RESUMO
The small intestinal tuft cell-ILC2 circuit mediates epithelial responses to intestinal helminths and protists by tuft cell chemosensory-like sensing and IL-25-mediated activation of lamina propria ILC2s. Small intestine ILC2s constitutively express the IL-25 receptor, which is negatively regulated by A20 (Tnfaip3). A20 deficiency in ILC2s spontaneously triggers the circuit and, unexpectedly, promotes adaptive small-intestinal lengthening and remodeling. Circuit activation occurs upon weaning and is enabled by dietary polysaccharides that render mice permissive for Tritrichomonas colonization, resulting in luminal accumulation of acetate and succinate, metabolites of the protist hydrogenosome. Tuft cells express GPR91, the succinate receptor, and dietary succinate, but not acetate, activates ILC2s via a tuft-, TRPM5-, and IL-25-dependent pathway. Also induced by parasitic helminths, circuit activation and small intestinal remodeling impairs infestation by new helminths, consistent with the phenomenon of concomitant immunity. We describe a metabolic sensing circuit that may have evolved to facilitate mutualistic responses to luminal pathosymbionts.
Assuntos
Intestino Delgado/fisiologia , Tritrichomonas/metabolismo , Acetatos/metabolismo , Animais , Fibras na Dieta/metabolismo , Metabolismo Energético , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/parasitologia , Interleucinas/genética , Interleucinas/metabolismo , Mucosa Intestinal/citologia , Intestino Delgado/microbiologia , Intestino Delgado/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microbiota , Plasmídeos/genética , Plasmídeos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Ácido Succínico/metabolismo , Canais de Cátion TRPM/metabolismo , Tritrichomonas/crescimento & desenvolvimento , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismoRESUMO
In the small intestine, type 2 responses are regulated by a signaling circuit that involves tuft cells and group 2 innate lymphoid cells (ILC2s). Here, we identified the microbial metabolite succinate as an activating ligand for small intestinal (SI) tuft cells. Sequencing analyses of tuft cells isolated from the small intestine, gall bladder, colon, thymus, and trachea revealed that expression of tuft cell chemosensory receptors is tissue specific. SI tuft cells expressed the succinate receptor (SUCNR1), and providing succinate in drinking water was sufficient to induce a multifaceted type 2 immune response via the tuft-ILC2 circuit. The helminth Nippostrongylus brasiliensis and a tritrichomonad protist both secreted succinate as a metabolite. In vivo sensing of the tritrichomonad required SUCNR1, whereas N. brasiliensis was SUCNR1 independent. These findings define a paradigm wherein tuft cells monitor microbial metabolites to initiate type 2 immunity and suggest the existence of other sensing pathways triggering the response to helminths.
Assuntos
Imunidade nas Mucosas/efeitos dos fármacos , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ácido Succínico/farmacologia , Animais , Linhagem Celular , Feminino , Mucosa Intestinal/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nippostrongylus/efeitos dos fármacos , Nippostrongylus/imunologia , Nippostrongylus/metabolismo , Especificidade de Órgãos , Infecções por Protozoários/imunologia , Receptores Acoplados a Proteínas G/imunologia , Transdução de Sinais/imunologia , Especificidade da Espécie , Infecções por Strongylida/imunologia , Canais de Cátion TRPM/metabolismo , Células Th2/imunologia , Tritrichomonas/efeitos dos fármacos , Tritrichomonas/imunologia , Tritrichomonas/metabolismoRESUMO
Commensal intestinal protozoa, unlike their pathogenic relatives, are neglected members of the mammalian microbiome. These microbes have a significant impact on the host's intestinal immune homeostasis, typically by elevating anti-microbial host defense. Tritrichomonas musculis, a protozoan gut commensal, strengthens the intestinal host defense against enteric Salmonella infections through Asc- and Il1r1-dependent Th1 and Th17 cell activation. However, the underlying inflammasomes mediating this effect remain unknown. In this study, we report that colonization with T. musculis results in an increase in luminal extracellular ATP that is followed by increased caspase activity, higher cell death, elevated levels of IL-1ß, and increased numbers of IL-18 receptor-expressing Th1 and Th17 cells in the colon. Mice deficient in either Nlrp1b or Nlrp3 failed to display these protozoan-driven immune changes and lost resistance to enteric Salmonella infections even in the presence of T. musculis These findings demonstrate that T. musculis-mediated host protection requires sensors of extracellular and intracellular ATP to confer resistance to enteric Salmonella infections.
Assuntos
Proteínas Reguladoras de Apoptose , Microbiota , Proteína 3 que Contém Domínio de Pirina da Família NLR , Tritrichomonas , Animais , Proteínas Reguladoras de Apoptose/imunologia , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Mamíferos/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Simbiose , Tritrichomonas/metabolismoRESUMO
Tritrichomonas muris is a common flagellated protist isolated from the cecum of wild rodents. This commensal protist has been shown previously to alter immune phenotypes in laboratory mice. Other trichomonads, referred to as Tritrichomonas musculis and Tritrichomonas rainier, also naturally colonize laboratory mice and cause immune alterations. This report formally describes two new trichomonads, Tritrichomonas musculus n. sp., and Tritrichomonas casperi n. sp., at the ultrastructural and molecular level. These two protists were isolated from laboratory mice and were differentiated by their size and the structure of their undulating membrane and posterior flagellum. Analysis at the 18S rRNA and trans-ITS genetic loci supported their designation as distinct species, related to T. muris. To assess the true extent of parabasalid diversity infecting laboratory mice, 135 mice bred at the National Institutes of Health (NIH) were screened using pan-parabasalid primers that amplify the trans-ITS region. Forty-four percent of mice were positive for parabasalids, encompassing a total of eight distinct sequence types. Tritrichomonas casperi and Trichomitus-like protists were dominant. T. musculus and T. rainier were also detected, but T. muris was not. Our work establishes a previously underappreciated diversity of commensal trichomonad flagellates that naturally colonize the enteric cavity of laboratory mice.
Assuntos
Parabasalídeos , Trichomonadida , Tritrichomonas , Animais , Camundongos , Tritrichomonas/ultraestrutura , Trichomonadida/genética , Eucariotos , Flagelos/ultraestruturaRESUMO
The mucus layer in the intestine plays a critical role in regulation of host-microbe interactions and maintaining homeostasis. Disruptions of the mucus layer due to genetic, environmental, or immune factors may lead to inflammatory bowel diseases (IBD). IBD frequently are accompanied with infections, and therefore are treated with antibiotics. Hence, it is important to evaluate risks of antibiotic treatment in individuals with vulnerable gut barrier and chronic inflammation. Mice with a knockout of the Muc2 gene, encoding the main glycoprotein component of the mucus, demonstrate a close contact of the microbes with the gut epithelium which leads to chronic inflammation resembling IBD. Here we demonstrate that the Muc2-/- mice harboring a gut protozoan infection Tritrichomonas sp. are susceptible to an antibiotic-induced depletion of the bacterial microbiota. Suppression of the protozoan infection with efficient metronidazole dosage or L-fucose administration resulted in amelioration of an illness observed in antibiotic-treated Muc2-/- mice. Fucose is a monosaccharide presented abundantly in gut glycoproteins, including Mucin2, and is known to be involved in host-microbe interactions, in particular in microbe adhesion. We suppose that further investigation of the role of fucose in protozoan adhesion to host cells may be of great value.
Assuntos
Fucose/metabolismo , Mucina-2/deficiência , Infecções por Protozoários/etiologia , Infecções por Protozoários/metabolismo , Tritrichomonas/fisiologia , Animais , Antibacterianos/farmacologia , Suscetibilidade a Doenças , Feminino , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Mortalidade , Infecções por Protozoários/tratamento farmacológico , Infecções por Protozoários/mortalidade , Tritrichomonas/classificaçãoRESUMO
Our current understanding of the host-microbiota interaction in the gut is dominated by studies focused primarily on prokaryotic bacterial communities. However, there is an underappreciated symbiotic eukaryotic protistic community that is an integral part of mammalian microbiota. How commensal protozoan bacteria might interact to form a stable microbial community remains poorly understood. Here, we describe a murine protistic commensal, phylogenetically assigned as Tritrichomonas musculis, whose colonization in the gut resulted in a reduction of gut bacterial abundance and diversity in wild-type C57BL/6 mice. Meanwhile, dietary nutrient and commensal bacteria also influenced the protozoan's intestinal colonization and stability. While mice fed a normal chow diet had abundant T. musculis organisms, switching to a Western-type high-fat diet led to the diminishment of the protozoan from the gut. Supplementation of inulin as a dietary fiber to the high-fat diet partially restored the protozoan's colonization. In addition, a cocktail of broad-spectrum antibiotics rendered permissive engraftment of T. musculis even under a high-fat, low-fiber diet. Furthermore, oral administration of Bifidobacterium spp. together with dietary supplementation of inulin in the high-fat diet impacted the protozoan's intestinal engraftment in a bifidobacterial species-dependent manner. Overall, our study described an example of dietary-nutrient-dependent murine commensal protozoan-bacterium cross talk as an important modulator of the host intestinal microbiome.IMPORTANCE Like commensal bacteria, commensal protozoa are an integral part of the vertebrate intestinal microbiome. How protozoa integrate into a commensal bacterium-enriched ecosystem remains poorly studied. Here, using the murine commensal Tritrichomonas musculis as a proof of concept, we studied potential factors involved in shaping the intestinal protozoal-bacterial community. Understanding the rules by which microbes form a multispecies community is crucial to prevent or correct microbial community dysfunctions in order to promote the host's health or to treat diseases.
Assuntos
Fenômenos Fisiológicos Bacterianos , Dieta Hiperlipídica , Microbioma Gastrointestinal/fisiologia , Interações entre Hospedeiro e Microrganismos , Tritrichomonas/fisiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nutrientes/fisiologiaRESUMO
We developed nested PCR protocols and performed a multiyear survey on the prevalence of several protozoan parasites in wild northern bobwhite (Colinus virginianus) and scaled quail (Callipepla squamata) in the Rolling Plains ecoregion of Texas and Oklahoma (i.e. fecal pellets, bird intestines and blood smears collected between 2010 and 2013). Coccidia, cryptosporidia, and microsporidia were detected in 46.2%, 11.7%, and 44.0% of the samples (n = 687), whereas histomona and hematozoa were undetected. Coccidia consisted of one major and two minor Eimeria species. Cryptosporidia were represented by a major unknown Cryptosporidium species and Cryptosporidium baileyi. Detected microsporidia species were highly diverse, in which only 11% were native avian parasites including Encephalitozoon hellem and Encephalitozoon cuniculi, whereas 33% were closely related to species from insects (e.g. Antonospora, Liebermannia, and Sporanauta). This survey suggests that coccidia infections are a significant risk factor in the health of wild quail while cryptosporidia and microsporidia may be much less significant than coccidiosis. In addition, the presence of E. hellem and E. cuniculi (known to cause opportunistic infections in humans) suggests that wild quail could serve as a reservoir for human microsporidian pathogens, and individuals with compromised or weakened immunity should probably take precautions while directly handling wild quail.
Assuntos
Doenças das Aves/parasitologia , Coccídios/isolamento & purificação , Cryptosporidium/isolamento & purificação , Microsporídios/isolamento & purificação , Microsporidiose/veterinária , Infecções Protozoárias em Animais/parasitologia , Codorniz/parasitologia , Trichomonadida/isolamento & purificação , Tritrichomonas/isolamento & purificação , Animais , Doenças das Aves/epidemiologia , Coccídios/genética , Colinus/parasitologia , Criptosporidiose/epidemiologia , Criptosporidiose/parasitologia , Cryptosporidium/genética , DNA de Protozoário/análise , DNA de Protozoário/genética , Fezes/parasitologia , Feminino , Masculino , Microsporídios/genética , Microsporidiose/epidemiologia , Microsporidiose/parasitologia , Oklahoma/epidemiologia , Reação em Cadeia da Polimerase/métodos , Infecções Protozoárias em Animais/diagnóstico , Infecções Protozoárias em Animais/epidemiologia , Codorniz/sangue , Fatores de Risco , Inquéritos e Questionários , Texas/epidemiologia , Trichomonadida/genética , Tritrichomonas/genéticaRESUMO
BACKGROUND: Tritrichomonads like porcine Tritrichomonas foetus (previously named Tritrichomonas suis), can commensally live in nasal cavity of pigs, but it is rare to cause pulmonary tritrichomonosis. CASE PRESENTATION: A 40-day-old piglet was presented for persistent labor breathing and diagnosed with parasite infections in the lung by analysis of bronchoalveolar lavage (BAL) under microscope. By taking advantage of next-generation sequencing approach, we found 9611 homologous tags belonging to 50 annotated genes of tritrichomonads by analysis of mRNA of the bronchoalveolar lavage with the parasite infection. Furthermore, RT-PCR and DNA sequencing analysis confirmed the presence of the tritrichomonad. FINDINGS: Here, we report a case of pulmonary tritrichomonosis in a pig. By taking advantage of next-generation sequencing approach, we found 9611 homologous tags belonging to 50 annotated genes of tritrichomonads by analysis of mRNA of the bronchoalveolar lavage with the parasite infections. Furthermore, RT-PCR and DNA sequencing analysis confirmed the presence of the tritrichomonad. CONCLUSION: Our results demonstrate that tritrichomonads like porcine Tritrichomonas foetus can cause lung infections of pigs and reveal that next-generation sequencing is potential to identify rare diseases like pulmonary tritrichomonosis in clinical.
Assuntos
Pneumopatias/veterinária , Infecções Protozoárias em Animais/diagnóstico , Doenças dos Suínos/diagnóstico , Tritrichomonas , Animais , Líquido da Lavagem Broncoalveolar/parasitologia , Genes de Protozoários/genética , Sequenciamento de Nucleotídeos em Larga Escala/veterinária , Pneumopatias/diagnóstico , Pneumopatias/parasitologia , Microscopia/veterinária , Infecções Protozoárias em Animais/parasitologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Suínos , Doenças dos Suínos/parasitologia , Tritrichomonas/genéticaRESUMO
Commensal protists and gut bacterial communities exhibit complex relationships, mediated at least in part through host immunity. To improve our understanding of this tripartite interplay, we investigated community and functional dynamics between the murine protist Tritrichomonas musculus and intestinal bacteria in healthy and B-cell-deficient mice. We identified dramatic, protist-driven remodeling of resident microbiome growth and activities, in parallel with Tritrichomonas musculus functional changes, which were accelerated in the absence of B cells. Metatranscriptomic data revealed nutrient-based competition between bacteria and the protist. Single-cell transcriptomics identified distinct Tritrichomonas musculus life stages, providing new evidence for trichomonad sexual replication and the formation of pseudocysts. Unique cell states were validated in situ through microscopy and flow cytometry. Our results reveal complex microbial dynamics during the establishment of a commensal protist in the gut, and provide valuable data sets to drive future mechanistic studies.
Assuntos
Microbioma Gastrointestinal , Microbiota , Tritrichomonas , Animais , Camundongos , Eucariotos , BactériasRESUMO
BACKGROUND: Trichomonosis is a common infection in small animals, mostly manifesting in gastrointestinal symptoms such as diarrhea. Although oral trichomonads are also known, the species found colonizing the large intestine are more frequently detected protozoa. METHODS: In the present study, four wildcats, 94 domestic cats, and 25 dogs, originating from 18 different locations in Hungary, were investigated for the presence of oral and large intestinal trichomonads based on the 18S rRNA gene and ITS2. RESULTS: All oral swabs were negative by polymerase chain reaction (PCR). However, Tritrichomonas foetus was detected in a high proportion among tested domestic cats (13.8%) and dogs (16%), and Pentatrichomonas hominis only in two domestic cats. In addition, a novel Tritrichomonas genotype was identified in one cat, probably representing a new species that was shown to be phylogenetically most closely related to Tritrichomonas casperi described recently from mice. All positive dogs and half of the positive cats showed symptoms, and among cats, the most frequent breed was the Ragdoll. CONCLUSIONS: With molecular methods, this study evaluated the prevalence of oral and intestinal trichomonads in clinical samples of dogs and cats from Hungary, providing the first evidence of T. foetus in dogs of this region. In contrast to literature data, P. hominis was more prevalent in cats than in dogs. Finally, a hitherto unknown large intestinal Tritrichomonas species (closely related to T. casperi) was shown to be present in a cat, raising two possibilities. First, this novel genotype might have been a rodent-associated pseudoparasite in the relevant cat. Otherwise, the cat was actually infected, thus suggesting the role of a predator-prey link in the evolution of this trichomonad.
Assuntos
Doenças do Gato , Doenças do Cão , Filogenia , Infecções Protozoárias em Animais , RNA Ribossômico 18S , Animais , Gatos , Cães , Doenças do Gato/parasitologia , Doenças do Gato/epidemiologia , Doenças do Cão/parasitologia , Doenças do Cão/epidemiologia , Infecções Protozoárias em Animais/parasitologia , Infecções Protozoárias em Animais/epidemiologia , Hungria/epidemiologia , RNA Ribossômico 18S/genética , Tritrichomonas/genética , DNA de Protozoário/genética , Feminino , Masculino , Genótipo , Prevalência , Reação em Cadeia da Polimerase , Tritrichomonas foetus/genética , Tritrichomonas foetus/isolamento & purificação , Tritrichomonas foetus/classificaçãoRESUMO
Feline trichomoniasis is an intestinal disease in cats resulting in chronic diarrhea, flatulence, tenesmus, and fecal incontinence. Bovine trichomoniasis is a sexually transmitted disease of cattle infecting the reproductive tract of cows causing pyometra and possible mid- to late-term abortions. The causative agent for both diseases has been reported to be the flagellated protozoan, Tritrichomonas foetus. However, several published reports support significant biological differences between T. foetus isolated from bovines and felines. In the present study, we describe Tritrichomonas blagburni n.sp. from the domestic cat (Felis catus) as the causative agent of feline intestinal trichomoniasis. We support our proposal based on results of experimental cross-infection studies between cats and cattle using both feline and bovine isolates of the parasite, differences in pathogenicity between the two parasites for the respective host species, and molecular gene sequencing differences between parasites obtained from domestic cats and parasites obtained from cattle.
Assuntos
Doenças do Gato/parasitologia , Enteropatias/veterinária , Infecções por Protozoários/parasitologia , Tritrichomonas/classificação , Tritrichomonas/isolamento & purificação , Animais , Gatos , Bovinos , Doenças dos Bovinos/parasitologia , Análise por Conglomerados , DNA de Protozoário/química , DNA de Protozoário/genética , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Genes de RNAr , Enteropatias/parasitologia , Dados de Sequência Molecular , Filogenia , RNA de Protozoário/genética , RNA Ribossômico/genética , RNA Ribossômico 18S/genética , Análise de Sequência de DNARESUMO
Tritrichomonas spp. are parasitic protozoans that proliferate on mucus membranes of the urogenital, gastro-intestinal or nasal tract. For instance, Tritrichomonas foetus is an important cause of reproductive failure in cattle. Some years ago, T. foetus was also identified as a causative agent of diarrhoea in cats. Previous studies on the morphological, physiological and molecular levels have raised doubts as to the phylogenetic relationship among some Tritrichomonas species, particularly in relation to T. foetus, Tritrichomonas suis, and Tritrichomonas mobilensis. With the advent of molecular genetic tools, it has become clear that these three tritrichomonad species are closely related or may even represent the same species. Indeed, since recently, T. suis and T. foetus are generally considered as one species, with T. mobilensis being a closely related sister taxon. To date, molecular studies have not yet been able to resolve the taxonomic (specific) status of T. foetus from cattle and cats. In the future, novel genomic approaches, particularly those involving next generation sequencing are poised to resolve the taxonomy of Tritrichomonas spp. Here, we review the literature on the current state of knowledge of the taxonomy of T. foetus, T. suis, and T. mobilensis with special reference to the relationship between T. foetus from cattle and cats.
Assuntos
DNA de Protozoário/química , Tritrichomonas/classificação , Animais , Gatos , Bovinos , Filogenia , Tritrichomonas/genéticaRESUMO
Recent findings position tuft cells as key mediators of intestinal immunity through their production of the cytokine interleukin (IL)-25 and activation of group 2 innate lymphoid cells (ILC2s). Though tuft cells are found in numerous epithelial tissues, their phenotype and function have been best characterized in the small intestine, where robust in vivo techniques have enabled the dissection of their cellular function, ontogeny, and key signaling pathways. We describe methods for the identification, quantification, and manipulation of tuft cells, focusing on analysis of ILC2s as a readout of tuft cell function. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Ex vivo analysis of small intestinal tuft cells and ILC2 by flow cytometry Alternate Protocol: Ex vivo analysis of small intestinal tuft cells and ILC2 by flow cytometry in the context of type 2 inflammation Basic Protocol 2: Ex vivo analysis of small intestinal tuft cells by imaging of intestinal Swiss roll Basic Protocol 3: Tuft-ILC2 circuit activation by oral gavage of adult Nippostrongylus brasiliensis worms Basic Protocol 4: Circuit activation by colonization with Tritrichomonas spp. Basic Protocol 5: Circuit activation by treatment with succinate in drinking water Basic Protocol 6: Circuit activation by treatment with recombinant IL-25.
Assuntos
Imunidade Inata , Tritrichomonas , Animais , Intestino Delgado , Linfócitos , NippostrongylusRESUMO
The role of p53 in tumor suppression has been extensively studied and well-established. However, the role of p53 in parasitic infections and the intestinal type 2 immunity is unclear. Here, we report that p53 is crucial for intestinal type 2 immunity in response to the infection of parasites, such as Tritrichomonas muris and Nippostrongylus brasiliensis. Mechanistically, p53 plays a critical role in the activation of the tuft cell-IL-25-type 2 innate lymphoid cell circuit, partly via transcriptional regulation of Lrmp in tuft cells. Lrmp modulates Ca2+ influx and IL-25 release, which are critical triggers of type 2 innate lymphoid cell response. Our results thus reveal a previously unrecognized function of p53 in regulating intestinal type 2 immunity to protect against parasitic infections, highlighting the role of p53 as a guardian of immune integrity.
Assuntos
Imunidade Inata/imunologia , Intestinos/imunologia , Nippostrongylus/imunologia , Doenças Parasitárias/imunologia , Tritrichomonas/imunologia , Proteína Supressora de Tumor p53/imunologia , Animais , Linhagem Celular Tumoral , Eosinófilos/imunologia , Eosinófilos/parasitologia , Regulação da Expressão Gênica , Células Caliciformes/imunologia , Células Caliciformes/parasitologia , Interações Hospedeiro-Parasita/imunologia , Humanos , Intestino Delgado/imunologia , Intestino Delgado/metabolismo , Intestino Delgado/parasitologia , Intestinos/parasitologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nippostrongylus/fisiologia , Doenças Parasitárias/metabolismo , Doenças Parasitárias/parasitologia , Tritrichomonas/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Purine metabolism in Giardia lamblia was investigated by monitoring incorporation of radiolabeled precursors into purine nucleotides in the log-phase trophozoites cultivated in vitro in axenic media and incubated in buffered saline glucose. The lack of incorporation of formate, glycine, hypoxanthine, inosine, and xanthine into the nucleotide pool suggests the absence of de novo purine nucleotide synthesis and the inability to form IMP as the precursor of AMP and GMP in G. lamblia. Only adenine, adenosine, guanine, and guanosine were incorporated. Further analysis of the labeled nucleotides by HPLC indicated that adenine and adenosine are converted only to adenine nucleotides whereas guanine and guanosine are only incorporated into guanine nucleotides. There is no competition of incorporation between adenine/adenosine and guanine/guanosine, and there is no interconversion between adenine and guanine nucleotides. Results from analyzing [5'-3H]guanosine incorporation indicate that the ribose moiety is not incorporated with the guanine base. Assays of purine salvage enzymic activities in the crude extracts of G. lamblia revealed the presence of only four major enzymes; adenosine and guanosine hydrolases and adenine and guanine phosphoribosyl transferases. Apparently, G. lamblia has an exceedingly simple purine salvage system; it converts adenosine and guanosine to corresponding purine bases and then forms AMP and GMP by the actions of corresponding purine phosphoribosyl transferases. The guanine phosphoribosyl transferase in G. lamblia is interesting because it does not recognize either hypoxanthine or xanthine as substrate. It thus must have a unique substrate specificity and may be regarded as a potential target to attack as a rational approach to chemotherapeutic control of giardiasis.
Assuntos
Giardia/metabolismo , Nucleotídeos de Purina/metabolismo , Purinas/metabolismo , Nucleotídeos de Adenina/metabolismo , Animais , Nucleotídeos de Guanina/metabolismo , Inosina Monofosfato/metabolismo , N-Glicosil Hidrolases/metabolismo , Pentosiltransferases/metabolismo , Trichomonas vaginalis/metabolismo , Tritrichomonas/metabolismoRESUMO
Tuft cells are an epithelial cell type critical for initiating type 2 immune responses to parasites and protozoa in the small intestine. To respond to these stimuli, intestinal tuft cells use taste chemosensory signaling pathways, but the role of taste receptors in type 2 immunity is poorly understood. In this study, we show that the taste receptor TAS1R3, which detects sweet and umami in the tongue, also regulates tuft cell responses in the distal small intestine. BALB/c mice, which have an inactive form of TAS1R3, as well as Tas1r3-deficient C57BL6/J mice both have severely impaired responses to tuft cell-inducing signals in the ileum, including the protozoa Tritrichomonas muris and succinate. In contrast, TAS1R3 is not required to mount an immune response to the helminth Heligmosomoides polygyrus, which infects the proximal small intestine. Examination of uninfected Tas1r3-/- mice revealed a modest reduction in the number of tuft cells in the proximal small intestine but a severe decrease in the distal small intestine at homeostasis. Together, these results suggest that TAS1R3 influences intestinal immunity by shaping the epithelial cell landscape at steady-state.
Assuntos
Células Epiteliais/imunologia , Mucosa Intestinal/imunologia , Intestino Delgado/imunologia , Receptores Acoplados a Proteínas G/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Células Epiteliais/metabolismo , Microbioma Gastrointestinal , Homeostase , Íleo/imunologia , Íleo/parasitologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitologia , Intestino Delgado/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nematospiroides dubius/imunologia , Receptores Acoplados a Proteínas G/deficiência , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologia , Paladar/fisiologia , Tritrichomonas/imunologiaRESUMO
To determine the localization of several enzymes in Tritrichomonas foetus, the axenic KV-1 strain was grown in Diamond's medium with bovine serum, homogenized in 0.25 M sucrose, and subjected to analytical differential and isopycnic centrifugation. The fractions were assayed for their enzymatic composition and examined electron microscopically. NADH and NADPH dehydrogenases, about 90% of the catalase, and two hydrolases, alpha-galactosidase and manganese-activated beta-galactosidase I are in the nonsedimentable part of the cytoplasm. alpha-Glycerophosphate and malate dehydrogenases are associated with a large particle, whose equilibrium density in sucrose gradients is 1.24. This particle corresponds to that population of the paracostal and paraxostylar granules which, having a uniform granular matrix surrounded by a single membrane, resemble microbodies from other organisms. The small sedimentable portion of catalase (about 10% of the total activity) is not associated with these granules and equilibrates at density 1.22. The nature of the subcellular entity carrying catalase could not be ascertained. Hydrolases with a pH optimum around 6-6.5 (protease, beta-N-acetylglucosaminidase, beta-N-acetylgalactosaminidase, and cation-independent beta-galactosidase II), as well as a large part of acid phosphatase, are associated with a population of large particles which equilibrate at densities from 1.15 to 1.20. The hydrolases in these granules lose their structure-bound latency easily after freezing and thawing. These particles correspond to another population of the paracostal and paraxostylar granules which have varied shape and inhomogeneous content with frequent myelin figures, indicating a digestive function. The rest of the phosphatase and most of the acid beta-glucuronidase activity are in a smaller granule fraction with an equilibrium density around 1.18. The latency of these enzymes is quite resistant to freezing and thawing. This particle population consists of smaller, very often flattened vesicles and granules, many of which are clearly fragments of the prominent Golgi apparatus of the cell.
Assuntos
Catalase/análise , Hidrolases/análise , Oxirredutases/análise , Tritrichomonas/enzimologia , Fosfatase Ácida/análise , Animais , Fracionamento Celular , Centrifugação com Gradiente de Concentração , Grânulos Citoplasmáticos/enzimologia , Congelamento , Galactosidases/análise , Vida Livre de Germes , Glucuronidase/análise , Glicerolfosfato Desidrogenase/análise , Complexo de Golgi/enzimologia , Hexosaminidases/análise , Histocitoquímica , Malato Desidrogenase/análise , Microscopia Eletrônica , NAD , NADP , Peptídeo Hidrolases/análise , Frações Subcelulares/enzimologia , Tritrichomonas/citologiaRESUMO
The costa is an intracellular organelle common to all trichomonads. Costae from Tritrichomonas foetus have been purified by a method which involves lysis of T. foetus with the heat-stable hemolysin produced by Pseudomonas aeruginosa, followed by differential centrifugation. Analysis of the purified costae demonstrated that the organelle is composed of 95 percent carbohydrate and 5 percent protein. The carbohydrate moiety, probably a polysaccharide, consisted of glucose (95 percent), mannose (0.4 percent), glucosamine (1.4 percent), ribose (0.6 percent), and an unidentified sugar (2.6 percent). The kinetosomal complex was attached to the costa after initial lysis of cells but was separated from the costa during purification.
Assuntos
Tritrichomonas/ultraestrutura , Animais , Carboidratos/análise , Bovinos/parasitologia , Fracionamento Celular , Organoides/análise , Proteínas/análise , Tritrichomonas/análiseRESUMO
Intestinal tuft cells are one of 4 secretory cell linages in the small intestine and the source of IL-25, a critical initiator of the type 2 immune response to parasite infection. When Raptor, a critical scaffold protein for mammalian target of rapamycin complex 1 (mTORC1), was acutely deleted in intestinal epithelium via Tamoxifen injection in Tritrichomonas muris (Tm) infected mice, tuft cells, IL-25 in epithelium and IL-13 in the mesenchyme were significantly reduced, but Tm burden was not affected. When Tm infected mice were treated with rapamycin, DCLK1 and IL-25 expression in enterocytes and IL-13 expression in mesenchyme were diminished. After massive small bowel resection, tuft cells and Tm were diminished due to the diet used postoperatively. The elimination of Tm and subsequent re-infection of mice with Tm led to type 2 immune response only in WT, but Tm colonization in both WT and Raptor deficient mice. When intestinal organoids were stimulated with IL-4, tuft cells and IL-25 were induced in both WT and Raptor deficient organoids. In summary, our study reveals that enterocyte specific Raptor is required for initiating a type 2 immune response which appears to function through the regulation of mTORC1 activity.
Assuntos
Enterócitos/citologia , Intestino Delgado/citologia , Infecções Protozoárias em Animais/imunologia , Proteína Regulatória Associada a mTOR/deficiência , Sirolimo/administração & dosagem , Tritrichomonas/imunologia , Animais , Quinases Semelhantes a Duplacortina , Regulação para Baixo , Enterócitos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Imunidade nas Mucosas/efeitos dos fármacos , Interleucina-13/genética , Interleucina-13/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Intestino Delgado/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Infecções Protozoárias em Animais/tratamento farmacológico , Sirolimo/farmacologia , Tamoxifeno/administração & dosagem , Tamoxifeno/farmacologiaRESUMO
The mammalian gastrointestinal tract hosts a diverse community of microbes including bacteria, fungi, protozoa, helminths, and viruses. Through coevolution, mammals and these microbes have developed a symbiosis that is sustained through the host's continuous sensing of microbial factors and the generation of a tolerant or pro-inflammatory response. While analyzing T cell-driven colitis in nonlittermate mouse strains, we serendipitously identified that a nongenetic transmissible factor dramatically increased disease susceptibility. We identified the protozoan Tritrichomonas muris as the disease-exacerbating element. Furthermore, experimental colonization with T. muris induced an elevated Th1 response in the cecum of naive wild-type mice and accelerated colitis in Rag1-/- mice after T cell transfer. Overall, we describe a novel cross-kingdom interaction within the murine gut that alters immune cell homeostasis and disease susceptibility. This example of unpredicted microbial priming of the immune response highlights the importance of studying trans-kingdom interactions and serves as a stark reminder of the importance of using littermate controls in all mouse research.