A genome-scale metabolic model of parasitic whipworm.

Genome-scale metabolic models are widely used to enhance our understanding of metabolic features of organisms, host-pathogen interactions and to identify therapeutics for diseases. Here we present iTMU798, the genome-scale metabolic model of the mouse whipworm Trichuris muris. The model demonstrates the metabolic features of T. muris and allows the prediction of metabolic steps essential for its survival. Specifically, that Thioredoxin Reductase (TrxR) enzyme is essential, a prediction we validate in vitro with the drug auranofin. Furthermore, our observation that the T. muris genome lacks gsr-1 encoding Glutathione Reductase (GR) but has GR activity that can be inhibited by auranofin indicates a mechanism for the reduction of glutathione by the TrxR enzyme in T. muris. In addition, iTMU798 predicts seven essential amino acids that cannot be synthesised by T. muris, a prediction we validate for the amino acid tryptophan. Overall, iTMU798 is as a powerful tool to study not only the T. muris metabolism but also other Trichuris spp. in understanding host parasite interactions and the rationale design of new intervention strategies.

High-fat diet-induced resistance to helminth infection via alternative induction of type 2 immunity.

Gastrointestinal nematode infections cause morbidity and socioeconomic loss in the most deprived communities. The shift in the context of obesity has led to spatial overlap with endemic gastrointestinal nematode regions resulting in the emergence of a novel comorbidity. Despite this, the impact of a high-fat diet (HFD) on immune-regulated protection against gastrointestinal infections remains largely unknown. We employed the murine model of nematode infection, Trichuris muris, to investigate the effect of an HFD on the immune response against chronic infection. Surprisingly, diet-induced obesity drove parasite expulsion in both single and repeated trickle low doses of T. muris eggs. Mechanistically, an HFD increased the expression of the ST2 receptor on CD4+ T cells, priming an enhanced type 2 helper T (Th2) cell cytokine production following interleukin (IL)-33 stimulation ex vivo. Despite IL-33-/- mice demonstrating that IL-33 is not critical for host protective immunity to T. muris under a conventional diet, HFD-fed T-cell deplete mice adoptively transferred with ST2-/- CD4 T cells were unable to expel a T. muris infection unlike those transferred with ST2-sufficient cells. Collectively, this study demonstrates that an HFD primes CD4+ T cells to utilize the IL-33-ST2 axis in a novel induction of type 2 immunity, providing insights into the emerging comorbidities of obesity and nematode infection.

Defining the early stages of intestinal colonisation by whipworms.

Whipworms are large metazoan parasites that inhabit multi-intracellular epithelial tunnels in the large intestine of their hosts, causing chronic disease in humans and other mammals. How first-stage larvae invade host epithelia and establish infection remains unclear. Here we investigate early infection events using both Trichuris muris infections of mice and murine caecaloids, the first in-vitro system for whipworm infection and organoid model for live helminths. We show that larvae degrade mucus layers to access epithelial cells. In early syncytial tunnels, larvae are completely intracellular, woven through multiple live dividing cells. Using single-cell RNA sequencing of infected mouse caecum, we reveal that progression of infection results in cell damage and an expansion of enterocytes expressing of Isg15, potentially instigating the host immune response to the whipworm and tissue repair. Our results unravel intestinal epithelium invasion by whipworms and reveal specific host-parasite interactions that allow the whipworm to establish its multi-intracellular niche.

Trichuris muris and comorbidities - within a mouse model context.

Trichuris muris is a mouse intestinal parasitic nematode that inhabits the large intestine of its host and induces a strong immune response. The effects of this strong anti-parasite response can be found locally within the intestinal niche and also systemically, having effects on multiple organs. Additionally, the anti-parasite response can have multiple effects on infectious organisms and on microbiota that the host is harbouring. It has been shown that Th1 responses induced by T. muris can affect progression of bowel inflammation, cause colitic-like intestinal inflammation, reduce barrier function and intestinal mucosal responses. In the brain, T. muris can exacerbate stroke outcome and other neurological conditions. In the lung, T. muris can suppress airway inflammation and alter immune responses to other parasites. Additionally, T. muris induced responses can inhibit anti-tumour immunity. Although this parasite maintains a localized niche in the large intestine, its effects can be far-reaching and substantially impact other infections through modulation of bystander immune responses.

Extracellular vesicles from Heligmosomoides bakeri and Trichuris muris contain distinct microRNA families and small RNAs that could underpin different functions in the host.

Extracellular vesicles (EVs) have emerged as a ubiquitous component of helminth excretory-secretory products that can deliver parasite molecules to host cells to elicit immunomodulatory effects. RNAs are one type of cargo molecule that can underpin EV functions, hence there is extensive interest in characterising the RNAs that are present in EVs from different helminth species. Here we outline methods for identifying all of the small RNAs (sRNA) in helminth EVs and address how different methodologies may influence the sRNAs detected. We show that different EV purification methods introduce relatively little variation in the sRNAs that are detected, and that different RNA library preparation methods yielded larger differences. We compared the EV sRNAs in the gastrointestinal nematode Heligmosomoides bakeri with those in EVs from the distantly related gastrointestinal nematode Trichuris muris, and found that many of the sRNAs in both organisms derive from repetitive elements or intergenic regions. However, only in H. bakeri do these RNAs contain a 5' triphosphate, and Guanine (G) starting nucleotide, consistent with their biogenesis by RNA-dependent RNA polymerases (RdRPs). Distinct microRNA (miRNA) families are carried in EVs from each parasite, with H. bakeri EVs specific for miR-71, miR-49, miR-63, miR-259 and miR-240 gene families, and T. muris EVs specific for miR-1, miR-1822 and miR-252, and enriched for miR-59, miR-72 and miR-44 families, with the miR-9, miR-10, miR-80 and let-7 families abundant in both. We found a larger proportion of miRNA reads derive from the mouse host in T. muris EVs, compared with H. bakeri EVs. Our report underscores potential biases in the sRNAs sequenced based on library preparation methods, suggests specific nematode lineages have evolved distinct sRNA synthesis/export pathways, and highlights specific differences in EV miRNAs from H. bakeri and T. muris that may underpin functional adaptation to their host niches.

ILC2s mediate systemic innate protection by priming mucus production at distal mucosal sites.

Host immunity to parasitic nematodes requires the generation of a robust type 2 cytokine response, characterized by the production of interleukin 13 (IL-13), which drives expulsion. Here, we show that infection with helminths in the intestine also induces an ILC2-driven, IL-13-dependent goblet cell hyperplasia and increased production of mucins (Muc5b and Muc5ac) at distal sites, including the lungs and other mucosal barrier sites. Critically, we show that type 2 priming of lung tissue through increased mucin production inhibits the progression of a subsequent lung migratory helminth infection and limits its transit through the airways. These data show that infection by gastrointestinal-dwelling helminths induces a systemic innate mucin response that primes peripheral barrier sites for protection against subsequent secondary helminth infections. These data suggest that innate-driven priming of mucus barriers may have evolved to protect from subsequent infections with multiple helminth species, which occur naturally in endemic areas.

The major secreted protein of the whipworm parasite tethers to matrix and inhibits interleukin-13 function.

Infection by soil transmitted parasitic helminths, such as Trichuris spp, are ubiquitous in humans and animals but the mechanisms determining persistence of chronic infections are poorly understood. Here we show that p43, the single most abundant protein in T. muris excretions/secretions, is non-immunogenic during infection and has an unusual sequence and structure containing subdomain homology to thrombospondin type 1 and interleukin (IL)-13 receptor (R) α2. Binding of p43 to IL-13, the key effector cytokine responsible for T. muris expulsion, inhibits IL-13 function both in vitro and in vivo. Tethering of p43 to matrix proteoglycans presents a bound source of p43 to facilitate interaction with IL-13, which may underpin chronic intestinal infection. Our results suggest that exploiting the biology of p43 may open up new approaches to modulating IL-13 function and control of Trichuris infections.

Trichuris muris whey acidic protein induces type 2 protective immunity against whipworm.

Human whipworm (Trichuris trichiura) infects approximately 1 in 15 people worldwide, representing the leading infectious cause of colitis and subsequent, inflammatory bowel disease (IBD). Current control measures focused on mass deworming have had limited success due to low drug efficacies. Vaccination would be an ideal, cost-effective strategy to induce protective immunity, leading to control of infection and transmission. Here we report the identification of whey acidic protein, a whipworm secretory protein, as a strong immunogen for inducing protective efficacy in a surrogate mouse T. muris infection model. The recombinant WAP protein (rTm-WAP49), as well as a single, highly conserved repeat within WAP (fragment 8) expressed as an Na-GST-1 fusion protein (rTm-WAP-F8+Na-GST-1), generate a strong T helper type 2 (Th2) immune response when delivered as subcutaneous vaccines formulated with Montanide ISA 720. Oral challenge with T. muris infective eggs following vaccination led to a significant reduction in worm burden of 48% by rTm-WAP49 and 33% by rTm-WAP-F8+Na-GST-1. The cellular immune correlates of protection included significant antigen-specific production of Th2 cytokines IL-4, IL-9, and IL-13 by cells isolated from the vaccine-draining inguinal lymph nodes, parasite-draining mesenteric lymph nodes, and spleen in mice vaccinated with either rTm-WAP49 or rTm-WAP-F8+Na-GST-1. The humoral immune correlates included a high antigen-specific ratio of IgG1 to IgG2a, without eliciting an IgE-mediated allergic response. Immunofluorescent staining of adult T. muris with WAP antisera identified the worm's pathogenic stichosome organ as the site of secretion of native Tm-WAP protein into the colonic mucosa. Given the high sequence conservation for the WAP proteins from T. muris and T. trichiura, the results presented here support the WAP protein to be further evaluated as a potential human whipworm vaccine candidate.

Manipulation of host and parasite microbiotas: Survival strategies during chronic nematode infection.

Intestinal dwelling parasites have evolved closely with the complex intestinal microbiota of their host, but the significance of the host microbiota for metazoan pathogens and the role of their own intestinal microbiota are still not fully known. We have found that the parasitic nematode Trichuris muris acquired a distinct intestinal microbiota from its host, which was required for nematode fitness. Infection of germ-free mice and mice monocolonized with Bacteroides thetaiotaomicron demonstrated that successful T. muris infections require a host microbiota. Following infection, T. muris-induced alterations in the host intestinal microbiota inhibited subsequent rounds of infection, controlling parasite numbers within the host intestine. This dual strategy could promote the long-term survival of the parasite within the intestinal niche necessary for successful chronic nematode infection.

Chronic Trichuris muris infection causes neoplastic change in the intestine and exacerbates tumour formation in APC min/+ mice.

Incidences of infection-related cancers are on the rise in developing countries where the prevalence of intestinal nematode worm infections are also high. Trichuris muris (T. muris) is a murine gut-dwelling nematode that is the direct model for human T. trichiura, one of the major soil-transmitted helminth infections of humans. In order to assess whether chronic infection with T. muris does indeed influence the development of cancer hallmarks, both wild type mice and colon cancer model (APC min/+) mice were infected with this parasite. Parasite infection in wild type mice led to the development of neoplastic change similar to that seen in mice that had been treated with the carcinogen azoxymethane. Additionally, both chronic and acute infection in the APCmin/+ mice led to an enhanced tumour development that was distinct to the site of infection suggesting systemic control. By blocking the parasite induced T regulatory response in these mice, the increase in the number of tumours following infection was abrogated. Thus T. muris infection alone causes an increase in gut pathologies that are known to be markers of cancer but also increases the incidence of tumour formation in a colon cancer model. The influence of parasitic worm infection on the development of cancer may therefore be significant.

Chronic Trichuris muris Infection in C57BL/6 Mice Causes Significant Changes in Host Microbiota and Metabolome: Effects Reversed by Pathogen Clearance.

Trichuris species are a globally important and prevalent group of intestinal helminth parasites, in which Trichuris muris (mouse whipworm) is an ideal model for this disease. This paper describes the first ever highly controlled and comprehensive investigation into the effects of T. muris infection on the faecal microbiota of mice and the effects on the microbiota following successful clearance of the infection. Communities were profiled using DGGE, 454 pyrosequencing, and metabolomics. Changes in microbial composition occurred between 14 and 28 days post infection, resulting in significant changes in α and ß- diversity. This impact was dominated by a reduction in the diversity and abundance of Bacteroidetes, specifically Prevotella and Parabacteroides. Metabolomic analysis of stool samples of infected mice at day 41 showed significant differences to uninfected controls with a significant increase in the levels of a number of essential amino acids and a reduction in breakdown of dietary plant derived carbohydrates. The significant reduction in weight gain by infected mice probably reflects these metabolic changes and the incomplete digestion of dietary polysaccharides. Following clearance of infection the intestinal microbiota underwent additional changes gradually transitioning by day 91 towards a microbiota of an uninfected animal. These data indicate that the changes in microbiota as a consequence of infection were transitory requiring the presence of the pathogen for maintenance. Interestingly this was not observed for all of the key immune cell populations associated with chronic T. muris infection. This reflects the highly regulated chronic response and potential lasting immunological consequences of dysbiosis in the microbiota. Thus infection of T. muris causes a significant and substantial impact on intestinal microbiota and digestive function of mice with affects in long term immune regulation.

Sex-dependent genetic effects on immune responses to a parasitic nematode.

BACKGROUND: Many disease aetiologies have sex specific effects, which have important implications for disease management. It is now becoming increasingly evident that such effects are the result of the differential expression of autosomal genes rather than sex-specific genes. Such sex-specific variation in the response to Trichuris muris, a murine parasitic nematode infection and model for the human parasitic nematode T. trichiura, has been well documented, however, the underlying genetic causes of these differences have been largely neglected. We used the BXD mouse set of recombinant inbred strains to identify sex-specific loci that contribute to immune phenotypes in T. muris infection. RESULTS: Response phenotypes to T. muris infection were found to be highly variable between different lines of BXD mice. A significant QTL on chromosome 5 (TM5) associated with IFN-γ production was found in male mice but not in female mice. This QTL was in the same location as a suggestive QTL for TNF-α and IL-6 production in male mice suggesting a common control of these pro-inflammatory cytokines. A second QTL was identified on chromosome 4 (TM4) affecting worm burden in both male and female cohorts. We have identified several genes as potential candidates for modifying responses to T. muris infection. CONCLUSIONS: We have used the largest mammalian genetic model system, the BXD mouse population, to identify candidate genes with sex-specific effects in immune responses to T. muris infection. Some of these genes may be differentially expressed in male and female mice leading to the difference in immune response between the sexes reported in previous studies. Our study further highlights the importance of considering sex as an important factor in investigations of immune response at the genome-wide level, in particular the bias that can be introduced when generalizing results obtained from only one sex or a mixed sex population. Rather, analyses of interaction effects between sex and genotype should be part of future studies.

Life on the edge: the balance between macrofauna, microflora and host immunity.

Mammals, microflora and gut-dwelling macrofauna have co-evolved over many millions of years until relatively recently when the geographical prevalence of macrofauna in humans has become restricted to the developing world. Immune homeostasis relies on a balance in the composition of intestinal microflora; long-lived macrofauna have also been shown to regulate immune function, and their absence in Western lifestyles is suggested to be a factor for the increasing frequency of allergy and autoimmunity. The intestinal nematode Trichuris muris was recently demonstrated to utilise microflora to initiate its life cycle. The interdependence on one another of all three factors is such that when the balance is perturbed it must be realigned or the consequences may be detrimental to the mammalian host.

Immune-mediated regulation of chronic intestinal nematode infection.

Gastrointestinal nematode infection is extremely prevalent worldwide in humans and animals. Infection levels vary between individuals in infected populations and exhibit a negative binomial distribution, and some individuals appear to be predisposed to certain infection levels. Moreover, infection tends to be chronic, despite evidence for the acquisition of some degree of acquired immunity. The host is subject to constant and repeated antigenic challenge, and individuals vary in the response they make. While a considerable amount of information is emerging on the immunoregulatory mechanisms operating during acute nematode infection from a variety of laboratory model systems, relatively little work has been carried out on the immune mechanisms underlying chronic infection. This review details some of the work that has addressed this important facet of gut nematode infection, highlighting studies from model systems that give insight into the induction of nonprotective immunity, while at the same time avoiding the induction of host-damaging pathology.