Solution structure of the N-terminal extension domain of a Schistosoma japonicum asparaginyl-tRNA synthetase.

Several secreted proteins from helminths (parasitic worms) have been shown to have immunomodulatory activities. Asparaginyl-tRNA synthetases are abundantly secreted in the filarial nematode Brugia malayi (BmAsnRS) and the parasitic flatworm Schistosoma japonicum (SjAsnRS), indicating a possible immune function. The suggestion is supported by BmAsnRS alleviating disease symptoms in a T-cell transfer mouse model of colitis. This immunomodulatory function is potentially related to an N-terminal extension domain present in eukaryotic AsnRS proteins but few structure/function studies have been done on this domain. Here we have determined the three-dimensional solution structure of the N-terminal extension domain of SjAsnRS. A protein containing the 114 N-terminal amino acids of SjAsnRS was recombinantly expressed with isotopic labelling to allow structure determination using 3D NMR spectroscopy, and analysis of dynamics using NMR relaxation experiments. Structural comparisons of the N-terminal extension domain of SjAsnRS with filarial and human homologues highlight a high degree of variability in the ß-hairpin region of these eukaryotic N-AsnRS proteins, but similarities in the disorder of the C-terminal regions. Limitations in PrDOS-based intrinsically disordered region (IDR) model predictions were also evident in this comparison. Empirical structural data such as that presented in our study for N-SjAsnRS will enhance the prediction of sequence-homology based structure modelling and prediction of IDRs in the future.Communicated by Ramaswamy H. Sarma.

Peptides derived from hookworm anti-inflammatory proteins suppress inducible colitis in mice and inflammatory cytokine production by human cells.

A decline in the prevalence of parasites such as hookworms appears to be correlated with the rise in non-communicable inflammatory conditions in people from high- and middle-income countries. This correlation has led to studies that have identified proteins produced by hookworms that can suppress inflammatory bowel disease (IBD) and asthma in animal models. Hookworms secrete a family of abundant netrin-domain containing proteins referred to as AIPs (Anti-Inflammatory Proteins), but there is no information on the structure-function relationships. Here we have applied a downsizing approach to the hookworm AIPs to derive peptides of 20 residues or less, some of which display anti-inflammatory effects when co-cultured with human peripheral blood mononuclear cells and oral therapeutic activity in a chemically induced mouse model of acute colitis. Our results indicate that a conserved helical region is responsible, at least in part, for the anti-inflammatory effects. This helical region has potential in the design of improved leads for treating IBD and possibly other inflammatory conditions.

Novel antiinflammatory biologics shaped by parasite-host coevolution.

Parasitic helminth infections, while a major cause of neglected tropical disease burden, negatively correlate with the incidence of immune-mediated inflammatory diseases such as inflammatory bowel diseases (IBD). To evade expulsion, helminths have developed sophisticated mechanisms to regulate their host's immune responses. Controlled experimental human helminth infections have been assessed clinically for treating inflammatory conditions; however, such a radical therapeutic modality has challenges. An alternative approach is to harness the immunomodulatory properties within the worm's excretory-secretory (ES) complement, its secretome. Here, we report a biologics discovery and validation pipeline to generate and screen in vivo a recombinant cell-free secretome library of helminth-derived immunomodulatory proteins. We successfully expressed 78 recombinant ES proteins from gastrointestinal hookworms and screened the crude in vitro translation reactions for anti-IBD properties in a mouse model of acute colitis. After statistical filtering and ranking, 20 proteins conferred significant protection against various parameters of colitis. Lead candidates from distinct protein families, including annexins, transthyretins, nematode-specific retinol-binding proteins, and SCP/TAPS were identified. Representative proteins were produced in mammalian cells and further validated, including ex vivo suppression of inflammatory cytokine secretion by T cells from IBD patient colon biopsies. Proteins identified herein offer promise as novel, safe, and mechanistically differentiated biologics for treating the globally increasing burden of inflammatory diseases.

Na-AIP-1 secreted by human hookworms suppresses collagen-induced arthritis.

Proteins from helminths have been posed as new immunomodulatory agents with exciting potential in the treatment of immune-mediated diseases including rheumatoid arthritis (RA). In this study we assess the effects of a helminthic excretory/secretory (ES) protein Na-AIP-1 as monotherapy and in combination with methotrexate (MTX) in the well-described collagen-induced arthritis (CIA) model of RA. CIA was induced in DBA/1 J mice which were treated after the onset of arthritis with Na-AIP-1 monotherapy, MTX or Na-AIP-1 + MTX. The clinical scores for weight, arthritis and paw width were recorded along with joint histology as outcome measures. For the clinical parameters of weight, paw score and paw width, none of the Na-AIP-1 monotherapy, MTX therapy or Na-AIP-1 + MTX combination therapy groups displayed any significant difference when compared to the arthritis control. However, a significant reduction in histological score was identified after both monotherapy (Na-AIP-1: 0.83 ± 0.24 vs Arthritis control: 5.58 ± 1.49, p = 0.0277) and combination therapy (Na-AIP-1 + MTX: 0.55 ± 0.28 vs Arthritis control: 5.58 ± 1.49, p = 0.0233) when compared to arthritis control. Furthermore, Na-AIP-1 as both monotherapy (Na-AIP-1: 0.83 ± 0.24 vs MTX: 5.73 ± 1.82 p = 0.0261) and combination therapy (Na-AIP-1 + MTX: 0.55 ± 0.28 vs MTX: 5.73 ± 1.82, p = 0.0221) also significantly reduced histological score when compared to MTX monotherapy. Na-AIP-1 significantly reduced joint pathology in CIA. The hookworm protein Na-AIP-1 seems to be effective in the treatment of RA as monotherapy and when dosed together with MTX, constituting a potential new candidate for drug development. Research should focus on elucidating the mechanism of Na-AIP-1 action as a means to identify novel targets for therapeutics and to further our current understanding of immunobiology in RA.

Monoclonal Antibodies Targeting an Opisthorchis viverrini Extracellular Vesicle Tetraspanin Protect Hamsters against Challenge Infection.

Opisthorchis viverrini causes severe pathology in the bile ducts of infected human hosts, and chronic infection can culminate in bile duct cancer. The prevention of infection by vaccination would decrease opisthorchiasis-induced morbidity and mortality. The tetraspanin protein, Ov-TSP-2, is located on the membrane of secreted extracellular vesicles (EVs), and is a candidate antigen for inclusion in a subunit vaccine. To address the role of anti-Ov-TSP-2 antibodies in protection, we assessed the protective capacity of anti-Ov-TSP-2 monoclonal antibodies (mAbs) against opisthorchiasis. Two anti-TSP-2 IgM mAbs, 1D6 and 3F5, and an isotype control were passively transferred to hamsters, followed by parasite challenge one day later. Hamsters that received 3F5 had 74.5% fewer adult flukes and 67.4% fewer eggs per gram of feces compared to hamsters that received the control IgM. Both 1D6 and 3F5 (but not the control IgM) blocked the uptake of fluke EVs by human bile duct epithelial cells in vitro. This is the first report of passive immunization against human liver fluke infection, and the findings portend the feasibility of antibody-directed therapies for liver fluke infection, bolstering the selection of TSPs as components of a subunit vaccine for opisthorchiasis and fluke infections generally.

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

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.

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

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 Schistosoma haematobium 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 Sm-TSP2, Sm23, 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 (S. mansoni) 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.

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

Schistosomiasis is a neglected tropical disease caused by parasitic blood flukes of the genus Schistosoma, 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 Schistosoma mansoni 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 in vitro with anti-schistosome cholinesterase (SmChE) IgG, both schistosomula and adult worms displayed significantly decreased ChE activity, which eventually resulted in parasite death. Vaccination with individual SmChEs, or a combination of all three SmChEs, 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 SmAChE1 in trial 1. Egg viability, as determined by egg hatching from liver homogenates, was significantly reduced in the groups vaccinated with the SmChE cocktail (40%) and SmAChE2 (46%). Furthermore, surviving worms from each vaccinated group were significantly stunted and depleted of glycogen stores, compared to controls. These results suggest that SmChEs could be incorporated into a vaccine against schistosomiasis to reduce the pathology and transmission of this debilitating disease.

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

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.

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

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.

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

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.

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

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-1. 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)-ß 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.

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

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 (Tregs) 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 Treg 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.

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

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.

Extracellular vesicles secreted by Schistosoma mansoni contain protein vaccine candidates.

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.

Lipopeptide Nanoparticles: Development of Vaccines against Hookworm Parasite.

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 ß-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.

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

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.

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

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.

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

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.

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

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.