RESUMEN
By developing a high-density murine immunophenotyping platform compatible with high-throughput genetic screening, we have established profound contributions of genetics and structure to immune variation (http://www.immunophenotype.org). Specifically, high-throughput phenotyping of 530 unique mouse gene knockouts identified 140 monogenic 'hits', of which most had no previous immunologic association. Furthermore, hits were collectively enriched in genes for which humans show poor tolerance to loss of function. The immunophenotyping platform also exposed dense correlation networks linking immune parameters with each other and with specific physiologic traits. Such linkages limit freedom of movement for individual immune parameters, thereby imposing genetically regulated 'immunologic structures', the integrity of which was associated with immunocompetence. Hence, we provide an expanded genetic resource and structural perspective for understanding and monitoring immune variation in health and disease.
Asunto(s)
Infecciones por Enterobacteriaceae/inmunología , Variación Genética/genética , Ensayos Analíticos de Alto Rendimiento/métodos , Inmunofenotipificación/métodos , Infecciones por Salmonella/inmunología , Animales , Citrobacter/inmunología , Infecciones por Enterobacteriaceae/microbiología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Salmonella/inmunología , Infecciones por Salmonella/microbiologíaRESUMEN
Trypanosomes are protozoan parasites that cause infectious diseases, including African trypanosomiasis (sleeping sickness) in humans and nagana in economically important livestock1,2. An effective vaccine against trypanosomes would be an important control tool, but the parasite has evolved sophisticated immunoprotective mechanisms-including antigenic variation3-that present an apparently insurmountable barrier to vaccination. Here we show, using a systematic genome-led vaccinology approach and a mouse model of Trypanosoma vivax infection4, that protective invariant subunit vaccine antigens can be identified. Vaccination with a single recombinant protein comprising the extracellular region of a conserved cell-surface protein that is localized to the flagellum membrane (which we term 'invariant flagellum antigen from T. vivax') induced long-lasting protection. Immunity was passively transferred with immune serum, and recombinant monoclonal antibodies to this protein could induce sterile protection and revealed several mechanisms of antibody-mediated immunity, including a major role for complement. Our discovery identifies a vaccine candidate for an important parasitic disease that has constrained socioeconomic development in countries in sub-Saharan Africa5, and provides evidence that highly protective vaccines against trypanosome infections can be achieved.
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Antígenos de Protozoos/inmunología , Vacunas Antiprotozoos/inmunología , Trypanosoma vivax/inmunología , Tripanosomiasis Africana/inmunología , Tripanosomiasis Africana/prevención & control , Animales , Antígenos de Protozoos/química , Proteínas del Sistema Complemento/inmunología , Secuencia Conservada/inmunología , Modelos Animales de Enfermedad , Femenino , Flagelos/química , Flagelos/inmunología , Ratones , Ratones Endogámicos BALB C , Vacunas Antiprotozoos/química , Factores de Tiempo , Trypanosoma vivax/química , Trypanosoma vivax/citología , Tripanosomiasis Africana/parasitología , Vacunas de Subunidad/química , Vacunas de Subunidad/inmunologíaRESUMEN
Leishmaniasis is an infectious disease caused by protozoan parasites belonging to the genus Leishmania for which there are no approved human vaccines. Infections localise to different tissues in a species-specific manner with the visceral form of the disease caused by Leishmania donovani and L. infantum being the most deadly in humans. Although Leishmania spp. parasites are predominantly intracellular, the visceral disease can be prevented in dogs by vaccinating with a complex mixture of secreted products from cultures of L. infantum promastigotes. With the logic that extracellular parasite proteins make good subunit vaccine candidates because they are directly accessible to vaccine-elicited host antibodies, here we attempt to discover proteins that are essential for in vitro growth and host infection with the goal of identifying subunit vaccine candidates. Using an in silico analysis of the Leishmania donovani genome, we identified 92 genes encoding proteins that are predicted to be secreted or externally anchored to the parasite membrane by a single transmembrane region or a GPI anchor. By selecting a transgenic L. donovani parasite that expresses both luciferase and the Cas9 nuclease, we systematically attempted to target all 92 genes by CRISPR genome editing and identified four that were required for in vitro growth. For fifty-five genes, we infected cohorts of mice with each mutant parasite and by longitudinally quantifying parasitaemia with bioluminescent imaging, showed that nine genes had evidence of an attenuated infection although all ultimately established an infection. Finally, we expressed two genes as full-length soluble recombinant proteins and tested them as subunit vaccine candidates in a murine preclinical infection model. Both proteins elicited significant levels of protection against the uncontrolled development of a splenic infection warranting further investigation as subunit vaccine candidates against this deadly infectious tropical disease.
Asunto(s)
Leishmania donovani , Leishmania infantum , Leishmaniasis Visceral , Leishmaniasis , Parásitos , Animales , Perros , Leishmania donovani/genética , RatonesRESUMEN
Animal African trypanosomiasis (AAT) is a severe, wasting disease of domestic livestock and diverse wildlife species. The disease in cattle kills millions of animals each year and inflicts a major economic cost on agriculture in sub-Saharan Africa. Cattle AAT is caused predominantly by the protozoan parasites Trypanosoma congolense and T. vivax, but laboratory research on the pathogenic stages of these organisms is severely inhibited by difficulties in making even minor genetic modifications. As a result, many of the important basic questions about the biology of these parasites cannot be addressed. Here we demonstrate that an in vitro culture of the T. congolense genomic reference strain can be modified directly in the bloodstream form reliably and at high efficiency. We describe a parental single marker line that expresses T. congolense-optimized T7 RNA polymerase and Tet repressor and show that minichromosome loci can be used as sites for stable, regulatable transgene expression with low background in non-induced cells. Using these tools, we describe organism-specific constructs for inducible RNA-interference (RNAi) and demonstrate knockdown of multiple essential and non-essential genes. We also show that a minichromosomal site can be exploited to create a stable bloodstream-form line that robustly provides >40,000 independent stable clones per transfection-enabling the production of high-complexity libraries of genome-scale. Finally, we show that modified forms of T. congolense are still infectious, create stable high-bioluminescence lines that can be used in models of AAT, and follow the course of infections in mice by in vivo imaging. These experiments establish a base set of tools to change T. congolense from a technically challenging organism to a routine model for functional genetics and allow us to begin to address some of the fundamental questions about the biology of this important parasite.
Asunto(s)
Genética Microbiana , Proteínas Protozoarias/genética , Transgenes , Trypanosoma congolense/genética , Trypanosoma congolense/patogenicidad , Tripanosomiasis Africana/parasitología , Animales , Femenino , Genoma de Protozoos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos BALB C , Tripanosomiasis Africana/genéticaRESUMEN
BACKGROUND: Schistosomiasis is a major global health problem caused by blood-dwelling parasitic worms, which is currently tackled primarily by mass administration of the drug praziquantel. Appropriate drug treatment strategies are informed by diagnostics that establish the prevalence and intensity of infection, which, in regions of low transmission, should be highly sensitive. METHODS: To identify sensitive new serological markers of Schistosoma mansoni infections, we have compiled a recombinant protein library of parasite cell-surface and secreted proteins expressed in mammalian cells. RESULTS: Together with a time series of sera samples from volunteers experimentally infected with a defined number of male parasites, we probed this protein library to identify several markers that can detect primary infections with as low as 10 parasites and as early as 5 weeks postinfection. CONCLUSIONS: These new markers could be further explored as valuable tools to detect ongoing and previous S mansoni infections, including in endemic regions where transmission is low.
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Esquistosomiasis mansoni , Esquistosomiasis , Animales , Biomarcadores , Humanos , Masculino , Mamíferos , Ratones , Praziquantel/uso terapéutico , Proteínas Recombinantes , Schistosoma mansoni , Esquistosomiasis/tratamiento farmacológico , Esquistosomiasis mansoni/diagnóstico , Esquistosomiasis mansoni/tratamiento farmacológico , Esquistosomiasis mansoni/parasitologíaRESUMEN
Innate lymphoid cells (ILCs) functionally resemble T lymphocytes in cytotoxicity and cytokine production but lack antigen-specific receptors, and they are important regulators of immune responses and tissue homeostasis. ILCs are generated from common lymphoid progenitors, which are subsequently committed to innate lymphoid lineages in the α-lymphoid progenitor, early innate lymphoid progenitor, common helper innate lymphoid progenitor and innate lymphoid cell progenitor compartments. ILCs consist of conventional natural killer cells and helper-like cells (ILC1, ILC2 and ILC3). Despite recent advances, the cellular heterogeneity, developmental trajectory and signalling dependence of ILC progenitors are not fully understood. Here, using single-cell RNA-sequencing (scRNA-seq) of mouse bone marrow progenitors, we reveal ILC precursor subsets, delineate distinct ILC development stages and pathways, and report that high expression of programmed death 1 (PD-1hi) marked a committed ILC progenitor that was essentially identical to an innate lymphoid cell progenitor. Our data defined PD-1hiIL-25Rhi as an early checkpoint in ILC2 development, which was abolished by deficiency in the zinc-finger protein Bcl11b but restored by IL-25R overexpression. Similar to T lymphocytes, PD-1 was upregulated on activated ILCs. Administration of a PD-1 antibody depleted PD-1hi ILCs and reduced cytokine levels in an influenza infection model in mice, and blocked papain-induced acute lung inflammation. These results provide a perspective for exploring PD-1 and its ligand (PD-L1) in immunotherapy, and allow effective manipulation of the immune system for disease prevention and therapy.
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Secuencia de Bases , Linaje de la Célula , Inmunidad Innata , Linfocitos/citología , Células Progenitoras Linfoides/citología , Receptor de Muerte Celular Programada 1/metabolismo , Análisis de la Célula Individual , Animales , Anticuerpos/inmunología , Diferenciación Celular , Linaje de la Célula/genética , Separación Celular , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Inmunoterapia/tendencias , Gripe Humana/inmunología , Gripe Humana/metabolismo , Células Asesinas Naturales/citología , Activación de Linfocitos , Linfocitos/inmunología , Linfocitos/metabolismo , Células Progenitoras Linfoides/metabolismo , Ratones , Ratones Endogámicos C57BL , Neumonía/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Receptores de Interleucina/metabolismo , Proteínas Represoras/deficiencia , Proteínas Represoras/metabolismo , Linfocitos T/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Relapsing C. difficile disease in humans is linked to a pathological imbalance within the intestinal microbiota, termed dysbiosis, which remains poorly understood. We show that mice infected with epidemic C. difficile (genotype 027/BI) develop highly contagious, chronic intestinal disease and persistent dysbiosis characterized by a distinct, simplified microbiota containing opportunistic pathogens and altered metabolite production. Chronic C. difficile 027/BI infection was refractory to vancomycin treatment leading to relapsing disease. In contrast, treatment of C. difficile 027/BI infected mice with feces from healthy mice rapidly restored a diverse, healthy microbiota and resolved C. difficile disease and contagiousness. We used this model to identify a simple mixture of six phylogenetically diverse intestinal bacteria, including novel species, which can re-establish a health-associated microbiota and clear C. difficile 027/BI infection from mice. Thus, targeting a dysbiotic microbiota with a defined mixture of phylogenetically diverse bacteria can trigger major shifts in the microbial community structure that displaces C. difficile and, as a result, resolves disease and contagiousness. Further, we demonstrate a rational approach to harness the therapeutic potential of health-associated microbial communities to treat C. difficile disease and potentially other forms of intestinal dysbiosis.
Asunto(s)
Clostridioides difficile/patogenicidad , Enterocolitis Seudomembranosa/terapia , Heces/microbiología , Intestinos/microbiología , Interacciones Microbianas , Probióticos/uso terapéutico , Animales , Clostridioides difficile/efectos de los fármacos , Farmacorresistencia Bacteriana , Enterocolitis Seudomembranosa/tratamiento farmacológico , Enterocolitis Seudomembranosa/microbiología , Femenino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Consorcios Microbianos , Datos de Secuencia Molecular , Recurrencia , Vancomicina/farmacologíaRESUMEN
Visceral leishmaniasis is a deadly infectious disease and is one of the world's major neglected health problems. Because the symptoms of infection are similar to other endemic diseases, accurate diagnosis is crucial for appropriate treatment. Definitive diagnosis using splenic or bone marrow aspirates is highly invasive, and so, serological assays are preferred, including the direct agglutination test (DAT) or rK39 strip test. These tests, however, are either difficult to perform in the field (DAT) or lack specificity in some endemic regions (rK39), making the development of new tests a research priority. The availability of Leishmania spp. genomes presents an opportunity to identify new diagnostic targets. Here, we use genome data and a mammalian protein expression system to create a panel of 93 proteins consisting of the extracellular ectodomains of the Leishmania donovani cell surface and secreted proteins. We use these panel and sera from murine experimental infection models and natural human and canine infections to identify new candidates for serological diagnosis. We observed a concordance between the most immunoreactive antigens in different host species and transmission settings. The antigen encoded by the LdBPK_323600.1 gene can diagnose Leishmania infections with high sensitivity and specificity in patient cohorts from different endemic regions including Bangladesh and Ethiopia. In longitudinal sampling of treated patients, we observed reductions in immunoreactivity to LdBPK_323600.1 suggesting it could be used to diagnose treatment success. In summary, we have identified new antigens that could contribute to improved serological diagnostic tests to help control the impact of this deadly tropical infectious disease. IMPORTANCE: Visceral leishmaniasis is fatal if left untreated with patients often displaying mild and non-specific symptoms during the early stages of infection making accurate diagnosis important. Current methods for diagnosis require highly trained medical staff to perform highly invasive biopsies of the liver or bone marrow which pose risks to the patient. Less invasive molecular tests are available but can suffer from regional variations in their ability to accurately diagnose an infection. To identify new diagnostic markers of visceral leishmaniasis, we produced and tested a panel of 93 proteins identified from the genome of the parasite responsible for this disease. We found that the pattern of host antibody reactivity to these proteins was broadly consistent across naturally acquired infections in both human patients and dogs, as well as experimental rodent infections. We identified a new protein called LdBPK_323600.1 that could accurately diagnose visceral leishmaniasis infections in humans.
Asunto(s)
Anticuerpos Antiprotozoarios , Antígenos de Protozoos , Leishmania donovani , Leishmaniasis Visceral , Proteínas Protozoarias , Pruebas Serológicas , Leishmania donovani/genética , Leishmania donovani/inmunología , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Animales , Humanos , Ratones , Perros , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Pruebas Serológicas/métodos , Biomarcadores/sangre , Femenino , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Ratones Endogámicos BALB C , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Sensibilidad y Especificidad , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/parasitologíaRESUMEN
Hundreds of millions of people worldwide are infected with the whipworm Trichuris trichiura. Novel treatments are urgently needed as current drugs, such as albendazole, have relatively low efficacy. We have investigated whether drugs approved for other human diseases could be repurposed as novel anti-whipworm drugs. In a previous comparative genomics analysis, we identified 409 drugs approved for human use that we predicted to target parasitic worm proteins. Here we tested these ex vivo by assessing motility of adult worms of Trichuris muris, the murine whipworm, an established model for human whipworm research. We identified 14 compounds with EC50 values of ≤50 µM against T. muris ex vivo, and selected nine for testing in vivo. However, the best worm burden reduction seen in mice was just 19%. The high number of ex vivo hits against T. muris shows that we were successful at predicting parasite proteins that could be targeted by approved drugs. In contrast, the low efficacy of these compounds in mice suggest challenges due to their chemical properties (e.g. lipophilicity, polarity, molecular weight) and pharmacokinetics (e.g. absorption, distribution, metabolism, and excretion) that may (i) promote absorption by the host gastrointestinal tract, thereby reducing availability to the worms embedded in the large intestine, and/or (ii) restrict drug uptake by the worms. This indicates that identifying structural analogues that have reduced absorption by the host, and increased uptake by worms, may be necessary for successful drug development against whipworms.
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Reposicionamiento de Medicamentos , Trichuris , Adulto , Humanos , Animales , Ratones , Trichuris/genética , Genómica , Albendazol/farmacología , Transporte BiológicoRESUMEN
Visceral leishmaniasis is a deadly infectious disease caused by Leishmania donovani, a kinetoplastid parasite for which no licensed vaccine is available. To identify potential vaccine candidates, we systematically identified genes encoding putative cell surface and secreted proteins essential for parasite viability and host infection. We identified a protein encoded by LdBPK_061160 which, when ablated, resulted in a remarkable increase in parasite adhesion to tissue culture flasks. Here, we show that this phenotype is caused by the loss of glycosylphosphatidylinositol (GPI)-anchored surface molecules and that LdBPK_061160 encodes a noncatalytic component of the L. donovani GPI-mannosyltransferase I (GPI-MT I) complex. GPI-anchored surface molecules were rescued in the LdBPK_061160 mutant by the ectopic expression of both human genes PIG-X and PIG-M, but neither gene could complement the phenotype alone. From further sequence comparisons, we conclude that LdBPK_061160 is the functional orthologue of yeast PBN1 and mammalian PIG-X, which encode the noncatalytic subunits of their respective GPI-MT I complexes, and we assign LdBPK_061160 as LdPBN1. The LdPBN1 mutants could not establish a visceral infection in mice, a phenotype that was rescued by constitutive expression of LdPBN1. Although mice infected with the null mutant did not develop an infection, exposure to these parasites provided significant protection against subsequent infection with a virulent strain. In summary, we have identified the orthologue of the PBN1/PIG-X noncatalytic subunit of GPI-MT I in trypanosomatids, shown that it is essential for infection in a murine model of visceral leishmaniasis, and demonstrated that the LdPBN1 mutant shows promise for the development of an attenuated live vaccine. IMPORTANCE Visceral leishmaniasis is a deadly infectious disease caused by the parasites Leishmania donovani and Leishmania infantum. It remains a major global health problem, and there is no licensed highly effective vaccine. Molecules that are displayed on the surface of parasites are involved in host-parasite interactions and have important roles in immune evasion, making vaccine development difficult. One major way in which parasite surface molecules are tethered to the surface is via glycophosphatidylinositol (GPI) anchors; however, the enzymes required for all the biosynthetic steps in these parasites are not known. Here, we identified the enzyme required for an essential step in the GPI anchor-biosynthetic pathway in L. donovani, and we show that while parasites lacking this gene are viable in vitro, they are unable to establish infections in mice, a property we show can be exploited to develop a live genetically attenuated parasite vaccine.
Asunto(s)
Enfermedades Transmisibles , Leishmania donovani , Vacunas contra la Leishmaniasis , Leishmaniasis Visceral , Animales , Glicosilfosfatidilinositoles , Leishmania donovani/genética , Vacunas contra la Leishmaniasis/genética , Leishmaniasis Visceral/parasitología , Mamíferos , Ratones , Vacunas AtenuadasRESUMEN
Increasing evidence shows that the host gut microbiota might be involved in the immunological cascade that culminates with the formation of tissue granulomas underlying the pathophysiology of hepato-intestinal schistosomiasis. In this study, we investigated the impact of Schistosoma mansoni infection on the gut microbial composition and functional potential of both wild type and microbiome-humanized mice. In spite of substantial differences in microbiome composition at baseline, selected pathways were consistently affected by parasite infection. The gut microbiomes of infected mice of both lines displayed, amongst other features, enhanced capacity for tryptophan and butyrate production, which might be linked to the activation of mechanisms aimed to prevent excessive injuries caused by migrating parasite eggs. Complementing data from previous studies, our findings suggest that the host gut microbiome might play a dual role in the pathophysiology of schistosomiasis, where intestinal bacteria may contribute to egg-associated pathology while, in turn, protect the host from uncontrolled tissue damage.
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Microbioma Gastrointestinal , Microbiota , Esquistosomiasis mansoni , Esquistosomiasis , Ratones , Animales , Roedores , BacteriasRESUMEN
Trypanosoma vivax is a unicellular hemoparasite, and a principal cause of animal African trypanosomiasis (AAT), a vector-borne and potentially fatal livestock disease across sub-Saharan Africa. Previously, we identified diverse T. vivax-specific genes that were predicted to encode cell surface proteins. Here, we examine the immune responses of naturally and experimentally infected hosts to these unique parasite antigens, to identify immunogens that could become vaccine candidates. Immunoprofiling of host serum shows that one particular family (Fam34) elicits a consistent IgG antibody response. This gene family, which we now call Vivaxin, encodes at least 124 transmembrane glycoproteins that display quite distinct expression profiles and patterns of genetic variation. We focused on one gene (viv-ß8) that encodes one particularly immunogenic vivaxin protein and which is highly expressed during infections but displays minimal polymorphism across the parasite population. Vaccination of mice with VIVß8 adjuvanted with Quil-A elicits a strong, balanced immune response and delays parasite proliferation in some animals but, ultimately, it does not prevent disease. Although VIVß8 is localized across the cell body and flagellar membrane, live immunostaining indicates that VIVß8 is largely inaccessible to antibody in vivo. However, our phylogenetic analysis shows that vivaxin includes other antigens shown recently to induce immunity against T. vivax. Thus, the introduction of vivaxin represents an important advance in our understanding of the T. vivax cell surface. Besides being a source of proven and promising vaccine antigens, the gene family is clearly an important component of the parasite glycocalyx, with potential to influence host-parasite interactions.
Asunto(s)
Trypanosoma vivax , Vacunas , Animales , Formación de Anticuerpos , Antígenos de Protozoos/genética , Inmunoglobulina G/genética , Ratones , Filogenia , Trypanosoma vivax/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genéticaRESUMEN
Experimental mouse models are central to basic biomedical research; however, variability exists across genetically identical mice and mouse facilities making comparisons difficult. Whether specific indigenous gut bacteria drive immunophenotypic variability in mouse models of human disease remains poorly understood. We performed a large-scale experiment using 579 genetically identical laboratory mice from a single animal facility, designed to identify the causes of disease variability in the widely used dextran sulphate sodium mouse model of inflammatory bowel disease. Commonly used treatment endpoint measures-weight loss and intestinal pathology-showed limited correlation and varied across mouse lineages. Analysis of the gut microbiome, coupled with machine learning and targeted anaerobic culturing, identified and isolated two previously undescribed species, Duncaniella muricolitica and Alistipes okayasuensis, and demonstrated that they exert dominant effects in the dextran sulphate sodium model leading to variable treatment endpoint measures. We show that the identified gut microbial species are common, but not ubiquitous, in mouse facilities around the world, and suggest that researchers monitor for these species to provide experimental design opportunities for improved mouse models of human intestinal diseases.
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Colitis , Microbioma Gastrointestinal , Enfermedades Inflamatorias del Intestino , Animales , Bacteroidetes , Colitis/inducido químicamente , Colitis/microbiología , Modelos Animales de Enfermedad , Enfermedades Inflamatorias del Intestino/microbiología , RatonesRESUMEN
EROS (essential for reactive oxygen species) protein is indispensable for expression of gp91phox, the catalytic core of the phagocyte NADPH oxidase. EROS deficiency in humans is a novel cause of the severe immunodeficiency, chronic granulomatous disease, but its mechanism of action was unknown until now. We elucidate the role of EROS, showing it acts at the earliest stages of gp91phox maturation. It binds the immature 58 kDa gp91phox directly, preventing gp91phox degradation and allowing glycosylation via the oligosaccharyltransferase machinery and the incorporation of the heme prosthetic groups essential for catalysis. EROS also regulates the purine receptors P2X7 and P2X1 through direct interactions, and P2X7 is almost absent in EROS-deficient mouse and human primary cells. Accordingly, lack of murine EROS results in markedly abnormal P2X7 signalling, inflammasome activation, and T cell responses. The loss of both ROS and P2X7 signalling leads to resistance to influenza infection in mice. Our work identifies EROS as a highly selective chaperone for key proteins in innate and adaptive immunity and a rheostat for immunity to infection. It has profound implications for our understanding of immune physiology, ROS dysregulation, and possibly gene therapy.
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Enfermedad Granulomatosa Crónica , NADPH Oxidasas , Humanos , Animales , Ratones , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Fagocitos/metabolismo , Transducción de Señal/fisiologíaRESUMEN
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.
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Helmintos , Tricuriasis , Animales , Mucosa Intestinal , Intestinos/parasitología , Mamíferos , Ratones , Trichuris/fisiologíaRESUMEN
Enterotoxigenic Escherichia coli (ETEC) expressing the colonization pili CFA/I are common causes of diarrhoeal infections in humans. Here, we use a combination of transposon mutagenesis and transcriptomic analysis to identify genes and pathways that contribute to ETEC persistence in water environments and colonization of a mammalian host. ETEC persisting in water exhibit a distinct RNA expression profile from those growing in richer media. Multiple pathways were identified that contribute to water survival, including lipopolysaccharide biosynthesis and stress response regulons. The analysis also indicated that ETEC growing in vivo in mice encounter a bottleneck driving down the diversity of colonizing ETEC populations.
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Escherichia coli Enterotoxigénica/crecimiento & desarrollo , Escherichia coli Enterotoxigénica/genética , Proteínas de Escherichia coli/genética , Proteínas Fimbrias/genética , Microbiología del Agua , Animales , Modelos Animales de Enfermedad , Escherichia coli Enterotoxigénica/aislamiento & purificación , Infecciones por Escherichia coli , Femenino , Proteínas Fimbrias/aislamiento & purificación , Fimbrias Bacterianas , Genes Bacterianos/genética , Ratones , Ratones Endogámicos C57BL , Fenotipo , AguaRESUMEN
Clostridium difficile is a major cause of antibiotic-associated diarrheal disease in many parts of the world. In recent years, distinct genetic variants of C. difficile that cause severe disease and persist within health care settings have emerged. Highly resistant and infectious C. difficile spores are proposed to be the main vectors of environmental persistence and host transmission, so methods to accurately monitor spores and their inactivation are urgently needed. Here we describe simple quantitative methods, based on purified C. difficile spores and a murine transmission model, for evaluating health care disinfection regimens. We demonstrate that disinfectants that contain strong oxidizing active ingredients, such as hydrogen peroxide, are very effective in inactivating pure spores and blocking spore-mediated transmission. Complete inactivation of 106 pure C. difficile spores on indicator strips, a six-log reduction, and a standard measure of stringent disinfection regimens require at least 5 min of exposure to hydrogen peroxide vapor (HPV; 400 ppm). In contrast, a 1-min treatment with HPV was required to disinfect an environment that was heavily contaminated with C. difficile spores (17 to 29 spores/cm²) and block host transmission. Thus, pure C. difficile spores facilitate practical methods for evaluating the efficacy of C. difficile spore disinfection regimens and bringing scientific acumen to C. difficile infection control.
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Clostridioides difficile/aislamiento & purificación , Desinfección/métodos , Microbiología Ambiental , Instituciones de Salud , Control de Infecciones/métodos , Esporas Bacterianas/aislamiento & purificación , Animales , Infecciones por Clostridium/transmisión , Modelos Animales de Enfermedad , Ratones , Control de CalidadRESUMEN
In spite of growing evidence supporting the occurrence of complex interactions between Schistosoma and gut bacteria in mice and humans, no data is yet available on whether worm-mediated changes in microbiota composition are dependent on the baseline gut microbial profile of the vertebrate host. In addition, the impact of such changes on the susceptibility to, and pathophysiology of, schistosomiasis remains largely unexplored. In this study, mice colonized with gut microbial populations from a human donor (HMA mice), as well as microbiota-wild type (WT) animals, were infected with Schistosoma mansoni, and alterations of their gut microbial profiles at 50 days post-infection were compared to those occurring in uninfected HMA and WT rodents, respectively. Significantly higher worm and egg burdens, together with increased specific antibody responses to parasite antigens, were observed in HMA compared to WT mice. These differences were associated to extensive dissimilarities between the gut microbial profiles of each HMA and WT groups of mice at baseline; in particular, the gut microbiota of HMA animals was characterized by low microbial alpha diversity and expanded Proteobacteria, as well as by the absence of putative immunomodulatory bacteria (e.g. Lactobacillus). Furthermore, differences in infection-associated changes in gut microbiota composition were observed between HMA and WT mice. Altogether, our findings support the hypothesis that susceptibility to S.mansoni infection in mice is partially dependent on the composition of the host baseline microbiota. Moreover, this study highlights the applicability of HMA mouse models to address key biological questions on host-parasite-microbiota relationships in human helminthiases.
Asunto(s)
Microbioma Gastrointestinal , Interacciones Huésped-Parásitos , Carga de Parásitos , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/parasitología , Animales , Anticuerpos Antiprotozoarios/inmunología , Bacterias/clasificación , Bacterias/genética , Biodiversidad , Biología Computacional/métodos , Modelos Animales de Enfermedad , Disbiosis , Heces/microbiología , Microbioma Gastrointestinal/inmunología , Interacciones Huésped-Parásitos/inmunología , Inmunomodulación , Metagenómica/métodos , Ratones , ARN Ribosómico 16S , SchistosomaRESUMEN
Background: Schistosomiasis is a major parasitic disease affecting people living in tropical and sup-tropical areas. Transmission of the parasite has been reported in 78 countries, causing significant morbidity and around 200,000 deaths per year in endemic regions. The disease is currently managed by the mass-administration of praziquantel to populations at risk of infection; however, the reliance on a single drug raises the prospect of parasite resistance to the only treatment widely available. The development of an effective vaccine would be a more powerful method of control, but none currently exists and the identification of new immunogens that can elicit protective immune responses therefore remains a priority. Because of the complex nature of the parasite life cycle, identification of new vaccine candidates has mostly relied on the use of animal models and on a limited set of recombinant proteins. Methods: In this study, we have established an infrastructure for testing a large number of vaccine candidates in mice and used it to screen 96 cell-surface and secreted recombinant proteins from Schistosoma mansoni. This approach, using standardised immunisation and percutaneous infection protocols, allowed us to compare an extensive set of antigens in a systematic manner. Results: Although some vaccine candidates were associated with a statistically significant reduction in the number of eggs in the initial screens, these observations could not be repeated in subsequent challenges and none of the proteins studied were associated with a strongly protective effect against infection. Conclusions: Although no antigens individually induced reproducible and strongly protective effects using our vaccination regime, we have established the experimental infrastructures to facilitate large-scale systematic subunit vaccine testing for schistosomiasis in a murine infection model.
RESUMEN
Interferon lambda (IFNλ) is a group of cytokines that belong to the IL-10 family. They exhibit antiviral activities against certain viruses during infection of the liver and mucosal tissues. Here we report that IFNλ restricts in vitro replication of the ß-herpesvirus murine cytomegalovirus (mCMV). However, IFNλR1-deficient (Ifnλr1-/-) mice were not preferentially susceptible to mCMV infection in vivo during acute infection after systemic or mucosal challenge, or during virus persistence in the mucosa. Instead, our studies revealed that IFNλ influences NK cell responses during mCMV infection. Ifnλr1-/- mice exhibited defective development of conventional interferon-gamma (IFNγ)-expressing NK cells in the spleen during mCMV infection whereas accumulation of granzyme B-expressing NK cells was unaltered. In vitro, development of splenic IFNγ+ NK cells following stimulation with IL-12 or, to a lesser extent, IL-18 was abrogated by IFNλR1-deficiency. Thus, IFNλ regulates NK cell responses during mCMV infection and restricts virus replication in vitro but is redundant in the control of acute and persistent mCMV replication within mucosal and non-mucosal tissues.