Publications by authors named "Darren Pickering"

23 Publications

  • Page 1 of 1

A netrin domain-containing protein secreted by the human hookworm Necator americanus protects against CD4 T cell transfer colitis.

Transl Res 2021 06 4;232:88-102. Epub 2021 Mar 4.

Centre for Molecular Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Queensland, Australia. Electronic address:

The symbiotic relationships shared between humans and their gastrointestinal parasites present opportunities to discover novel therapies for inflammatory diseases. A prime example of this phenomenon is the interaction of humans and roundworms such as the hookworm, Necator americanus. Epidemiological observations, animal studies and clinical trials using experimental human hookworm infection show that hookworms can suppress inflammation in a safe and well-tolerated way, and that the key to their immunomodulatory properties lies within their secreted proteome. Herein we describe the identification of 2 netrin domain-containing proteins from the N. americanus secretome, and explore their potential in treating intestinal inflammation in mouse models of ulcerative colitis. One of these proteins, subsequently named Na-AIP-1, was effective at suppressing disease when administered prophylactically in the acute TNBS-induced model of colitis. This protective effect was validated in the more robust CD4 T cell transfer model of chronic colitis, where prophylactic Na-AIP-1 reduced T-cell-dependent type-1 cytokine responses in the intestine and the associated intestinal pathology. Mechanistic studies revealed that depletion of CD11c+ cells abrogated the protective anticolitic effect of Na-AIP-1. Next generation sequencing of colon tissue in the T-cell transfer model of colitis revealed that Na-AIP-1 induced a transcriptomic profile associated with the downregulation of metabolic and signaling pathways involved in type-1 inflammation, notably TNF. Finally, co-culture of Na-AIP-1 with a human monocyte-derived M1 macrophage cell line resulted in significantly reduced secretion of TNF. Na-AIP-1 is now a candidate for clinical development as a novel therapeutic for the treatment of human inflammatory bowel diseases.
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http://dx.doi.org/10.1016/j.trsl.2021.02.012DOI Listing
June 2021

Extracellular Vesicle Proteins Confer Protection in a Heterologous Model of Schistosomiasis.

Vaccines (Basel) 2020 Jul 24;8(3). Epub 2020 Jul 24.

Centre for Molecular Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns 4878, Queensland, Australia.

Helminth parasites release extracellular vesicles which interact with the surrounding host tissues, mediating host-parasite communication and other fundamental processes of parasitism. As such, vesicle proteins present attractive targets for the development of novel intervention strategies to control these parasites and the diseases they cause. Herein, we describe the first proteomic analysis by LC-MS/MS of two types of extracellular vesicles (exosome-like, 120 k pellet vesicles and microvesicle-like, 15 k pellet vesicles) from adult worms. A total of 57 and 330 proteins were identified in the 120 k pellet vesicles and larger 15 k pellet vesicles, respectively, and some of the most abundant molecules included homologues of known helminth vaccine and diagnostic candidates such as -TSP2, 23, glutathione S-transferase, saponins and aminopeptidases. Tetraspanins were highly represented in the analysis and found in both vesicle types. Vaccination of mice with recombinant versions of three of these tetraspanins induced protection in a heterologous challenge () model of infection, resulting in significant reductions (averaged across two independent trials) in liver (47%, 38% and 41%) and intestinal (47%, 45% and 41%) egg burdens. These findings offer insight into the mechanisms by which anti-tetraspanin antibodies confer protection and highlight the potential that extracellular vesicle surface proteins offer as anti-helminth vaccines.
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http://dx.doi.org/10.3390/vaccines8030416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563238PMC
July 2020

Vaccination with Cholinesterases Reduces the Parasite Burden and Egg Viability in a Mouse Model of Schistosomiasis.

Vaccines (Basel) 2020 Apr 3;8(2). Epub 2020 Apr 3.

Centre for Molecular Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD 4878, Australia.

Schistosomiasis is a neglected tropical disease caused by parasitic blood flukes of the genus , which kills 300,000 people every year in developing countries, and there is no vaccine. Recently, we have shown that cholinesterases (ChEs)-enzymes that regulate neurotransmission-from are expressed on the outer tegument surface and present in the excretory/secretory products of larval schistosomula and adult worms, and are essential for parasite survival in the definitive host, highlighting their utility as potential schistosomiasis vaccine targets. When treated with anti-schistosome cholinesterase (ChE) IgG, both schistosomula and adult worms displayed significantly decreased ChE activity, which eventually resulted in parasite death. Vaccination with individual ChEs, or a combination of all three ChEs, significantly reduced worm burdens in two independent trials compared to controls. Average adult worm numbers and liver egg burdens were significantly decreased for all vaccinated mice across both trials, with values of 29-39% and 13-46%, respectively, except for those vaccinated with AChE1 in trial 1. Egg viability, as determined by egg hatching from liver homogenates, was significantly reduced in the groups vaccinated with the ChE cocktail (40%) and AChE2 (46%). Furthermore, surviving worms from each vaccinated group were significantly stunted and depleted of glycogen stores, compared to controls. These results suggest that ChEs could be incorporated into a vaccine against schistosomiasis to reduce the pathology and transmission of this debilitating disease.
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http://dx.doi.org/10.3390/vaccines8020162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349746PMC
April 2020

Proteomic analysis of two populations of Schistosoma mansoni-derived extracellular vesicles: 15k pellet and 120k pellet vesicles.

Mol Biochem Parasitol 2020 03 31;236:111264. Epub 2020 Jan 31.

Centre for Molecular Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, 4878, Queensland, Australia; Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain. Electronic address:

Helminth parasites secrete extracellular vesicles (EVs) into their environment that have potential roles in host-parasite communication, and thus represent potentially useful targets for novel control strategies. Here, we carried out a comprehensive proteomic analysis of two different populations of EVs - 15k pellet and 120k pellet EVs - from Schistosoma mansoni adult worms. We characterised the proteins present in the membranes of the EVs (including external trypsin-liberated peptides, integral membrane proteins (IMPs) and peripheral membrane proteins (PMPs)), as well as cargo proteins, using LC-MS/MS. A total of 286 and 716 proteins were identified in 15k and 120k pellets, respectively. Some of the most abundant proteins identified from both 15k and 120k pellets include known vaccine candidates such as Sm-TSP-2, saponin B domain-containing proteins, calpain glutathione-S-transferase, Sm29 and cathepsin domain-containing proteins. Other abundant proteins that have not been tested as vaccines include DM9 domain-containing protein, 13 kDa tegumental antigen and histone H4-like protein. Sm23, a member of the tetraspanin family with known vaccine efficacy, was identified in the cargo and IMP compartments of only 15k pellet vesicles. Moreover, a collection of proteins with known or potential relevance in host-parasite communication including proteases, antioxidants and EV biogenesis/trafficking of both vesicle types were identified. Our results provide the first report of a comprehensive compartmental proteomic analysis of adult S. mansoni-derived EVs. Future research should investigate recombinant forms of these proteins as vaccine and serodiagnostic antigens as well as the roles of EV proteins in host-parasite communication.
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http://dx.doi.org/10.1016/j.molbiopara.2020.111264DOI Listing
March 2020

Novel cholinesterase paralogs of Schistosoma mansoni have perceived roles in cholinergic signalling and drug detoxification and are essential for parasite survival.

PLoS Pathog 2019 12 6;15(12):e1008213. Epub 2019 Dec 6.

Centre for Molecular Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Queensland, Australia.

Cholinesterase (ChE) function in schistosomes is essential for orchestration of parasite neurotransmission but has been poorly defined with respect to the molecules responsible. Interrogation of the S. mansoni genome has revealed the presence of three ChE domain-containing genes (Smche)s, which we have shown to encode two functional acetylcholinesterases (AChE)s (Smache1 -smp_154600 and Smache2 -smp_136690) and a butyrylcholinesterase (BChE) (Smbche1 -smp_125350). Antibodies to recombinant forms of each SmChE localized the proteins to the tegument of adults and schistosomula and developmental expression profiling differed among the three molecules, suggestive of functions extending beyond traditional cholinergic signaling. For the first time in schistosomes, we identified ChE enzymatic activity in fluke excretory/secretory (ES) products and, using proteomic approaches, attributed this activity to the presence of SmAChE1 and SmBChE1. Parasite survival in vitro and in vivo was significantly impaired by silencing of each smche, either individually or in combination, attesting to the essential roles of these molecules. Lastly, in the first characterization study of a BChE from helminths, evidence is provided that SmBChE1 may act as a bio-scavenger of AChE inhibitors as the addition of recombinant SmBChE1 to parasite cultures mitigated the effect of the anti-schistosome AChE inhibitor 2,2- dichlorovinyl dimethyl phosphate-dichlorvos (DDVP), whereas smbche1-silenced parasites displayed increased sensitivity to DDVP.
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http://dx.doi.org/10.1371/journal.ppat.1008213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919630PMC
December 2019

Polypyridylruthenium(II) complexes exert anti-schistosome activity and inhibit parasite acetylcholinesterases.

PLoS Negl Trop Dis 2017 12 14;11(12):e0006134. Epub 2017 Dec 14.

Centre for Biodiscovery and Molecular Development of Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Queensland, Australia.

Background: Schistosomiasis affects over 200 million people and there are concerns whether the current chemotherapeutic control strategy (periodic mass drug administration with praziquantel (PZQ)-the only licenced anti-schistosome compound) is sustainable, necessitating the development of new drugs.

Methodology/principal Findings: We investigated the anti-schistosome efficacy of polypyridylruthenium(II) complexes and showed they were active against all intra-mammalian stages of S. mansoni. Two compounds, Rubb12-tri and Rubb7-tnl, which were among the most potent in their ability to kill schistosomula and adult worms and inhibit egg hatching in vitro, were assessed for their efficacy in a mouse model of schistosomiasis using 5 consecutive daily i.v. doses of 2 mg/kg (Rubb12-tri) and 10 mg/kg (Rubb7-tnl). Mice treated with Rubb12-tri showed an average 42% reduction (P = 0.009), over two independent trials, in adult worm burden. Liver egg burdens were not significantly decreased in either drug-treated group but ova from both of these groups showed significant decreases in hatching ability (Rubb12-tri-68%, Rubb7-tnl-56%) and were significantly morphologically altered (Rubb12-tri-62% abnormal, Rubb7-tnl-35% abnormal). We hypothesize that the drugs exerted their activity, at least partially, through inhibition of both neuronal and tegumental acetylcholinesterases (AChEs), as worms treated in vitro showed significant decreases in activity of these enzymes. Further, treated parasites exhibited a significantly decreased ability to uptake glucose, significantly depleted glycogen stores and withered tubercules (a site of glycogen storage), implying drug-mediated interference in this nutrient acquisition pathway.

Conclusions/significance: Our data provide compelling evidence that ruthenium complexes are effective against all intra-mammalian stages of schistosomes, including schistosomula (refractory to PZQ) and eggs (agents of disease transmissibility). Further, the results of this study suggest that schistosome AChE is a target of ruthenium drugs, a finding that can inform modification of current compounds to identify analogues which are even more effective and selective against schistosomes.
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http://dx.doi.org/10.1371/journal.pntd.0006134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746282PMC
December 2017

Suppression of inflammation and tissue damage by a hookworm recombinant protein in experimental colitis.

Clin Transl Immunology 2017 Oct 6;6(10):e157. Epub 2017 Oct 6.

Centre for the Biodiversity and Molecular Development of Therapeutics, Australian Institute of Tropical Health Medicine, James Cook University, Cairns, Queensland, Australia.

Gastrointestinal parasites, hookworms in particular, have evolved to cause minimal harm to their hosts when present in small numbers, allowing them to establish chronic infections for decades. They do so by creating an immunoregulatory environment that promotes their own survival, but paradoxically also benefits the host by protecting against the onset of many inflammatory diseases. To harness the therapeutic value of hookworms without using live parasites, we have examined the protective properties of the recombinant protein anti-inflammatory protein (AIP)-1, secreted in abundance by hookworms within the intestinal mucosa, in experimental colitis. Colitic inflammation assessed by weight loss, colon atrophy, oedema, ulceration and necrosis, as well as abdominal adhesion was significantly suppressed in mice treated with a single intraperitoneal dose of AIP-1 at 1 mg kg. Local infiltration of inflammatory cells was also significantly reduced, with minimal goblet cell loss and preserved mucosal architecture. Treatment with AIP-1 promoted the production of colon interleukin (IL)-10, transforming growth factor (TGF)-β and thymic stromal lymphopoietin (TSLP), resulting in the suppression of tumour necrosis factor (TNF)-α, IL-13 and IL-17 A cytokines and granulocyte macrophage colony-stimulating factor (GM-CSF), CX motif chemokine (CXCL)-11 and cyclooxygenase synthase (COX)-2 mRNA transcripts. AIP-1 promoted the accumulation of regulatory T cells in the colon likely allowing rapid healing of the colon mucosa. Hookworm recombinant AIP-1 is a novel therapeutic candidate for the treatment of inflammatory bowel diseases that can be explored for the prevention of acute inflammatory relapses, an important cause of colorectal cancer.
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http://dx.doi.org/10.1038/cti.2017.42DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5671989PMC
October 2017

Hookworm recombinant protein promotes regulatory T cell responses that suppress experimental asthma.

Sci Transl Med 2016 10;8(362):362ra143

Centre for Biodiscovery and Molecular Development of Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Queensland, Australia.

In the developed world, declining prevalence of some parasitic infections correlates with increased incidence of allergic and autoimmune disorders. Moreover, experimental human infection with some parasitic worms confers protection against inflammatory diseases in phase 2 clinical trials. Parasitic worms manipulate the immune system by secreting immunoregulatory molecules that offer promise as a novel therapeutic modality for inflammatory diseases. We identify a protein secreted by hookworms, anti-inflammatory protein-2 (AIP-2), that suppressed airway inflammation in a mouse model of asthma, reduced expression of costimulatory markers on human dendritic cells (DCs), and suppressed proliferation ex vivo of T cells from human subjects with house dust mite allergy. In mice, AIP-2 was primarily captured by mesenteric CD103 DCs and suppression of airway inflammation was dependent on both DCs and Foxp3 regulatory T cells (T) that originated in the mesenteric lymph nodes (MLNs) and accumulated in distant mucosal sites. Transplantation of MLNs from AIP-2-treated mice into naïve hosts revealed a lymphoid tissue conditioning that promoted T induction and long-term maintenance. Our findings indicate that recombinant AIP-2 could serve as a novel curative therapeutic for allergic asthma and potentially other inflammatory diseases.
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http://dx.doi.org/10.1126/scitranslmed.aaf8807DOI Listing
October 2016

Compounds Derived from the Bhutanese Daisy, Ajania nubigena, Demonstrate Dual Anthelmintic Activity against Schistosoma mansoni and Trichuris muris.

PLoS Negl Trop Dis 2016 08 4;10(8):e0004908. Epub 2016 Aug 4.

Centre for Biodiscovery and Molecular Development of Therapeutics, Queensland Tropical Health Alliance, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns Campus, Cairns, Australia.

Background: Whipworms and blood flukes combined infect almost one billion people in developing countries. Only a handful of anthelmintic drugs are currently available to treat these infections effectively; there is therefore an urgent need for new generations of anthelmintic compounds. Medicinal plants have presented as a viable source of new parasiticides. Ajania nubigena, the Bhutanese daisy, has been used in Bhutanese traditional medicine for treating various diseases and our previous studies revealed that small molecules from this plant have antimalarial properties. Encouraged by these findings, we screened four major compounds isolated from A. nubigena for their anthelmintic properties.

Methodology/principal Findings: Here we studied four major compounds derived from A. nubigena for their anthelmintic properties against the nematode whipworm Trichuris muris and the platyhelminth blood fluke Schistosoma mansoni using the xWORM assay technique. Of four compounds tested, two compounds-luteolin (3) and (3R,6R)-linalool oxide acetate (1)-showed dual anthelmintic activity against S. mansoni (IC50 range = 5.8-36.9 μg/mL) and T. muris (IC50 range = 9.7-20.4 μg/mL). Using scanning electron microscopy, we determined luteolin as the most efficacious compound against both parasites and additionally was found effective against the schistosomula, the infective stage of S. mansoni (IC50 = 13.3 μg/mL). Luteolin induced tegumental damage to S. mansoni and affected the cuticle, bacillary bands and bacillary glands of T. muris. Our in vivo assessment of luteolin (3) against T. muris infection at a single oral dosing of 100 mg/kg, despite being significantly (27.6%) better than the untreated control group, was markedly weaker than mebendazole (93.1%) in reducing the worm burden in mice.

Conclusions/significance: Among the four compounds tested, luteolin demonstrated the best broad-spectrum activity against two different helminths-T. muris and S. mansoni-and was effective against juvenile schistosomes, the stage that is refractory to the current gold standard drug, praziquantel. Medicinal chemistry optimisation including cytotoxicity analysis, analogue development and structure-activity relationship studies are warranted and could lead to the identification of more potent chemical entities for the control of parasitic helminths of humans and animals.
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http://dx.doi.org/10.1371/journal.pntd.0004908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4973903PMC
August 2016

Extracellular vesicles secreted by Schistosoma mansoni contain protein vaccine candidates.

Int J Parasitol 2016 Jan 13;46(1):1-5. Epub 2015 Oct 13.

Centre for Biodiscovery and Molecular Development of Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD, Australia. Electronic address:

Herein we show for the first time that Schistosoma mansoni adult worms secrete exosome-like extracellular vesicles ranging from 50 to 130nm in size. Extracellular vesicles were collected from the excretory/secretory products of cultured adult flukes and purified by Optiprep density gradient, resulting in highly pure extracellular vesicle preparations as confirmed by transmission electron microscopy and Nanosight tracking analysis. Extracellular vesicle proteomic analysis showed numerous known vaccine candidates, potential virulence factors and molecules implicated in feeding. These findings provide new avenues for the exploration of host-schistosome interactions and offer a potential mechanism by which some vaccine antigens exert their protective efficacy.
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http://dx.doi.org/10.1016/j.ijpara.2015.09.002DOI Listing
January 2016

Lipopeptide Nanoparticles: Development of Vaccines against Hookworm Parasite.

ChemMedChem 2015 Oct 12;10(10):1647-54. Epub 2015 Aug 12.

School of Chemistry and Molecular Biosciences, The University of Queensland (St. Lucia), Brisbane, Queensland 4072 (Australia).

Necator americanus (hookworm) infects over half a billion people worldwide. Anthelminthic drugs are commonly used to treat the infection; however, vaccination is a more favorable strategy to combat this parasite. We designed new B-cell peptide epitopes based on the aspartic protease of N. americanus (Na-APR-1). The peptides were conjugated to self-adjuvanting lipid core peptide (LCP) systems via stepwise solid-phase peptide synthesis (SPPS) and copper catalyst azide-alkyne cycloaddition (CuAAC) reactions. The LCP vaccine candidates were able to self-assemble into nanoparticles, were administered to mice without the use of additional adjuvant, and generated antibodies that recognized the parent epitope. However, only one LCP derivative was able to produce a high titer of antibodies specific to Na-APR-1; circular dichroism analyses of this compound showed a β-sheet conformation for the incorporated epitope. This study provides important insight in epitope and delivery system design for the development of a vaccine against hookworm infections.
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http://dx.doi.org/10.1002/cmdc.201500227DOI Listing
October 2015

The use of a conformational cathepsin D-derived epitope for vaccine development against Schistosoma mansoni.

Bioorg Med Chem 2015 Mar 30;23(6):1307-12. Epub 2015 Jan 30.

School of Chemistry and Molecular Biosciences, The University of Queensland (St Lucia), Chemistry Blg #68, St Lucia, Brisbane, Queensland 4072, Australia; School of Pharmacy, The University of Queensland (St Lucia), Woolloongabba, Queensland 4012, Australia. Electronic address:

Schistosomiasis is caused by the infection from Schistosoma species. Among these, Schistosoma mansoni is one of the major species that infects millions of people worldwide. The use of praziquantel is effective in clearing the infestation but treatment of a large and widespread population in endemic areas is unsustainable. Thus, synergistic approach of using drug and vaccination can serve as an alternative to the current treatment. In this study, we have developed vaccine candidates that composed of three components: a B-cell epitope derived from S. mansoni cathepsin D protein (Sm-CatD) flanked by GCN4 helix promoting peptide; a promiscuous T-helper epitope (P25); and a lipid core peptide system, in attempt to develop self-adjuvanting vaccine candidates against the schistosome. Physicochemical properties of the vaccine candidates were analysed and antibodies to each construct were raised in BALB/c mice. The vaccine candidates were able to self-assemble into particles that induced high titres of IgG without the use of additional adjuvant. The antibody levels were comparable to that induced by peptide formulated with strong but toxic Freund's adjuvant. The integration of a GCN4 sequence induced the helical conformation of the epitope, while the addition of the T helper peptide was very effective in inducing consistent IgG-specific antibodies response amongst mice. These findings are particularly encouraging for the development of efficient and immunogenic vaccine against schistosomiasis.
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http://dx.doi.org/10.1016/j.bmc.2015.01.033DOI Listing
March 2015

Probing of a human proteome microarray with a recombinant pathogen protein reveals a novel mechanism by which hookworms suppress B-cell receptor signaling.

J Infect Dis 2015 Feb 19;211(3):416-25. Epub 2014 Aug 19.

Centre for Biodiscovery and Molecular Development of Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns.

Na-ASP-2 is an efficacious hookworm vaccine antigen. However, despite elucidation of its crystal structure and studies addressing its immunobiology, the function of Na-ASP-2 has remained elusive. We probed a 9000-protein human proteome microarray with Na-ASP-2 and showed binding to CD79A, a component of the B-cell antigen receptor complex. Na-ASP-2 bound to human B lymphocytes ex vivo and downregulated the transcription of approximately 1000 B-cell messenger RNAs (mRNAs), while only approximately 100 mRNAs were upregulated, compared with control-treated cells. The expression of a range of molecules was affected by Na-ASP-2, including factors involved in leukocyte transendothelial migration pathways and the B-cell signaling receptor pathway. Of note was the downregulated transcription of lyn and pi3k, molecules that are known to interact with CD79A and control B-cell receptor signaling processes. Together, these results highlight a previously unknown interaction between a hookworm-secreted protein and B cells, which has implications for helminth-driven immunomodulation and vaccine development. Further, the novel use of human protein microarrays to identify host-pathogen interactions, coupled with ex vivo binding studies and subsequent analyses of global gene expression in human host cells, demonstrates a new pipeline by which to explore the molecular basis of infectious diseases.
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http://dx.doi.org/10.1093/infdis/jiu451DOI Listing
February 2015

Secreted proteomes of different developmental stages of the gastrointestinal nematode Nippostrongylus brasiliensis.

Mol Cell Proteomics 2014 Oct 3;13(10):2736-51. Epub 2014 Jul 3.

From the ‡Centre for Biodiscovery and Molecular Development of Therapeutics, Australian Institute for Tropical Health and Medicine, James Cook University, Cairns, Queensland, Australia;

Hookworms infect more than 700 million people worldwide and cause more morbidity than most other human parasitic infections. Nippostrongylus brasiliensis (the rat hookworm) has been used as an experimental model for human hookworm because of its similar life cycle and ease of maintenance in laboratory rodents. Adult N. brasiliensis, like the human hookworm, lives in the intestine of the host and releases excretory/secretory products (ESP), which represent the major host-parasite interface. We performed a comparative proteomic analysis of infective larval (L3) and adult worm stages of N. brasiliensis to gain insights into the molecular bases of host-parasite relationships and determine whether N. brasiliensis could indeed serve as an appropriate model for studying human hookworm infections. Proteomic data were matched to a transcriptomic database assembled from 245,874,892 Illumina reads from different developmental stages (eggs, L3, L4, and adult) of N. brasiliensis yielding∼18,426 unigenes with 39,063 possible isoform transcripts. From this analysis, 313 proteins were identified from ESPs by LC-MS/MS-52 in the L3 and 261 in the adult worm. Most of the proteins identified in the study were stage-specific (only 13 proteins were shared by both stages); in particular, two families of proteins-astacin metalloproteases and CAP-domain containing SCP/TAPS-were highly represented in both L3 and adult ESP. These protein families are present in most nematode groups, and where studied, appear to play roles in larval migration and evasion of the host's immune response. Phylogenetic analyses of defined protein families and global gene similarity analyses showed that N. brasiliensis has a greater degree of conservation with human hookworm than other model nematodes examined. These findings validate the use of N. brasiliensis as a suitable parasite for the study of human hookworm infections in a tractable animal model.
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http://dx.doi.org/10.1074/mcp.M114.038950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4188999PMC
October 2014

Solution structure, membrane interactions, and protein binding partners of the tetraspanin Sm-TSP-2, a vaccine antigen from the human blood fluke Schistosoma mansoni.

J Biol Chem 2014 Mar 15;289(10):7151-7163. Epub 2014 Jan 15.

Queensland Institute of Medical Research, Brisbane, QLD 4006, Australia; The University of Queensland, School of Biomedical Sciences, Brisbane, QLD 4072, Australia. Electronic address:

The tetraspanins (TSPs) are a family of integral membrane proteins that are ubiquitously expressed at the surface of eukaryotic cells. TSPs mediate a range of processes at the surface of the plasma membrane by providing a scaffold for the assembly of protein complexes known as tetraspanin-enriched microdomains (TEMs). We report here the structure of the surface-exposed EC2 domain from Sm-TSP-2, a TSP from Schistosoma mansoni and one of the better prospects for the development of a vaccine against schistosomiasis. This is the first solution structure of this domain, and our investigations of its interactions with lipid micelles provide a general model for interactions between TSPs, membranes, and other proteins. Using chemical cross-linking, eight potential protein constituents of Sm-TSP-2-mediated TEMs were also identified. These include proteins important for membrane maintenance and repair, providing further evidence for the functional role of Sm-TSP-2- and Sm-TSP-2-mediated TEMs. The identification of calpain, Sm29, and fructose-bisphosphate aldolase, themselves potential vaccine antigens, suggests that the Sm-TSP-2-mediated TEMs could be disrupted via multiple targets. The identification of further Sm-TSP-2-mediated TEM proteins increases the available candidates for multiplex vaccines and/or novel drugs targeting TEMs in the schistosome tegument.
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http://dx.doi.org/10.1074/jbc.M113.531558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3945375PMC
March 2014

TIMPs of parasitic helminths - a large-scale analysis of high-throughput sequence datasets.

Parasit Vectors 2013 May 30;6:156. Epub 2013 May 30.

Center for Biodiscovery and Molecular Development of Therapeutics, Queensland Tropical Health Alliance, James Cook University, Cairns, Queensland, Australia.

Background: Tissue inhibitors of metalloproteases (TIMPs) are a multifunctional family of proteins that orchestrate extracellular matrix turnover, tissue remodelling and other cellular processes. In parasitic helminths, such as hookworms, TIMPs have been proposed to play key roles in the host-parasite interplay, including invasion of and establishment in the vertebrate animal hosts. Currently, knowledge of helminth TIMPs is limited to a small number of studies on canine hookworms, whereas no information is available on the occurrence of TIMPs in other parasitic helminths causing neglected diseases.

Methods: In the present study, we conducted a large-scale investigation of TIMP proteins of a range of neglected human parasites including the hookworm Necator americanus, the roundworm Ascaris suum, the liver flukes Clonorchis sinensis and Opisthorchis viverrini, as well as the schistosome blood flukes. This entailed mining available transcriptomic and/or genomic sequence datasets for the presence of homologues of known TIMPs, predicting secondary structures of defined protein sequences, systematic phylogenetic analyses and assessment of differential expression of genes encoding putative TIMPs in the developmental stages of A. suum, N. americanus and Schistosoma haematobium which infect the mammalian hosts.

Results: A total of 15 protein sequences with high homology to known eukaryotic TIMPs were predicted from the complement of sequence data available for parasitic helminths and subjected to in-depth bioinformatic analyses.

Conclusions: Supported by the availability of gene manipulation technologies such as RNA interference and/or transgenesis, this work provides a basis for future functional explorations of helminth TIMPs and, in particular, of their role/s in fundamental biological pathways linked to long-term establishment in the vertebrate hosts, with a view towards the development of novel approaches for the control of neglected helminthiases.
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http://dx.doi.org/10.1186/1756-3305-6-156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3679795PMC
May 2013

Complement inhibitors from scabies mites promote streptococcal growth--a novel mechanism in infected epidermis?

PLoS Negl Trop Dis 2012 17;6(7):e1563. Epub 2012 Jul 17.

Infectious Diseases Program, Biology Department, Queensland Institute of Medical Research, Herston, Brisbane, Australia.

Background: Scabies is highly prevalent in socially disadvantaged communities such as indigenous populations and in developing countries. Generalized itching causes discomfort to the patient; however, serious complications can occur as a result of secondary bacterial pyoderma, commonly caused by Streptococcus pyogenes (GAS) or Staphylococcus aureus. In the tropics, skin damage due to scabies mite infestations has been postulated to be an important link in the pathogenesis of disease associated with acute rheumatic fever and heart disease, poststreptococcal glomerulonephritis and systemic sepsis. Treatment of scabies decreases the prevalence of infections by bacteria. This study aims to identify the molecular mechanisms underlying the link between scabies and GAS infections.

Methodology/principal Findings: GAS bacteria were pre-incubated with blood containing active complement, phagocytes and antibodies against the bacteria, and subsequently tested for viability by plate counts. Initial experiments were done with serum from an individual previously exposed to GAS with naturally acquired anti-GAS antibodies. The protocol was optimized for large-scale testing of low-opsonic whole blood from non-exposed human donors by supplementing with a standard dose of heat inactivated human sera previously exposed to GAS. This allowed an extension of the dataset to two additional donors and four proteins tested at a range of concentrations. Shown first is the effect of scabies mite complement inhibitors on human complement using ELISA-based complement activation assays. Six purified recombinant mite proteins tested at a concentration of 50 µg/ml blocked all three complement activation pathways. Further we demonstrate in human whole blood assays that each of four scabies mite complement inhibitors tested increased GAS survival rates by 2-15 fold.

Conclusions/significance: We propose that local complement inhibition plays an important role in the development of pyoderma in scabies infested skin. This molecular link between scabies and bacterial infections may provide new avenues to develop alternative treatment options against this neglected disease.
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http://dx.doi.org/10.1371/journal.pntd.0001563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3398963PMC
November 2012

Novel scabies mite serpins inhibit the three pathways of the human complement system.

PLoS One 2012 11;7(7):e40489. Epub 2012 Jul 11.

Infectious Diseases Program, Biology Department, Queensland Institute of Medical Research, Brisbane, Queensland, Australia.

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040489PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3394726PMC
April 2013

Enhanced protective efficacy of a chimeric form of the schistosomiasis vaccine antigen Sm-TSP-2.

PLoS Negl Trop Dis 2012 13;6(3):e1564. Epub 2012 Mar 13.

Queensland Tropical Health Alliance and School of Public Health and Tropical Medicine, James Cook University, Cairns, Queensland, Australia.

The large extracellular loop of the Schistosoma mansoni tetraspanin, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. Moreover, Sm-TSP-2 is uniquely recognised by IgG(1) and IgG(3) from putatively resistant individuals resident in S. mansoni endemic areas in Brazil. In the present study, we expressed Sm-TSP-2 at high yield and in soluble form in E. coli without the need for a solubility enhancing fusion partner. We also expressed in E. coli a chimera called Sm-TSP-2/5B, which consisted of Sm-TSP-2 fused to the immunogenic 5B region of the hookworm aspartic protease and vaccine antigen, Na-APR-1. Sm-TSP-2 formulated with alum/CpG showed significant reductions in adult worm and liver egg burdens in two separate murine schistosomiasis challenge studies. Sm-TSP-2/5B afforded significantly greater protection than Sm-TSP-2 alone when both antigens were formulated with alum/CpG. The enhanced protection obtained with the chimeric fusion protein was associated with increased production of anti-Sm-TSP-2 antibodies and IL-4, IL-10 and IFN-γ from spleen cells of vaccinated animals. Sera from 666 individuals from Brazil who were infected with S. mansoni were screened for potentially deleterious IgE responses to Sm-TSP-2. Anti-Sm-TSP-2 IgE to this protein was not detected (also shown previously for Na-APR-1), suggesting that the chimeric antigen Sm-TSP-2/5B could be used to safely and effectively vaccinate people in areas where schistosomes and hookworms are endemic.
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http://dx.doi.org/10.1371/journal.pntd.0001564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302818PMC
July 2012

Insights into the membrane interactions of the saposin-like proteins Na-SLP-1 and Ac-SLP-1 from human and dog hookworm.

PLoS One 2011 3;6(10):e25369. Epub 2011 Oct 3.

Parasite Cell Biology, Queensland Institute of Medical Research, Herston, Queensland, Australia.

Saposin-like proteins (SAPLIPs) from soil-transmitted helminths play pivotal roles in host-pathogen interactions and have a high potential as targets for vaccination against parasitic diseases. We have identified two non-orthologous SAPLIPs from human and dog hookworm, Na-SLP-1 and Ac-SLP-1, and solved their three-dimensional crystal structures. Both proteins share the property of membrane binding as monitored by liposome co-pelleting assays and monolayer adsorption. Neither SAPLIP displayed any significant haemolytic or bactericidal activity. Based on the structural information, as well as the results from monolayer adsorption, we propose models of membrane interactions for both SAPLIPs. Initial membrane contact of the monomeric Na-SLP-1 is most likely by electrostatic interactions between the membrane surface and a prominent basic surface patch. In case of the dimeric Ac-SLP-1, membrane interactions are most likely initiated by a unique tryptophan residue that has previously been implicated in membrane interactions in other SAPLIPs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0025369PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184995PMC
February 2012

Neutralizing antibodies to the hookworm hemoglobinase Na-APR-1: implications for a multivalent vaccine against hookworm infection and schistosomiasis.

J Infect Dis 2010 May;201(10):1561-9

Division of Infectious Diseases, Queensland Institute of Medical Research, The Australian Centre for Vaccine Development, Queensland, Australia.

The aspartic protease of Necator americanus, Na-APR-1, is a vaccine antigen that induces antibodies that neutralize hemoglobin proteolysis in the gut of the worm. To define the epitopes recognized by these antibodies, monoclonal antibodies (mAbs) were raised and assessed for neutralizing activity. Three immunoglobulin (Ig) G1 mAbs bound to the intestine of N. americanus and inhibited Na-APR-1 enzymatic activity. Overlapping fragments of Na-APR-1 were expressed, and one (APR-1/5B) was recognized by all 3 mAbs; the epitope was further characterized as AGPKAQVEAIQKY (A(291)Y). This same peptide with a Phe/Tyr(303) substitution was recognized by mAbs in APR-1 orthologues from Ancylostoma species hookworms. IgG from humans infected with hookworms did not recognize A(291)Y but, rather, recognized the S(107)L epitope. APR-1/5B was fused to other helminth vaccine antigens, including Schistosoma mansoni Sm-TSP-2 and N. americanus Na-GST-1; antibodies against both chimeras neutralized the enzymatic activity of Na-APR-1. These findings support the incorporation of Na-APR-1 into a multivalent vaccine against hookworm and/or schistosomiasis.
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http://dx.doi.org/10.1086/651953DOI Listing
May 2010

An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection.

FASEB J 2009 Sep 20;23(9):3007-19. Epub 2009 Apr 20.

Division of Infectious Diseases, Queensland Institute of Medical Research and The Australian Centre for Vaccine Development, Brisbane, Australia.

Hookworms digest hemoglobin from erythrocytes via a proteolytic cascade that begins with the aspartic protease, APR-1. Ac-APR-1 from the dog hookworm, Ancylostoma caninum, protects dogs against hookworm infection via antibodies that neutralize enzymatic activity and interrupt blood-feeding. Toward developing a human hookworm vaccine, we expressed both wild-type (Na-APR-1(wt)) and mutant (Na-APR-1(mut)-mutagenesis of the catalytic aspartic acids) forms of Na-APR-1 from the human hookworm, Necator americanus. Refolded Na-APR-1(wt) was catalytically active, and Na-APR-1(mut) was catalytically inactive but still bound substrates. Vaccination of canines with Na-APR-1(mut) and heterologous challenge with A. caninum resulted in significantly reduced parasite egg burdens (P=0.034) and weight loss (P=0.022). Vaccinated dogs also had less gut pathology, fewer adult worms, and reduced blood loss compared to controls but these did not reach statistical significance. Vaccination with Na-APR-1(mut) induced antibodies that bound the native enzyme in the parasite gut and neutralized enzymatic activity of Na-APR-1(wt) and APR-1 orthologues from three other hookworm species that infect humans. IgG1 against Na-APR-1(mut) was the most prominently detected antibody in sera from people resident in high-transmission areas for N. americanus, indicating that natural boosting may occur in exposed humans. Na-APR-1(mut) is now a lead antigen for the development of an antihematophagy vaccine for human hookworm disease.
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http://dx.doi.org/10.1096/fj.09-131433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2735369PMC
September 2009

High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever.

J Infect Dis 2002 Oct 16;186(8):1165-8. Epub 2002 Sep 16.

Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, 55 Lake Avenue N., Worcester, MA 01655, USA.

Infection with any 1 of 4 dengue viruses produces a spectrum of clinical illness ranging from a mild undifferentiated febrile illness to dengue fever (DF) to dengue hemorrhagic fever (DHF), a potentially life-threatening disease. The morbidity and mortality of DHF can be reduced by early hospitalization and careful supportive care. To determine its usefulness as a predictor of DHF, plasma levels of the secreted dengue virus nonstructural protein NS1 (sNS1) were measured daily in 32 children with dengue-2 virus infections participating in a prospective, hospital-based study. Free sNS1 levels in plasma correlated with viremia levels and were higher in patients with DHF than in those with DF. An elevated free sNS1 level (> or =600 ng/mL) within 72 h of illness onset identified patients at risk for developing DHF.
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http://dx.doi.org/10.1086/343813DOI Listing
October 2002
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