RESUMEN
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
B cells play a central role in humoral immunity but also have antibody-independent functions. Studies to date have focused on B cells in blood and secondary lymphoid organs but whether B cells reside in non-lymphoid organs (NLO) in homeostasis is unknown. Here we identify, using intravenous labeling and parabiosis, a bona-fide tissue-resident B cell population in lung, liver, kidney and urinary bladder, a substantial proportion of which are B-1a cells. Tissue-resident B cells are present in neonatal tissues and also in germ-free mice NLOs, albeit in lower numbers than in specific pathogen-free mice and following co-housing with 'pet-store' mice. They spatially co-localise with macrophages and regulate their polarization and function, promoting an anti-inflammatory phenotype, in-part via interleukin-10 production, with effects on bacterial clearance during urinary tract infection. Thus, our data reveal a critical role for tissue-resident B cells in determining the homeostatic 'inflammatory set-point' of myeloid cells, with important consequences for tissue immunity.
Asunto(s)
Linfocitos B , Macrófagos , Ratones , Animales , Anticuerpos , Hígado , PulmónRESUMEN
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.
Asunto(s)
Enfermedad Granulomatosa Crónica , NADPH Oxidasas , Humanos , Animales , Ratones , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Fagocitos/metabolismo , Transducción de Señal/fisiologíaRESUMEN
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.
Asunto(s)
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.
Asunto(s)
Colitis , Microbioma Gastrointestinal , Enfermedades Inflamatorias del Intestino , Animales , Bacteroidetes , Colitis/inducido químicamente , Colitis/microbiología , Modelos Animales de Enfermedad , Enfermedades Inflamatorias del Intestino/microbiología , RatonesRESUMEN
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
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
Anti-TNFα and anti-IL-23 antibodies are highly effective therapies for Crohn's disease or ulcerative colitis in a proportion of patients. V56B2 is a novel bispecific domain antibody in which a llama-derived IL-23p19-specific domain antibody, humanised and engineered for intestinal protease resistance, V900, was combined with a previously-described TNFα-specific domain antibody, V565. V56B2 contains a central protease-labile linker to create a single molecule for oral administration. Incubation of V56B2 with trypsin or human faecal supernatant resulted in a complete separation of the V565 and V900 monomers without loss of neutralising potency. Following oral administration of V900 and V565 in mice, high levels of each domain antibody were detected in the faeces, demonstrating stability in the intestinal milieu. In ex vivo cultures of colonic biopsies from IBD patients, treatment with V565 or V900 inhibited tissue phosphoprotein levels and with a combination of the two, inhibition was even greater. These results support further development of V56B2 as an oral therapy for IBD with improved safety and efficacy in a greater proportion of patients as well as greater convenience for patients compared with traditional monoclonal antibody therapies.
Asunto(s)
Anticuerpos Biespecíficos , Anticuerpos Monoclonales/administración & dosificación , Evaluación Preclínica de Medicamentos/métodos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Interleucina-23/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Anticuerpos Biespecíficos/administración & dosificación , Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales/farmacología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
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.
Asunto(s)
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
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
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
The central nervous system has historically been viewed as an immune-privileged site, but recent data have shown that the meninges-the membranes that surround the brain and spinal cord-contain a diverse population of immune cells1. So far, studies have focused on macrophages and T cells, but have not included a detailed analysis of meningeal humoral immunity. Here we show that, during homeostasis, the mouse and human meninges contain IgA-secreting plasma cells. These cells are positioned adjacent to dural venous sinuses: regions of slow blood flow with fenestrations that can potentially permit blood-borne pathogens to access the brain2. Peri-sinus IgA plasma cells increased with age and following a breach of the intestinal barrier. Conversely, they were scarce in germ-free mice, but their presence was restored by gut re-colonization. B cell receptor sequencing confirmed that meningeal IgA+ cells originated in the intestine. Specific depletion of meningeal plasma cells or IgA deficiency resulted in reduced fungal entrapment in the peri-sinus region and increased spread into the brain following intravenous challenge, showing that meningeal IgA is essential for defending the central nervous system at this vulnerable venous barrier surface.
Asunto(s)
Senos Craneales/inmunología , Microbioma Gastrointestinal/inmunología , Inmunoglobulina A Secretora/inmunología , Intestinos/inmunología , Meninges/inmunología , Células Plasmáticas/inmunología , Anciano , Envejecimiento/inmunología , Animales , Barrera Hematoencefálica/inmunología , Femenino , Hongos/inmunología , Vida Libre de Gérmenes , Humanos , Intestinos/citología , Intestinos/microbiología , Masculino , Meninges/irrigación sanguínea , Meninges/citología , Ratones , Ratones Endogámicos C57BL , Células Plasmáticas/citologíaRESUMEN
Macrophages play a central role in intestinal immunity, but inappropriate macrophage activation is associated with inflammatory bowel disease (IBD). Here, we identify granulocyte-macrophage colony stimulating factor (GM-CSF) as a critical regulator of intestinal macrophage activation in patients with IBD and mice with dextran sodium sulfate (DSS)-induced colitis. We find that GM-CSF drives the maturation and polarization of inflammatory intestinal macrophages, promoting anti-microbial functions while suppressing wound-healing transcriptional programs. Group 3 innate lymphoid cells (ILC3s) are a major source of GM-CSF in intestinal inflammation, with a strong positive correlation observed between ILC or CSF2 transcripts and M1 macrophage signatures in IBD mucosal biopsies. Furthermore, GM-CSF-dependent macrophage polarization results in a positive feedback loop that augmented ILC3 activation and type 17 immunity. Together, our data reveal an important role for GM-CSF-mediated ILC-macrophage crosstalk in calibrating intestinal macrophage phenotype to enhance anti-bacterial responses, while inhibiting pro-repair functions associated with fibrosis and stricturing, with important clinical implications.
Asunto(s)
Infecciones por Enterobacteriaceae/patología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Inflamación/patología , Intestinos/patología , Macrófagos/patología , Cicatrización de Heridas , Animales , Polaridad Celular , Citrobacter rodentium/fisiología , Colitis/complicaciones , Colitis/inmunología , Colitis/patología , Humanos , Inmunidad Innata , Linfocitos/inmunología , Activación de Macrófagos , Ratones Endogámicos C57BL , FenotipoRESUMEN
Signalling lymphocyte activation molecule family member 9 (SLAMF9) is an orphan receptor of the CD2/SLAM family of leucocyte surface proteins. Examination of SLAMF9 expression and function indicates that SLAMF9 promotes inflammation by specialized subsets of antigen-presenting cells. Within healthy liver and circulating mouse peripheral blood mononuclear cells, SLAMF9 is expressed on CD11b+ , Ly6C- , CD11clow , F4/80low , MHC-II+ , CX3 CR1+ mononuclear phagocytes as well as plasmacytoid dendritic cells. In addition, SLAMF9 can be found on peritoneal B1 cells and small (F4/80low ), but not large (F4/80high ), peritoneal macrophages. Upon systemic challenge with Salmonella enterica Typhimurium, Slamf9-/- mice were impaired in their ability to clear the infection from the liver. In humans, SLAMF9 is up-regulated upon differentiation of monocytes into macrophages, and lipopolysaccharide stimulation of PMA-differentiated, SLAMF9 knockdown THP-1 cells showed an essential role of SLAMF9 in production of granulocyte-macrophage colony-stimulating factor, tumour necrosis factor-α, and interleukin-1ß. Taken together, these data implicate SLAMF9 in the initiation of inflammation and clearance of bacterial infection.
Asunto(s)
Células Dendríticas/inmunología , Interacciones Huésped-Patógeno/inmunología , Hígado/inmunología , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/microbiología , Diferenciación Celular , Células Dendríticas/efectos de los fármacos , Células Dendríticas/microbiología , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Interacciones Huésped-Patógeno/genética , Humanos , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Lipopolisacáridos/farmacología , Hígado/microbiología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/microbiología , Infecciones por Salmonella/genética , Infecciones por Salmonella/microbiología , Salmonella typhimurium/patogenicidad , Transducción de Señal , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/deficiencia , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/genética , Células THP-1 , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
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
The discovery of a Salmonella-targeting phage from the waterways of the United Kingdom provided an opportunity to address the mechanism by which Chi-like bacteriophage (phage) engages with bacterial flagellae. The long tail fibre seen on Chi-like phages has been proposed to assist the phage particle in docking to a host cell flagellum, but the identity of the protein that generates this fibre was unknown. We present the results from genome sequencing of this phage, YSD1, confirming its close relationship to the original Chi phage and suggesting candidate proteins to form the tail structure. Immunogold labelling in electron micrographs revealed that YSD1_22 forms the main shaft of the tail tube, while YSD1_25 forms the distal part contributing to the tail spike complex. The long curling tail fibre is formed by the protein YSD1_29, and treatment of phage with the antibodies that bind YSD1_29 inhibits phage infection of Salmonella. The host range for YSD1 across Salmonella serovars is broad, but not comprehensive, being limited by antigenic features of the flagellin subunits that make up the Salmonella flagellum, with which YSD1_29 engages to initiate infection.
Asunto(s)
Flagelos/genética , Fagos de Salmonella/genética , Fagos de Salmonella/aislamiento & purificación , Bacteriófagos/genética , ADN Viral/genética , Flagelos/metabolismo , Flagelos/fisiología , Genoma Viral/genética , Especificidad del Huésped , Filogenia , Fagos de Salmonella/metabolismo , Salmonella typhi/genética , Salmonella typhi/metabolismo , Análisis de Secuencia de ADN/métodos , Reino UnidoRESUMEN
Bloodstream infections by Salmonella enterica serovar Typhimurium constitute a major health burden in sub-Saharan Africa (SSA). These invasive non-typhoidal (iNTS) infections are dominated by isolates of the antibiotic resistance-associated sequence type (ST) 313. Here, we report emergence of ST313 sublineage II.1 in the Democratic Republic of the Congo. Sublineage II.1 exhibits extensive drug resistance, involving a combination of multidrug resistance, extended spectrum ß-lactamase production and azithromycin resistance. ST313 lineage II.1 isolates harbour an IncHI2 plasmid we name pSTm-ST313-II.1, with one isolate also exhibiting decreased ciprofloxacin susceptibility. Whole genome sequencing reveals that ST313 II.1 isolates have accumulated genetic signatures potentially associated with altered pathogenicity and host adaptation, related to changes observed in biofilm formation and metabolic capacity. Sublineage II.1 emerged at the beginning of the 21st century and is involved in on-going outbreaks. Our data provide evidence of further evolution within the ST313 clade associated with iNTS in SSA.
Asunto(s)
Adaptación Fisiológica/efectos de los fármacos , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Adaptación Fisiológica/genética , Animales , Azitromicina/farmacología , Biopelículas/crecimiento & desarrollo , Línea Celular , Ciprofloxacina/farmacología , República Democrática del Congo , Humanos , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Salmonella typhimurium/aislamiento & purificación , Células THP-1 , Secuenciación Completa del GenomaRESUMEN
Bacterial speciation is a fundamental evolutionary process characterized by diverging genotypic and phenotypic properties. However, the selective forces that affect genetic adaptations and how they relate to the biological changes that underpin the formation of a new bacterial species remain poorly understood. Here, we show that the spore-forming, healthcare-associated enteropathogen Clostridium difficile is actively undergoing speciation. Through large-scale genomic analysis of 906 strains, we demonstrate that the ongoing speciation process is linked to positive selection on core genes in the newly forming species that are involved in sporulation and the metabolism of simple dietary sugars. Functional validation shows that the new C. difficile produces spores that are more resistant and have increased sporulation and host colonization capacity when glucose or fructose is available for metabolism. Thus, we report the formation of an emerging C. difficile species, selected for metabolizing simple dietary sugars and producing high levels of resistant spores, that is adapted for healthcare-mediated transmission.
Asunto(s)
Aclimatación/genética , Clostridioides difficile/genética , Infecciones por Clostridium/transmisión , Especiación Genética , Esporas Bacterianas/crecimiento & desarrollo , Azúcares/metabolismo , Virulencia/genética , Antibacterianos/farmacología , Clostridioides difficile/aislamiento & purificación , Infecciones por Clostridium/metabolismo , Infecciones por Clostridium/microbiología , Genoma Bacteriano , Genómica , Humanos , Esporas Bacterianas/efectos de los fármacos , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismoRESUMEN
FBXO7 encodes an F box containing protein that interacts with multiple partners to facilitate numerous cellular processes and has a canonical role as part of an SCF E3 ubiquitin ligase complex. Mutation of FBXO7 is responsible for an early onset Parkinsonian pyramidal syndrome and genome-wide association studies have linked variants in FBXO7 to erythroid traits. A putative orthologue in Drosophila, nutcracker, has been shown to regulate the proteasome, and deficiency of nutcracker results in male infertility. Therefore, we reasoned that modulating Fbxo7 levels in a murine model could provide insights into the role of this protein in mammals. We used a targeted gene trap model which retained 4-16% residual gene expression and assessed the sensitivity of phenotypic traits to gene dosage. Fbxo7 hypomorphs showed regenerative anaemia associated with a shorter erythrocyte half-life, and male mice were infertile. Alterations to T cell phenotypes were also observed, which intriguingly were both T cell intrinsic and extrinsic. Hypomorphic mice were also sensitive to infection with Salmonella, succumbing to a normally sublethal challenge. Despite these phenotypes, Fbxo7 hypomorphs were produced at a normal Mendelian ratio with a normal lifespan and no evidence of neurological symptoms. These data suggest that erythrocyte survival, T cell development and spermatogenesis are particularly sensitive to Fbxo7 gene dosage.