Publications
155 results found
Brown A, Selkirk ME, Sarkies P, 2023, Identification of proteins that bind extracellular microRNAs secreted by the parasitic nematode Trichinella spiralis, Biology Open, Vol: 12, ISSN: 2046-6390
Small non-coding RNAs such as microRNAs (miRNAs) are conserved across eukaryotes and play key roles in regulating gene expression. In many organisms, miRNAs are also secreted from cells, often encased within vesicles such as exosomes, and sometimes extravesicular. The mechanisms of miRNA secretion, how they are stabilised outside of cells and their functional importance are poorly understood. Recently, we characterised the parasitic nematode Trichinella spiralis as a model to study miRNA secretion. T. spiralis muscle-stage larvae (MSL) secrete abundant miRNAs which are largely extravesicular. Here, we investigated how T. spiralis miRNAs might remain stable outside of cells. Using proteomics, we identified two RNA binding proteins secreted by T. spiralis larvae and characterised their RNA binding properties. One, a homologue of the known RNA binding protein KSRP, binds miRNA in a selective and sequence-specific fashion. Another protein, which is likely a novel RNA binding protein, binds to miRNA without exhibiting sequence specificity. Our results suggest a possible mechanism for miRNA secretion by T. spiralis and may have relevance for understanding the biology of extracellular miRNA more widely.
Pillay R, Mkhize-Kwitshana ZL, Horsnell WGC, et al., 2023, Excretory-secretory products from adult helminth Nippostrongylus brasiliensis have in vitro bactericidal activity., J Med Microbiol, Vol: 72
Introduction. Intestinal helminths and microbiota share the same anatomical niche during infection and are likely to interact either directly or indirectly. Whether intestinal helminths employ bactericidal strategies that influence their microbial environment is not completely understood.Hypothesis. In the present study, the hypothesis that the adult hookworm Nippostrongylus brasiliensis produces molecules that impair bacterial growth in vitro, is tested.Aim. To investigate the in vitro bactericidal activity of Nippostrongylus brasiliensis against commensal and pathogenic bacteria.Methodology. The bactericidal effect of somatic extract and excretory-secretory products of adult Nippostrongylus brasiliensis on Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Salmonella enterica serovar Typhimurium, and Klebsiella pneumoniae) bacteria was assessed using growth assays. Minimum inhibitory concentration and minimum bactericidal concentration assays were performed using excretory-secretory products released from the pathogen.Results. Broad-spectrum in vitro bactericidal activity in excretory-secretory products, but not somatic extract of adult Nippostrongylus brasiliensis was detected. The bactericidal activity of excretory-secretory products was concentration-dependent, maintained after heat treatment, and preserved after repeated freezing and thawing.Conclusion. The results of this study demonstrate that helminths such as Nippostrongylus brasiliensis release molecules via their excretory-secretory pathway that have broad-spectrum bactericidal activity. The mechanisms responsible for this bactericidal activity remain to be determined and further studies aimed at isolating and identifying active bactericidal molecules are needed.
Hagen J, Ghosh S, Sarkies P, et al., 2023, Gene editing in the nematode parasite Nippostrongylus brasiliensis using extracellular vesicles to deliver active Cas9/guide RNA complexes, Frontiers in Parasitology, Vol: 2, Pages: 1-14, ISSN: 2813-2424
Despite recent advances, animal-parasitic nematodes have thus far been largely refractory to genetic manipulation. We describe here a new approach providing proof of principle that CRISPR/Cas9-mediated gene editing of parasitic nematodes is achievable using vesicular stomatitis virus glycoprotein-pseudotyped extracellular vesicles for the delivery of Cas9-single guide ribonucleoprotein complexes. We demonstrate that extracellular vesicle-delivered ribonucleoproteins can be used to disrupt a secreted deoxyribonuclease in Nippostrogylus brasiliensis. Introduction of a repair template encoding multiple stop codons led to measurable reduction in expression of the targeted gene. Altered transcripts corresponding to the edited locus were detected by RT-PCR, demonstrating that vesicles can access cells of tissues actively expressing the gene of interest. These data provide evidence that this technique can be employed for targeted gene editing in N. brasiliensis, making this species genetically tractable for the first time, although further refinement will be necessary for routine and robust interrogation of gene function.
Nash B, Gregory WF, White RR, et al., 2023, Large-scale proteomic analysis of T. spiralis muscle-stage ESPs identifies a novel upstream motif for in silico prediction of secreted products, Frontiers in Parasitology, Vol: 2, Pages: 1-13, ISSN: 2813-2424
The Trichinella genus contains parasitic nematodes capable of infecting a wide range of hosts including mammals, birds and reptiles. Like other helminths, T. spiralis secretes a complex mixture of bioactive molecules capable of modulating its immediate surroundings and creating a hospitable environment for growth, survival and ultimately transmission. The constitution of these excretory-secretory products (ESPs) changes depending on the tissue niche and the specific stage of parasite development. Unique to T. spiralis is a true intracellular stage wherein larvae develop inside striated myotubes. Remarkably, the parasite larvae do not destroy the host cell but rather reprogram it to support their presence and growth. This transformation is largely mediated through stage-specific secretions released into the host cell cytoplasm. In this study, we apply state of the art proteomics and computational approaches to elucidate the composition and functions of muscle-stage T. spiralis ESPs. Moreover, we define a recurring, upstream motif associated with the stichosome, the main secretory organ of this worm, and can be used to predict secreted proteins across experimentally less tractable T. spiralis life cycle stages.
Almeida-Santos J, Berkachy R, Tye CA, et al., 2022, Temporal profiling of CD4 T-cell activation and differentiation upon SARS-CoV-2 spike protein immunisation
<jats:title>ABSTRACT</jats:title><jats:p>CD4 T-cells require T-cell receptor (TCR) signalling for their activation and differentiation. Foxp3+ regulatory T-cells (Treg) are dependent on TCR signals for their differentiation and suppressive function. However, it is not fully known how TCR signalling controls the differentiation of polyclonal CD4 T-cells upon antigen recognition at the single-cell level in vivo. In this study, using Nr4a3-Tocky (<jats:underline>T</jats:underline>imer-<jats:underline>o</jats:underline>f-<jats:underline>c</jats:underline>ell-<jats:underline>k</jats:underline>inetics-and-activit<jats:underline>y</jats:underline>), which analyses temporal changes of antigen-reactive T-cells following TCR signalling, we investigated T-cell response to Spike protein fragments (S1a, S1b, S2a, and S2b) upon immunisation. We show that S1a and S2a induced the differentiation of PD1<jats:sup>hi</jats:sup>CXCR5<jats:sup>+</jats:sup> T follicular helper (Tfh) cells, which is related to CD4 T-cell immunogenicity. In contrast, S1b induced CD25<jats:sup>hi</jats:sup>GITR<jats:sup>hi</jats:sup>PD-1<jats:sup>int</jats:sup> Treg, which intermittently received TCR signalling. Using Foxp3-Tocky, which analyses <jats:italic>Foxp3</jats:italic> transcriptional dynamics, the S1b-reactive Treg sustained <jats:italic>Foxp3</jats:italic> transcription over time, which is a hallmark of activated Treg. Foxp3 fate-mapping showed that the S1b-reactive Treg were derived not from pre-existing thymic Treg, suggesting Foxp3 induction in non-Treg cells. Thus, the current study reveals temporally dynamic differentiation of CD4 T-cells and Treg upon immunisation in the polyclonal TCR repertoire.</jats:p>
Roberts L, Berkachy R, Wane M, et al., 2022, Differential regulation of allergic airway inflammation by acetylcholine, Frontiers in Immunology, Vol: 13, ISSN: 1664-3224
Acetylcholine (ACh) from neuronal and non-neuronal sources plays important roles in the regulation of immune responses and is associated with the development of several disease pathologies. We have previously demonstrated that group 2 innate lymphoid cell (ILC2)-derived ACh is required for optimal type 2 responses to parasitic infection, and therefore sought to determine whether this also plays a role in allergic inflammation. RoraCre+ChatLoxP mice (in which ILC2s cannot synthesize ACh) were exposed to an allergenic extract of the fungus Alternaria alternata, and immune responses in the airways and lung tissues analysed. Airway neutrophilia and expression of the neutrophil chemoattractants CXCL1 and CXCL2 were enhanced 24 hours after exposure, suggesting that ILC2-derived ACh plays a role in limiting excessive pulmonary neutrophilic inflammation. The effect of non-selective depletion of ACh was examined by intranasal administration of a stable parasite-secreted acetylcholinesterase. Depletion of airway ACh in this manner resulted in more profound enhancement of neutrophilia and chemokine expression, suggesting multiple cellular sources for release of ACh. In contrast, depletion of ACh inhibited Alternaria-induced activation of ILC2s, suppressing expression of IL-5, IL-13 and subsequent eosinophilia. Depletion of ACh resulted in a reduction of macrophages with an alternatively activated M2 phenotype, and an increase in M1 macrophage marker expression. These data suggest that ACh regulates allergic airway inflammation in several ways, enhancing ILC2-driven eosinophilia, but suppressing neutrophilia through reduced chemokine expression.
Culerier E, Togbe D, Maillet I, et al., 2022, Respiratory research days (J2R) (vol 39, pg 108-131, 2022), REVUE DES MALADIES RESPIRATOIRES, Vol: 39, ISSN: 0761-8425
Roberts LB, Jowett GM, Read E, et al., 2021, MicroRNA-142 is a critical regulator of group 2 innate lymphoid cell homeostasis and function, Publisher: WILEY, Pages: 42-42, ISSN: 0014-2980
Roberts LB, Jowett GM, Read E, et al., 2021, MicroRNA-142 critically regulates group 2 innate lymphoid cell homeostasis and function, Journal of Immunology, Vol: 206, Pages: 2725-2739, ISSN: 0022-1767
Innate lymphoid cells are central to the regulation of immunity at mucosal barrier sites, with group 2 innate lymphoid cells (ILC2s) being particularly important in type 2 immunity. In this study, we demonstrate that microRNA(miR)-142 plays a critical, cell-intrinsic role in the homeostasis and function of ILC2s. Mice deficient for miR-142 expression demonstrate an ILC2 progenitor–biased development in the bone marrow, and along with peripheral ILC2s at mucosal sites, these cells display a greatly altered phenotype based on surface marker expression. ILC2 proliferative and effector functions are severely dysfunctional following Nippostrongylus brasiliensis infection, revealing a critical role for miR-142 isoforms in ILC2-mediated immune responses. Mechanistically, Socs1 and Gfi1 expression are regulated by miR-142 isoforms in ILC2s, impacting ILC2 phenotypes as well as the proliferative and effector capacity of these cells. The identification of these novel pathways opens potential new avenues to modulate ILC2-dependent immune functions.
Darby M, Roberts LB, Mackowiak C, et al., 2021, ILC3-derived acetylcholine promotes protease-driven allergic lung pathology, Journal of Allergy and Clinical Immunology, Vol: 147, Pages: 1513-1516.E4, ISSN: 0091-6749
Roberts LB, Schnoeller C, Berkachy R, et al., 2021, Acetylcholine production by group 2 innate lymphoid cells promotes mucosal immunity to helminths, Science Immunology, Vol: 6, ISSN: 2470-9468
Innate lymphoid cells (ILCs) are critical mediators of immunological and physiological responses at mucosal barrier sites. Whereas neurotransmitters can stimulate ILCs, the synthesis of small-molecule neurotransmitters by these cells has only recently been appreciated. Group 2 ILCs (ILC2s) are shown here to synthesize and release acetylcholine (ACh) during parasitic nematode infection. The cholinergic phenotype of pulmonary ILC2s was associated with their activation state, could be induced by in vivo exposure to extracts of Alternaria alternata or the alarmin cytokines interleukin-33 (IL-33) and IL-25, and was augmented by IL-2 in vitro. Genetic disruption of ACh synthesis by murine ILC2s resulted in increased parasite burdens, lower numbers of ILC2s, and reduced lung and gut barrier responses to Nippostrongylus brasiliensis infection. These data demonstrate a functional role for ILC2-derived ACh in the expansion of ILC2s for maximal induction of type 2 immunity.
Hagen J, Sarkies P, Selkirk ME, 2021, Lentiviral transduction facilitates RNA interference in the nematode parasite Nippostrongylus brasiliensis, PLoS Pathogens, Vol: 17, ISSN: 1553-7366
Animal-parasitic nematodes have thus far been largely refractory to genetic manipulation, and methods employed to effect RNA interference (RNAi) have been ineffective or inconsistent in most cases. We describe here a new approach for genetic manipulation of Nippostrongylus brasiliensis, a widely used laboratory model of gastrointestinal nematode infection. N. brasiliensis was successfully transduced with Vesicular Stomatitis Virus glycoprotein G (VSV-G)-pseudotyped lentivirus. The virus was taken up via the nematode intestine, RNA reverse transcribed into proviral DNA, and transgene transcripts produced stably in infective larvae, which resulted in expression of the reporter protein mCherry. Improved transgene expression was achieved by incorporating the C. elegans hlh11 promoter and the tbb2 3´-UTR into viral constructs. MicroRNA-adapted short hairpin RNAs delivered in this manner were processed correctly and resulted in partial knockdown of β-tubulin isotype-1 (tbb-iso-1) and secreted acetylcholinesterase B (ache-B). The system was further refined by lentiviral delivery of double stranded RNAs, which acted as a trigger for RNAi following processing and generation of 22G-RNAs. Virus-encoded sequences were detectable in F1 eggs and third stage larvae, demonstrating that proviral DNA entered the germline and was heritable. Lentiviral transduction thus provides a new means for genetic manipulation of parasitic nematodes, including gene silencing and expression of exogenous genes.
Berkachy R, Smyth DJ, Schnoeller C, et al., 2021, Characterisation of the secreted apyrase family of Heligmosomoides polygyrus, International Journal for Parasitology, Vol: 51, Pages: 39-48, ISSN: 0020-7519
Apyrases are a recurrent feature of secretomes from numerous species of parasitic nematodes. Here we characterise the five apyrases secreted by Heligmosomoides polygyrus, a natural parasite of mice and a widely used laboratory model for intestinal nematode infection. All five enzymes are closely related to soluble calcium-activated nucleotidases described in a variety of organisms, and distinct from the CD39 family of ecto-nucleotidases. Expression is maximal in adult worms and restricted to adults and L4s. Recombinant apyrases were produced and purified from Pichia pastoris. The five enzymes showed very similar biochemical properties, with strict calcium dependence and a broad substrate specificity, catalysing the hydrolysis of all nucleoside tri- and diphosphates, with no activity against nucleoside monophosphates. Natural infection of mice provoked very low antibodies to any enzyme, but immunisation with an apyrase cocktail showed partial protection against reinfection, with reduced egg output and parasite recovery. The most likely role for nematode secreted apyrases is hydrolysis of extracellular ATP, which acts as an alarmin for cellular release of IL-33 and initiation of type 2 immunity.
de Lange A, Prodjinotho UF, Tomes H, et al., 2020, Taenia larvae possess distinct acetylcholinesterase profiles with implications for host cholinergic signalling, PLoS Neglected Tropical Diseases, Vol: 14, Pages: 1-21, ISSN: 1935-2727
Larvae of the cestodes Taenia solium and Taenia crassiceps infect the central nervous system of humans. Taenia solium larvae in the brain cause neurocysticercosis, the leading cause of adult-acquired epilepsy worldwide. Relatively little is understood about how cestode-derived products modulate host neural and immune signalling. Acetylcholinesterases, a class of enzyme that breaks down acetylcholine, are produced by a host of parasitic worms to aid their survival in the host. Acetylcholine is an important signalling molecule in both the human nervous and immune systems, with powerful modulatory effects on the excitability of cortical networks. Therefore, it is important to establish whether cestode derived acetylcholinesterases may alter host neuronal cholinergic signalling. Here we make use of multiple techniques to profile acetylcholinesterase activity in different extracts of both Taenia crassiceps and Taenia solium larvae. We find that the larvae of both species contain substantial acetylcholinesterase activity. However, acetylcholinesterase activity is lower in Taenia solium as compared to Taenia crassiceps larvae. Further, whilst we observed acetylcholinesterase activity in all fractions of Taenia crassiceps larvae, including on the membrane surface and in the excreted/secreted extracts, we could not identify acetylcholinesterases on the membrane surface or in the excreted/secreted extracts of Taenia solium larvae. Bioinformatic analysis revealed conservation of the functional protein domains in the Taenia solium acetylcholinesterases, when compared to the homologous human sequence. Finally, using whole-cell patch clamp recordings in rat hippocampal brain slice cultures, we demonstrate that Taenia larval derived acetylcholinesterases can break down acetylcholine at a concentration which induces changes in neuronal signalling. Together, these findings highlight the possibility that Taenia larval acetylcholinesterases can interfere with cholinergic signalling in
Srey MT, Taccogna A, Oksov Y, et al., 2020, Vaccination with novel low-molecular weight proteins secreted from Trichinella spiralis inhibits establishment of infection, PLoS Neglected Tropical Diseases, Vol: 14, ISSN: 1935-2727
Trichinella spiralis muscle stage larvae (mL1) produce excretory-secreted products (ESPs), a complex mixture of protein, which are believed to be important for establishing or maintaining an infection niche within skeletal muscle and the intestine. Studies of both whole ESPs and individual cloned proteins have shown that some ESPs are potent immunogens capable of eliciting protective immune responses. Here we describe two novel proteins, Secreted from Muscle stage Larvae SML-4 and SML-5 which are 15 kDa and 12 kDa respectively. The genes encoding these proteins are highly conserved within the Trichinellids, are constituents of mL1 ESP and localized in the parasite stichosome. While SML-5 is only expressed in mL1 and early stages of adult nematode development, SML-4 is a tyvosylated glycoprotein also produced by adult nematodes, indicating it may have a function in the enteral phase of the infection. Vaccination with these proteins resulted in an impaired establishment of adult stages and consequently a reduction in the burden of mL1 in BALB/c mice. This suggests that both proteins may be important for establishment of parasite infection of the intestine and are prophylactic vaccine candidates.
Taylor PJ, Hagen J, Faruqu FN, et al., 2020, Trichinella spiralis secretes abundant unencapsulated small RNAs with potential effects on host gene expression, International Journal for Parasitology, Vol: 50, Pages: 697-705, ISSN: 0020-7519
Many organisms, including parasitic nematodes, secrete small RNAs into the extracellular environment, largely encapsulated within small vesicles. Parasite-secreted material often contains microRNAs (miRNAs), raising the possibility that they might regulate host genes in target cells. Here we characterise secreted RNAs from the parasitic nematode Trichinella spiralis at two different life stages. We show that adult T. spiralis, which inhabit intestinal mucosa, secrete miRNAs within vesicles. Unexpectedly, T. spiralis muscle stage larvae, which live intracellularly within skeletal muscle cells, secrete miRNAs that appear not to be encapsulated. Notably, secreted miRNAs include a homologue of mammalian miRNA-31, which has an important role in muscle development. Our work therefore suggests that RNAs may be secreted without encapsulation in vesicles, with implications for the biology of T. spiralis infection.
Rolot M, Dougall AM, Chetty A, et al., 2018, Helminth-induced IL-4 expands bystander memory CD8(+) T cells for early control of viral infection, Nature Communications, Vol: 9, ISSN: 2041-1723
Infection with parasitic helminths can imprint the immune system to modulate bystander inflammatory processes. Bystander or virtual memory CD8+ T cells (TVM) are non-conventional T cells displaying memory properties that can be generated through responsiveness to interleukin (IL)-4. However, it is not clear if helminth-induced type 2 immunity functionally affects the TVM compartment. Here, we show that helminths expand CD44hiCD62LhiCXCR3hiCD49dlo TVM cells through direct IL-4 signaling in CD8+ T cells. Importantly, helminth-mediated conditioning of TVM cells provided enhanced control of acute respiratory infection with the murid gammaherpesvirus 4 (MuHV-4). This enhanced control of MuHV-4 infection could further be explained by an increase in antigen-specific CD8+ T cell effector responses in the lung and was directly dependent on IL-4 signaling. These results demonstrate that IL-4 during helminth infection can non-specifically condition CD8+ T cells, leading to a subsequently raised antigen-specific CD8+ T cell activation that enhances control of viral infection.
Selkirk ME, Davis RE, Gounaris K, et al., 2018, Special issue: molecular and cellular biology of helminth parasites XI Preface, International Journal for Parasitology, Vol: 48, Pages: 319-320, ISSN: 0020-7519
Rosic S, Amouroux R, Requena C, et al., 2018, Evolutionary analysis indicates that DNA alkylation damage is a byproduct of cytosine DNA methyltransferase activity, Nature Genetics, Vol: 50, Pages: 452-459, ISSN: 1061-4036
Methylation at the 5 position of cytosine in DNA (5meC) is a key epigenetic mark in eukaryotes. Once introduced, 5meC can be maintained through DNA replication by the activity of ‘maintenance’ DNA methyltransferases (DNMTs). Despite their ancient origin, DNA methylation pathways differ widely across animals, such that 5meC is either confined to transcribed genes or lost altogether in several lineages. We used comparative epigenomics to investigate the evolution of DNA methylation. Although the model nematode Caenorhabditis elegans lacks DNA methylation, more basal nematodes retain cytosine DNA methylation, which is targeted to repeat loci. We found that DNA methylation coevolved with the DNA alkylation repair enzyme ALKB2 across eukaryotes. In addition, we found that DNMTs introduced the toxic lesion 3-methylcytosine into DNA both in vitro and in vivo. Alkylation damage is therefore intrinsically associated with DNMT activity, and this may promote the loss of DNA methylation in many species.
Chatonnet A, Lenfant N, Marchot P, et al., 2017, Natural genomic amplification of cholinesterase genes in animals., Journal of Neurochemistry, Vol: 142, Pages: 73-81, ISSN: 1471-4159
Tight control of the concentration of acetylcholine at cholinergic synapses requires precise regulation of the number and state of the acetylcholine receptors, and of the synthesis and degradation of the neurotransmitter. In particular, the cholinesterase activity has to be controlled exquisitely. In the genome of the first experimental models used (man, mouse, zebrafish and drosophila), there are only one or two genes coding for cholinesterases, whereas there are more genes for their closest relatives the carboxylesterases. Natural amplification of cholinesterase genes was first found to occur in some cancer cells and in insect species subjected to evolutionary pressure by insecticides. Analysis of the complete genome sequences of numerous representatives of the various metazoan phyla show that moderate amplification of cholinesterase genes is not uncommon in molluscs, echinoderms, hemichordates, prochordates or lepidosauria. Amplification of acetylcholinesterase genes is also a feature of parasitic nematodes or ticks. In these parasites, over-production of cholinesterase-like proteins in secreted products and the saliva are presumed to have effector roles related to host infection. These amplification events raise questions about the role of the amplified gene products, and the adaptation processes necessary to preserve efficient cholinergic transmission.
White RR, Ponsford AH, Weekes MP, et al., 2016, Ubiquitin-Dependent Modification of Skeletal Muscle by the Parasitic Nematode, Trichinella spiralis, PLoS Pathogens, Vol: 12, ISSN: 1553-7366
Trichinella spiralis is a muscle-specific parasitic worm that is uniquely intracellular. T. spiralisreprograms terminally differentiated skeletal muscle cells causing them to de-differentiateand re-enter the cell cycle, a process that cannot occur naturally in mammalian skeletalmuscle cells, but one that holds great therapeutic potential. Although the host ubiquitin pathwayis a common target for viruses and bacteria during infection, its role in parasite pathogenesishas been largely overlooked. Here we demonstrate that the secreted proteins of T.spiralis contain E2 Ub-conjugating and E3 Ub-ligase activity. The E2 activity is attributed toTsUBE2L3, a novel and conserved T. spiralis enzyme located in the secretory organ of theparasite during the muscle stages of infection. TsUBE2L3 cannot function with any T.spiralissecreted E3, but specifically binds to a panel of human RING E3 ligases, including the RBRE3 ARIH2 with which it interacts with a higher affinity than the mammalian ortholog UbcH7/UBE2L3. Expression of TsUBE2L3 in skeletal muscle cells causes a global downregulationin protein ubiquitination, most predominantly affecting motor, sarcomeric and extracellularmatrix proteins, thus mediating their stabilization with regards to proteasomal degradation.This effect is not observed in the presence of the mammalian ortholog, suggesting functionaldivergence in the evolution of the parasite protein. These findings demonstrate the firstexample of host-parasite interactions via a parasite-derived Ub conjugating enzyme; an E2that demonstrates a novel muscle protein stabilization function.
Vaux R, Schnoeller C, Berkachy R, et al., 2016, Modulation of the immune response by nematode secreted acetylcholinesterase revealed by heterologous expression in Trypanosoma musculi, PLOS Pathogens, Vol: 12, Pages: 1-18, ISSN: 1553-7366
Nematode parasites secrete molecules which regulate the mammalian immune system, but their genetic intractability is a major impediment to identifying and characterising the biological effects of these molecules. We describe here a novel system for heterologous expression of helminth secreted proteins in the natural parasite of mice, Trypanosoma musculi, which can be used to analyse putative immunomodulatory functions. Trypanosomes were engineered to express a secreted acetylcholinesterase from Nippostrongylus brasiliensis. Infection of mice with transgenic parasites expressing acetylcholinesterase resulted in truncated infection, with trypanosomes cleared early from the circulation. Analysis of cellular phenotypes indicated that exposure to acetylcholinesterase in vivo promoted classical activation of macrophages (M1), with elevated production of nitric oxide and lowered arginase activity. This most likely occurred due to the altered cytokine environment, as splenocytes from mice infected with T. musculi expressing acetylcholinesterase showed enhanced production of IFNγ and TNFα, with diminished IL-4, IL-13 and IL-5. These results suggest that one of the functions of nematode secreted acetylcholinesterase may be to alter the cytokine environment in order to inhibit development of M2 macrophages which are deleterious to parasite survival. Transgenic T. musculi represents a valuable new vehicle to screen for novel immunoregulatory proteins by extracellular delivery in vivo to the murine host.
Hunt VL, Tsai IJ, Selkirk ME, et al., 2016, The genome of Strongyloides spp. gives insights into protein families with a putative role in nematode parasitism., Parasitology, Vol: 144, Pages: 343-358, ISSN: 1469-8161
Parasitic nematodes are important and abundant parasites adapted to live a parasitic lifestyle, with these adaptations all aimed at facilitating their survival and reproduction in their hosts. The recently sequenced genomes of four Strongyloides species, gastrointestinal parasites of humans and other animals, alongside transcriptomic and proteomic analysis of free-living and parasitic stages of their life cycles have revealed a number of protein families with a putative role in their parasitism. Many of these protein families have also been associated with parasitism in other parasitic nematode species, suggesting that these proteins may play a fundamental role in nematode parasitism more generally. Here, we review key protein families that have a putative role in Strongyloides' parasitism - acetylcholinesterases, astacins, aspartic proteases, prolyl oligopeptidases, proteinase inhibitors (trypsin inhibitors and cystatins), SCP/TAPS and transthyretin-like proteins - and the evidence for their key, yet diverse, roles in the parasitic lifestyle.
Schuijs MJ, Hartmann S, Selkirk ME, et al., 2016, The Helminth-Derived Immunomodulator AvCystatin Reduces Virus Enhanced Inflammation by Induction of Regulatory IL-10+ T Cells., PLOS One, Vol: 11, ISSN: 1932-6203
Respiratory Syncytial Virus (RSV) is a major pathogen causing low respiratory tract disease (bronchiolitis), primarily in infants. Helminthic infections may alter host immune responses to both helminths and to unrelated immune triggers. For example, we have previously shown that filarial cystatin (AvCystatin/Av17) ameliorates allergic airway inflammation. However, helminthic immunomodulators have so far not been tested in virus-induced disease. We now report that AvCystatin prevents Th2-based immunopathology in vaccine-enhanced RSV lung inflammation, a murine model for bronchiolitis. AvCystatin ablated eosinophil influx, reducing both weight loss and neutrophil recruitment without impairing anti-viral immune responses. AvCystatin also protected mice from excessive inflammation following primary RSV infection, significantly reducing neutrophil influx and cytokine production in the airways. Interestingly, we found that AvCystatin induced an influx of CD4+ FoxP3+ interleukin-10-producing T cells in the airway and lungs, correlating with immunoprotection, and the corresponding cells could also be induced by adoptive transfer of AvCystatin-primed F4/80+ macrophages. Thus, AvCystatin ameliorates enhanced RSV pathology without increasing susceptibility to, or persistence of, viral infection and warrants further investigation as a possible therapy for virus-induced airway disease.
Thawer S, Auret J, Schnoeller C, et al., 2016, Surfactant Protein-D Is Essential for Immunity to Helminth Infection., PLOS Pathogens, Vol: 12, ISSN: 1553-7366
Pulmonary epithelial cell responses can enhance type 2 immunity and contribute to control of nematode infections. An important epithelial product is the collectin Surfactant Protein D (SP-D). We found that SP-D concentrations increased in the lung following Nippostrongylus brasiliensis infection; this increase was dependent on key components of the type 2 immune response. We carried out loss and gain of function studies of SP-D to establish if SP-D was required for optimal immunity to the parasite. N. brasiliensis infection of SP-D-/- mice resulted in profound impairment of host innate immunity and ability to resolve infection. Raising pulmonary SP-D levels prior to infection enhanced parasite expulsion and type 2 immune responses, including increased numbers of IL-13 producing type 2 innate lymphoid cells (ILC2), elevated expression of markers of alternative activation by alveolar macrophages (alvM) and increased production of the type 2 cytokines IL-4 and IL-13. Adoptive transfer of alvM from SP-D-treated parasite infected mice into naïve recipients enhanced immunity to N. brasiliensis. Protection was associated with selective binding by the SP-D carbohydrate recognition domain (CRD) to L4 parasites to enhance their killing by alvM. These findings are the first demonstration that the collectin SP-D is an essential component of host innate immunity to helminths.
Sarkies P, Selkirk M E, Jones J T, et al., 2015, Ancient and novel small RNA pathways compensate for the loss of piRNAs in multiple independent nematode lineages, PLoS Biology, Vol: 13, ISSN: 1544-9173
Culley FJ, Darby M, Vira A, et al., 2015, The M3 muscarinic receptor Is required for optimal adaptive immunity to Helminth and bacterial infection, Plos Pathogens, Vol: 11, ISSN: 1553-7374
Innate immunity is regulated by cholinergic signalling through nicotinic acetylcholine receptors. We show here that signalling through the M3 muscarinic acetylcholine receptor (M3R) plays an important role in adaptive immunity to both Nippostrongylus brasiliensis and Salmonella enterica serovar Typhimurium, as M3R-/- mice were impaired in their ability to resolve infection with either pathogen. CD4 T cell activation and cytokine production were reduced in M3R-/- mice. Immunity to secondary infection with N. brasiliensis was severely impaired, with reduced cytokine responses in M3R-/- mice accompanied by lower numbers of mucus-producing goblet cells and alternatively activated macrophages in the lungs. Ex vivo lymphocyte stimulation of cells from intact BALB/c mice infected with N. brasiliensis and S. typhimurium with muscarinic agonists resulted in enhanced production of IL-13 and IFN-γ respectively, which was blocked by an M3R-selective antagonist. Our data therefore indicate that cholinergic signalling via the M3R is essential for optimal Th1 and Th2 adaptive immunity to infection.
Rashidi NM, Scott MK, Scherf N, et al., 2014, In vivo time-lapse imaging shows diverse niche engagement by quiescent and naturally activated hematopoietic stem cells, BLOOD, Vol: 124, Pages: 79-83, ISSN: 0006-4971
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Selkirk ME, Huang SC, Knox DP, et al., 2012, The development of RNA interference (RNAi) in gastrointestinal nematodes, PARASITOLOGY, Vol: 139, Pages: 605-612, ISSN: 0031-1820
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Pezzementi L, Krejci E, Chatonnet A, et al., 2012, A tetrameric acetylcholinesterase from the parasitic nematode <i>Dictyocaulus viviparus</i> associates with the vertebrate tail proteins PRiMA and ColQ, MOLECULAR AND BIOCHEMICAL PARASITOLOGY, Vol: 181, Pages: 40-48, ISSN: 0166-6851
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