases as well as novel trypsin-like serine proteases and carboxypeptidases. Proteomics analysis of proteins secreted by infective larvae, immature flukes, and adult F. hepatica showed that these proteases are developmentally regulated and correlate with the passage of the parasite through host tissues and its encounters with different host macromolecules. Proteases such as FhCL3 and cathepsin B have specific functions in larvae activation and intestinal wall penetration, whereas FhCL1, FhCL2, and FhCL5 are required for liver penetration and tissue and blood feeding. Besides proteases, the parasites secrete an array of antioxidants that are also highly regulated according to their migration through host tissues. However, whereas the proteases of F. hepatica are secreted into the parasite gut via a classical endoplasmic reticulum/Golgi pathway, we speculate that the antioxidants, which all lack a signal sequence, are released via a non-classical trans-tegumental pathway.
Walters, SN, Donnelly, S, Robinson, M, Dalton, J & Grey, ST 2009, 'Identification of novel parasite-derived compounds that promote long-term islet allograft survival', XENOTRANSPLANTATION, vol. 16, no. 5, pp. 402-402.
Goh, F, Irvine, K, Lovelace, E, Donnelly, SM, Jones, MK, Brion, K, Hume, D, Kotze, A, Dalton, JP, Ingham, A & Sweet, M 2008, 'Selective induction of the Notch ligand Jagged-1 in macrophages by soluble egg antigen from Schistosoma mansoni involves ERK signalling', Immunology, vol. 127, no. 3, pp. 326-337.View/Download from: Publisher's site
Soluble egg antigen (SEA) from the helminth Schistosoma mansoni promotes T helper type 2 (Th2) responses by modulating antigen-presenting cell function. The Jagged/Notch pathway has recently been implicated in driving Th2 development. We show here that SEA rapidly up-regulated mRNA and protein expression of the Notch ligand Jagged-1 in both murine bone marrow-derived macrophages (BMMs) and human monocyte-derived macrophages (HMDMs). Another potential Th2-promoting factor, interleukin (IL)-33, was not transcriptionally induced by SEA in BMMs. Up-regulation of Jagged-1 mRNA by SEA was also apparent in conventional dendritic cells (DCs), although the effect was less striking than in BMMs. Conversely, SEA-pulsed DCs, but not BMMs, promoted IL-4 production upon T-cell activation, suggesting that Jagged-1 induction alone is insufficient for instructing Th2 development. A comparison of the responses initiated in BMMs by SEA and the bacterial endotoxin lipopolysaccharide (LPS) revealed common activation of extracellular signal-regulated kinase-1/2 (ERK-1/2) and p38 phosphorylation, as well as induction of Jagged-1 mRNA. However, only LPS triggered I kappa B degradation, phosphorylation of c-Jun N-terminal kinase (Jnk) and signal transducer and activator of transcription 1 (Stat1) Tyr701, and IL-33 and IL-12p40 mRNA up-regulation. Inducible gene expression was modified by the presence of the macrophage growth factor colony-stimulating factor (CSF)-1, which inhibited Jagged-1 induction by SEA and LPS, but enhanced LPS-induced IL-12p40 expression. Unlike LPS, SEA robustly activated signalling in HEK293 cells expressing either Toll-like receptor 2 (TLR2) or TLR4/MD2. Pharmacological inhibition of the ERK-1/2 pathway impaired SEA- and LPS-inducible Jagged-1 expression in BMMs. Taken together, our data suggest that Jagged-1 is an ERK-dependent target of TLR signalling that has a macrophage-specific function in the response to SEA.
Robinson, MW, Dalton, JP & Donnelly, SM 2008, 'Helminth pathogen cathepsin proteases: it's a family affair.', Trends in Biochemical Sciences, vol. 33, no. 12, pp. 601-608.View/Download from: Publisher's site
Helminth pathogens express papain-like cysteine peptidases, termed cathepsins, which have important roles in virulence, including host entry, tissue migration and the suppression of host immune responses. The liver fluke Fasciola hepatica, an emerging human pathogen, expresses the largest cathepsin L cysteine protease family yet described. Recent phylogenetic, biochemical and structural studies indicate that this family contains five separate clades, which exhibit overlapping but distinct substrate specificities created by a process of gene duplication followed by subtle residue divergence within the protease active site. The developmentally regulated expression of these proteases correlates with the passage of the parasite through host tissues and its encounters with different host macromolecules.
Robinson, MW, Tort, JF, Lowther, J, Donnelly, SM, Wong, E, Xu, W, Stack, CM, Padula, M, Herbert, BR & Dalton, JP 2008, 'Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica', Molecular & Cellular Proteomics, vol. 7, no. 6, pp. 1111-1123.View/Download from: Publisher's site
Cathepsin L proteases secreted by the helminth pathogen Fasciola hepatica have functions in parasite virulence including tissue invasion and suppression of host immune responses. Using proteomics methods alongside phylogenetic studies we characterized th
Stack, CM, Caffrey, C, Donnelly, SM, Seshaadri, A, Lowther, J, Tort, JF, Collins, PR, Robinson, MW, Xu, W, Mckerrow, J, Craik, C, Geiger, S, Marion, R, Brinen, L & Dalton, JP 2008, 'Structural and functional relationships in the virulence-associated cathepsin L proteases of the parasitic liver fluke, Fasciola hepatica', Journal Of Biological Chemistry, vol. 283, no. 15, pp. 9896-9908.View/Download from: Publisher's site
The helminth parasite Fasciola hepatica secretes cysteine proteases to facilitate tissue invasion, migration, and development within the mammalian host. The major proteases cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) were recombinantly produced and b
Donnelly, SM, Stack, CM, O'Neill, SA, Sayed, AA, Williams, DL & Dalton, JP 2008, 'Helminth 2-Cys peroxiredoxin drives Th2 responses through a mechanism involving alternatively activated macrophages', FASEB Journal, vol. 22, no. 11, pp. 4022-4032.View/Download from: Publisher's site
During helminth infections, alternatively activated macrophages (AAMacs) are key to promoting Th2 responses and suppressing Th1-driven inflammatory pathology. Th2 cytokines IL-4 and/or IL-13 are believed to be important in the induction and activation of
Kanaji, S, Tanaka, Y, Sakata, Y, Takeshita, K, Arima, K, Ohta, S, Hansell, E, Caffrey, C, Mottram, J, Lowther, J, Donnelly, SM, Stack, CM, Kadowaki, T, Yamamoto, K, Mckerrow, J, Dalton, JP, Coombs, G & Izuhara, K 2007, 'Squamous Cell Carcinoma Antigen 1 Is An Inhibitor Of Parasite-derived Cysteine Proteases', Febs Letters, vol. 581, no. 22, pp. 4260-4264.View/Download from: Publisher's site
The physiological significance of the squamous cell carcinoma antigens 1 (SCCA1) and SCCA2, members of the ovalbumin serpin family, remains unresolved. In this study, we examined whether SCCA1 or SCCA2 inhibits protozoa- or helminth-derived cysteine prot
Stack, CM, Donnelly, SM, Lowther, J, Xu, W, Collins, PR, Brinen, L & Dalton, JP 2007, 'The major secreted cathepsin L1 protease of the liver fluke, Fasciola hepatica - A Leu(-12) to Pro(-12) replacement in the nonconserved C-terminal region of the prosegment prevents complete enzyme autoactivation and allows definition of the molecular', Journal Of Biological Chemistry, vol. 282, no. 22, pp. 16532-16543.View/Download from: Publisher's site
A protease secreted by the parasitic helminth Fasciola hepatica, a 37-kDa procathepsin L1 (FheproCL1), autocatalytically processes and activates to its mature enzyme (FheCL1) over a wide pH range of 7.3 to 4.0, although activation is more rapid at low pH
Stack, CM, Lowther, J, Cunningham, E, Donnelly, SM, Gardiner, D, Trenholme, K, Skinner-Adams, TS, Teuscher, F, Grembecka, J, Mucha, A, Kafarski, P, Lua, L, Bell, A & Dalton, JP 2007, 'Characterization of the Plasmodium falciparum M17 leucyl aminopeptidase - A protease involved in amino acid regulation with potential for antimalarial drug development', Journal Of Biological Chemistry, vol. 282, no. 3, pp. 2069-2080.View/Download from: Publisher's site
Amino acids generated from the catabolism of hemoglobin by intra-erythrocytic malaria parasites are not only essential for protein synthesis but also function in maintaining an osmotically stable environment, and creating a gradient by which amino acids that are rare or not present in hemoglobin are drawn into the parasite from gost serum. We have proposed that a Plasmodium flaciparum M17 lecyl aminopeptidase (PfLAP) generates and regulates the internal pool of free amino acids and therefore respesents a target for novel antimalarial drugs. This enzyme has been expressed in insect cells as a functional 320-kDa homo-hexamer that is optimally active at neutral or alkaline pH, is dependent on metal ions for activity, and exhibits a substrate preference for N-terminally exposed hydrophobic amino acids, particularly leucine. pfLAP is produced by all stages in the intra-erythrocytic developmental cycle of malaria byt was most highly expressed by trophozoites, a stage at which hemoglobin degradation and parasite protein synthesis are elevated. The enzyme was located by immunohistochemical methods and by transfecting malaria cells with a pfLAP-green fluorescent protein construct, to the cytosolic compartment of the cell at all developmental stages, including segregated merozoites. Amino acid dipeptide analogs, such as bestatin and its derivatives, are potent inhibitors f the protease and also block the growth of p. falciparum malaria parasites in culture. This study provides a biochemical basis for the antimalarial activity of aminopeptidase inhibitors. Availability of functionally active recombitant PfLAP, coupled witha simple enzymatic readout, will aid medicinal chemistry and/or high throughout approaches for the future design./discovery of new antimalarial drugs.
Teuscher, F, Lowther, J, Skinner-Adams, TS, Spielmann, T, Dixon, M, Stack, CM, Donnelly, SM, Mucha, A, Kafarski, P, Vassiliou, S, Gardiner, D, Dalton, JP & Trenholme, K 2007, 'The M18 Aspartyl Aminopeptidase Of The Human Malaria Parasite Plasmodium Falciparum', Journal Of Biological Chemistry, vol. 282, no. 42, pp. 30817-30826.View/Download from: Publisher's site
A member of the M18 family of aspartyl aminopeptidases is expressed by all intra-erythrocytic stages of the human malaria parasite Plasmodium falciparum ( PfM18AAP), with highest expression levels in rings. Functionally active recombinant enzyme, rPfM18A
Donnelly, SM, Dalton, JP & Loukas, A 2006, 'Proteases in Helminth- and Allergen- Induced Inflammatory Responses', Chemical Immunology and Allergy, vol. 90, pp. 45-64.
Proteolytic activity is a central biochemical property that endows molecules with intrinsic allergenicity. Thus, the cysteine protease of dust mite, Der p1, the aspartic protease of cockroach, Bla g 2, the serine protease of Aspergillus fumigatus and the bacterial subtilisins are all major allergenic molecules responsible for the increase in asthma and atopic conditions worldwide. These proteases induce Th2-driven inflammatory responses in the airways by disrupting the epithelial cell junctions so that these, and other molecules, gain access to, and alter the function of, underlying cells of the innate immune system (dendritic cells, mast cells, basophils and macrophages) and B and T cells. Helminth parasites secrete proteases to gain entry into their hosts, and to feed on and migrate through tissues. Their action leads to tissue damage and the activation of inflammatory responses dominated by elevated IgE, eosinophilia and Th2 cells, much like allergenic responses. In certain situations, such as in acute infections (especially with zoonotic helminths), proteases secreted by helminths may sensitise individuals to allergens. However, the anti-inflammatory responses observed in chronic helminthiases, involving IL-10 and TGF?, that are primarily responsible for controlling immune-mediated damage to the host that is initiated by secreted proteases, coincidentally protects against similar inflammatory damage by allergens.
Sekiya, M, Mulcahy, G, Irwin, JA, Stack, CM, Donnelly, SM, Xu, W, Collins, P & Dalton, JP 2006, 'Biochemical characterisation of the recombinant peroxiredoxin (FhePrx) of the liver fluke, Fasciola hepatica', Febs Letters, vol. 580, no. 21, pp. 5016-5022.View/Download from: Publisher's site
The parasitic helminth Fasciola hepatica secretes a 2-Cys peroxiredoxin (Prx) that may play important functions in host-parasite interaction. Recombinant peroxiredoxin (FhePrx) prevented metal-catalyzed oxidative nicking of plasmid DNA and detoxilied hyd
Donnelly, SM, O'Neill, S, Sekiya, M, Mulcahy, G & Dalton, JP 2005, 'Thioredoxin peroxidase secreted by Fasciola hepatica induces the alternative activation of macrophages', Infection And Immunity, vol. 73, no. 1, pp. 166-173.View/Download from: Publisher's site
Alternatively activated macrophages (AAMphi) are primarily associated with the chronic stages of parasitic infections and the development of a polarized Th2 response. We have shown that Fasciola hepatica infection of BALB/c mice induces a polarized Th2 r
Stack, CM, Dalton, JP, Cunneen, M & Donnelly, SM 2005, 'De-glycosylation of Pichia pastoris-produced Schistosoma mansoni cathepsin B eliminates non-specific reactivity with IgG in normal human serum', Journal Of Immunological Methods, vol. 304, no. 1-2, pp. 151-157.
Production of diagnostic reagents in the yeast Pichia pastoris is particularly attractive since this organism is capable of expressing complex eukaryotic proteins in their correctly folded form and is amenable to large-scale fermentation at low cost. The potential of Schistosoma mansoni cathepsin B as a diagnostic antigen for human schistosomiasis has been previously established using both native and E. coli-derived recombinant proteins. However, when produced in P. pastoris we found that recombinant wild-type cathepsin B was preferentially secreted as a heterogeneously glycosylated molecule that migrated at 39 kDa, 41 kDa and a smear of >50 kDa on SDS-PAGE, and was susceptible to treatment with Endo H and PGNase F. The addition of yeast sugars to the cathepsin B caused it to react with IgG in the serum of both normal (non-infected) and schistosome-infected humans in immunoblotting and enzyme linked immunosorbent assays (ELISA). To avoid this non-specific reactivity, a non-glycosylated mutant form of cathepsin B, engineered by disrupting its potential glycosylation site, was produced. The non-glycosylated recombinant cathepsin B migrated as a single band of 39 kDa on SDS-PAGE. Most importantly, the molecule was not reactive with IgG in normal sera and, hence, could be employed in immunoblots or ELISA to specifically detect antibodies in schistosome-infected patients. Addition of oligosaccharides by P. pastoris is a potential drawback that needs to be considered before using P. pastoris-produced proteins as diagnostic reagents.
Collins, PR, Stack, CM, O'Neill, S, Doyle, S, Ryan, T, Brennan, GP, Mousley, A, Stewart, M, Maule, AG, Dalton, JP & Donnelly, SM 2004, 'Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica) virulence - Propeptide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells', Journal Of Biological Chemistry, vol. 279, pp. 17038-17046.View/Download from: Publisher's site
McCarthy, E, Stack, CM, Donnelly, SM, Doyle, S, Mann, VH, Brindley, PJ, Stewart, M, Day, TA, Maule, AG & Dalton, JP 2004, 'Leucine aminopeptidase of the human blood flukes| Schistosoma mansoni and Schistosoma japonicum', International Journal For Parasitology, vol. 34, no. 6, pp. 703-714.View/Download from: Publisher's site
An array of schistosome endoproteases involved in the digestion of host hemoglobin to absorbable peptides has been described, but the exoprotease responsible for catabolising these peptides to amino acids has yet to be identified. By searching the public databases we found that Schistosoma mansoni and Schistosoma japonicum express a gene encoding a member of the M17 family of leucine aminopeptidases (LAPs). A functional recombinant S. mansoni LAP produced in insect cells shared biochemical properties, including pH optimum for activity, substrate specificity and reliance on metal cations for activity, with the major aminopeptidase activity in soluble extracts of adult worms. The pH range in which the enzyme functions and the lack of a signal peptide indicate that the enzyme functions intracellularly. Immunolocalisation studies showed that the S. mansoni LAP is synthesised in the gastrodermal cells surrounding the gut lumen. Accordingly, we propose that peptides generated in the lumen of the schistosome gut are absorbed into the gastrodermal cells and are cleaved by LAP to free amino acids before being distributed to the internal tissues of the parasite. Since LAP was also localised to the surface tegument it may play an additional role in surface membrane re-modelling.
Mulcahy, G, O'Neill, S, Donnelly, SM & Dalton, JP 2004, 'Helminths at mucosal barriers - interaction with the immune system', Advanced Drug Delivery Reviews, vol. 56, pp. 853-868.View/Download from: Publisher's site
Dalton, JP, Brindley, PJ, Knox, DP, Brady, CP, Hotez, PJ, Donnelly, SM, O'Neill, S, Mulcahy, G & Loukas, AC 2003, 'Helminth Vaccines: From Mining Genomic Information For Vaccine Targets To Systems Used For Protein Expression', International Journal For Parasitology, vol. 33, no. 5-6, pp. 621-640.View/Download from: Publisher's site
The control of helminth diseases of people and livestock continues to rely on the widespread use of anti-helminthic drugs. However, concerns with the appearance of drug resistant parasites and the presence of pesticide residues in food and the environmen
Dalton, JP, O'Neill, S, Stack, CM, Collins, PR, Walshe, AL, Sekiya, M, Doyle, S, Mulcahy, G, Hoyle, D, Khaznadji, E, Moire, N, Brennan, GP, Mousley, A, Kreshchenko, N, Maule, AG & Donnelly, SM 2003, 'Fasciola Hepatica Cathepsin L-like Proteases: Biology, Function, And Potential In The Development Of First Generation Liver Fluke Vaccines', International Journal For Parasitology, vol. 33, no. 11, pp. 1173-1181.View/Download from: Publisher's site
Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and catheps
Donnelly, S, Loscher, CE, Lynch, MA & Mills, KHG 2001, 'Whole-cell but not acellular pertussis vaccines induce convulsive activity in mice: Evidence of a role for toxin-induced interleukin-1 beta in a new murine model for analysis of neuronal side effects of vaccination', INFECTION AND IMMUNITY, vol. 69, no. 7, pp. 4217-4223.View/Download from: Publisher's site
Loscher, CE, Donnelly, S, Lynch, MA & Mills, KHG 2000, 'Induction of inflammatory cytokines in the brain following respiratory infection with Bordetella pertussis', JOURNAL OF NEUROIMMUNOLOGY, vol. 102, no. 2, pp. 172-181.View/Download from: Publisher's site
Loscher, CE, Donnelly, S, Mills, KHG & Lynch, MA 2000, 'Interleukin-1 beta-dependent changes in the hippocampus following parenteral immunization with a whole cell pertussis vaccine', JOURNAL OF NEUROIMMUNOLOGY, vol. 111, no. 1-2, pp. 68-76.View/Download from: Publisher's site
Donnelly, S, Loscher, C, Mills, KHG & Lynch, MA 1999, 'Glycerol-induced seizure: involvement of IL-1 beta and glutamate', NEUROREPORT, vol. 10, no. 9, pp. 1821-1825.View/Download from: Publisher's site
Loscher, CE, Donnelly, S, McBennett, S, Lynch, MA & Mills, KHG 1998, 'Proinflammatory cytokines in the adverse systemic and neurologic effects associated with parenteral injection of a whole cell pertussis vaccine', MOLECULAR MECHANISMS OF FEVER, vol. 856, pp. 274-277.View/Download from: Publisher's site
Donnelly, SM, Sheahan, BJ & Atkins, GJ 1997, 'Long-term effects of Semliki Forest virus infection in the mouse central nervous system', NEUROPATHOLOGY AND APPLIED NEUROBIOLOGY, vol. 23, no. 3, pp. 235-241.
Donnelly, SM, Sheahan, BJ & Atkins, GJ 1997, 'Long-term effects of Semliki Forest virus infection in the mouse central nervous system', Neuropathology and Applied Neurobiology, vol. 23, no. 3, pp. 235-241.View/Download from: Publisher's site
Semliki Forest virus (SFV) infection of mice is used as a model to study pathogenic processes occurring in viral encephalitis and demyelinating disease. In this study, the long-term effects of infection by the avirulent M9 mutant of SFV on the central nervous system (CNS) of BALB/c and SJL mice were determined. The presence of infectious virus, viral RNA and cytokine mRNA in the brains of individual mice and the presence of lesions in the spinal cords of the same mice up to 360 days post-infection (d.p.i.) were analysed in order to detect any correlation between these parameters of pathogenesis. Infectious virus could not be detected beyond 7 d.p.i. for either mouse strain. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the presence of the E2 and nsP1 regions of the virus genome and mRNA for interferon-γ and tumour necrosis factor-α. Viral RNA could be detected up to 90 d.p.i. for both mouse strains. Cytokine mRNA could be detected up to 28 d.p.i. for BALB/c mice but up to 360 d.p.i. for SJL mice. Inflammatory lesions, which were associated with cytokine mRNA expression, were not detected in BALB/c mice beyond 28 d.p.i. but were detected in two SJL mice at 90 d.p.i. It is concluded that M9-SFV infection induces long-term prolonged expression of pro-inflammatory cytokines in the CNS of the majority of SJL (but not BALB/c) mice which is not associated with persistence of the virus genome. M9-SFV infection of SJL mice may be a relevant model for the pathogenesis of multiple sclerosis in man.
Robinson, MW, Dalton, JP, O'Niell, SM & Donnelly, SM 2012, 'Mechanisms of immune modulation by Fasciola hepatica: Importance for vaccine development and for novel immunotherapeutics' in Caffrey, CR (ed), Parasitic Helminths: Targets, Screens, Drugs and Vaccines, Wiley-VCH Verlag GmbH & Co. KGaA, Germany, pp. 451-463.View/Download from: Publisher's site
The liver fluke Fasciola hepatica can live for long periods in its definitive mammalian host. This longevity is related to the parasite's ability to modulate host immune responses to benefit its survival (i.e., suppression of Th1/Th17 responses and the promotion of strong Th2/Treg-mediated responses). Various reports indicate that this immune regulation may reduce the capacity of animals to resist other bystander infections (e.g., F. hepatica-infected mice exhibit reduced protective immune responses to the respiratory bacterium, Bordetella pertussis). Experiments in cattle infected with F. hepatica revealed reductions in interferon-? responses to coinfections with Mycobacterium bovis. Molecules secreted by the parasite such as cathepsin L cysteine peptidases, the antioxidant peroxiredoxin, and a cathelicidin-like defense molecule play central roles in manipulating the function of host innate immune cells, and thus the development of protective adaptive immune responses. While these molecules influence innate immune cells in distinct ways, they likely function in concert to establish the potent Th2/Treg-mediated immune environment in the host. Vaccines that prevent the action of these immunomodulatory molecules may not only protect animals against liver fluke disease, but reduce their susceptibility to coincident parasitic or microbial infections. Taking a broader view, understanding how the liver fluke influences host immunity via specific cell surface receptors and intracellular signaling pathways could reveal strategies to selectively suppress certain inflammatory processes, and eventually lead to immunotherapeutic treatments for conditions such as arthritis, inflammatory bowel disease, and diabetes
Dixit, A, Donnelly, S, Lund, MA, Dalton, JP & Greer, JM 2016, 'Protection against development of a mouse model of multiple sclerosis by a parasite-derived 68-mer peptide', EUROPEAN JOURNAL OF IMMUNOLOGY, pp. 1042-1042.
Burke, ML, Donnelly, S, McManus, DP, Dalton, JP & Gobert, GN 2010, 'TRANSCRIPTIONAL PROFILING OF SCHISTOSOMA JAPONICUM-STIMULATED ALTERNATIVELY ACTIVATED MACROPHAGES', AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 59th Annual Meeting of the American-Society-of-Tropical-Medicine-and-Hygiene (ASTMH), AMER SOC TROP MED & HYGIENE, Atlanta, GA, pp. 124-124.
Background: Autoimmune diseases are characterised by a TH1-type immune response, mediated by inflammatory-M1 macrophages, whereas regulatory-M2 macrophages promote the TH2-type immune response to allergens or parasitic worm infections. Understanding how these macrophage phenotypes are activated could lead to novel therapeutic approaches. Micro(mi)RNAs are small non-coding RNAs which exhibit post-translational/post-transcriptional mechanisms to regulate the differentiation of immune cells. However, few studies have investigated the role of miRNAs in the activation of macrophage phenotypes in vivo.
Hypothesis: Parasitic infections alter host miRNAs to stimulate the activation of M2 macrophages, resulting in a TH2 immune response.
Methods: Using a murine model of a parasitic infection, we established a correlation between the expression of miRNAs and the changing phenotype of macrophages over time within the context of a T¬H2 immune response. Peritoneal macrophages were isolated from Balb/c mice at various time points over a 5 day period of infection with Fasciola hepatica and analysed for the abundance of miRNA by RNAseq analysis, where the entire expression profile of all miRNAs in peritoneal macrophages at various time points after infection was obtained. From this, the temporal profile of expression of the top 5 most abundant, along with various mRNA, was validated by qPCR. Additionally, the cytokine levels within the peritoneal lavage fluid, from the same mice, were measured by ELISA.
Results: Two significant patterns emerged; a change of expression at 6h post-infection which immediately returned to baseline and a gradual increase in expression reaching a maximum at day 5. The latter profile matched the expression patterns of genetic M2 markers but occurred prior to the production of TH2-type cytokines.
Conclusion: Specific miRNAs are altered immediately after infection to activate M2 macrophages which then instigate an adaptive immune response. However, fu...
- National University of Galway, Ireland
- The Centre for Clinical Research, University of Queensland
- The Westmead Millennium Institute
- University of New South Wales
- Dublin City University, Ireland