RESUMO
Ultraviolet radiation's germicidal efficacy depends on several parameters, including wavelength, radiant exposure, microbial physiology, biological matrices, and surfaces. In this work, several ultraviolet radiation sources (a low-pressure mercury lamp, a KrCl excimer, and four UV LEDs) emitting continuous or pulsed irradiation were compared. The greatest log reductions in E. coli cells and B. subtilis endospores were 4.1 ± 0.2 (18 mJ cm-2) and 4.5 ± 0.1 (42 mJ cm-2) with continuous 222 nm, respectively. The highest MS2 log reduction observed was 2.7 ± 0.1 (277 nm at 3809 mJ cm-2). Log reductions of SARS-CoV-2 with continuous 222 nm and 277 nm were ≥ 3.4 ± 0.7, with 13.3 mJ cm-2 and 60 mJ cm-2, respectively. There was no statistical difference between continuous and pulsed irradiation (0.83-16.7% [222 nm and 277 nm] or 0.83-20% [280 nm] duty rates) on E. coli inactivation. Pulsed 260 nm radiation (0.5% duty rate) at 260 nm yielded significantly greater log reduction for both bacteria than continuous 260 nm radiation. There was no statistical difference in SARS-CoV-2 inactivation between continuous and pulsed 222 nm UV-C radiation and pulsed 277 nm radiation demonstrated greater germicidal efficacy than continuous 277 nm radiation. Greater radiant exposure for all radiation sources was required to inactivate MS2 bacteriophage. Findings demonstrate that pulsed irradiation could be more useful than continuous UV radiation in human-occupied spaces, but threshold limit values should be respected. Pathogen-specific sensitivities, experimental setup, and quantification methods for determining germicidal efficacy remain important factors when optimizing ultraviolet radiation for surface decontamination or other applications.
Assuntos
COVID-19 , Raios Ultravioleta , Humanos , SARS-CoV-2 , Escherichia coli/efeitos da radiação , Desinfecção/métodosRESUMO
Salmonella comprises more than 2,600 serovars. Very few environmental and uncommon serovars have been characterized for their potential role in virulence and human infections. A complementary in vitro and in vivo systematic high-throughput analysis of virulence was used to elucidate the association between genetic and phenotypic variations across Salmonella isolates. The goal was to develop a strategy for the classification of isolates as a benchmark and predict virulence levels of isolates. Thirty-five phylogenetically distant strains of unknown virulence were selected from the Salmonella Foodborne Syst-OMICS (SalFoS) collection, representing 34 different serovars isolated from various sources. Isolates were evaluated for virulence in 4 complementary models of infection to compare virulence traits with the genomics data, including interactions with human intestinal epithelial cells, human macrophages, and amoeba. In vivo testing was conducted using the mouse model of Salmonella systemic infection. Significant correlations were identified between the different models. We identified a collection of novel hypothetical and conserved proteins associated with isolates that generate a high burden. We also showed that blind prediction of virulence of 33 additional strains based on the pan-genome was high in the mouse model of systemic infection (82% agreement) and in the human epithelial cell model (74% agreement). These complementary approaches enabled us to define virulence potential in different isolates and present a novel strategy for risk assessment of specific strains and for better monitoring and source tracking during outbreaks.IMPORTANCESalmonella species are bacteria that are a major source of foodborne disease through contamination of a diversity of foods, including meat, eggs, fruits, nuts, and vegetables. More than 2,600 different Salmonella enterica serovars have been identified, and only a few of them are associated with illness in humans. Despite the fact that they are genetically closely related, there is enormous variation in the virulence of different isolates of Salmonella enterica Identification of foodborne pathogens is a lengthy process based on microbiological, biochemical, and immunological methods. Here, we worked toward new ways of integrating whole-genome sequencing (WGS) approaches into food safety practices. We used WGS to build associations between virulence and genetic diversity within 83 Salmonella isolates representing 77 different Salmonella serovars. Our work demonstrates the potential of combining a genomics approach and virulence tests to improve the diagnostics and assess risk of human illness associated with specific Salmonella isolates.
Assuntos
Células Epiteliais/microbiologia , Genoma Bacteriano , Salmonelose Animal/microbiologia , Salmonella/genética , Virulência , Acanthamoeba/microbiologia , Animais , Modelos Animais de Doenças , Feminino , Genômica , Humanos , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Filogenia , Salmonella/classificação , Salmonella/patogenicidade , Salmonelose Animal/sangue , Sorogrupo , Células THP-1 , Sequenciamento Completo do GenomaRESUMO
Salmonella is an intracellular bacterium found in the gastrointestinal tract of mammalian, avian, and reptilian hosts. Mouse models have been extensively used to model in vivo distinct aspects of human Salmonella infections and have led to the identification of several host susceptibility genes. We have investigated the susceptibility of Collaborative Cross strains to intravenous infection with Salmonella enterica serovar Typhimurium as a model of human systemic invasive infection. In this model, strain CC042/GeniUnc (CC042) mice displayed extreme susceptibility with very high bacterial loads and mortality. CC042 mice showed lower spleen weights and decreased splenocyte numbers before and after infection, affecting mostly CD8+ T cells, B cells, and all myeloid cell populations, compared with control C57BL/6J mice. CC042 mice also had lower thymus weights with a reduced total number of thymocytes and double-negative and double-positive (CD4+, CD8+) thymocytes compared to C57BL/6J mice. Analysis of bone marrow-resident hematopoietic progenitors showed a strong bias against lymphoid-primed multipotent progenitors. An F2 cross between CC042 and C57BL/6N mice identified two loci on chromosome 7 (Stsl6 and Stsl7) associated with differences in bacterial loads. In the Stsl7 region, CC042 carried a loss-of-function variant, unique to this strain, in the integrin alpha L (Itgal) gene, the causative role of which was confirmed by a quantitative complementation test. Notably, Itgal loss of function increased the susceptibility to S. Typhimurium in a (C57BL/6J × CC042)F1 mouse background but not in a C57BL/6J mouse inbred background. These results further emphasize the utility of the Collaborative Cross to identify new host genetic variants controlling susceptibility to infections and improve our understanding of the function of the Itgal gene.
Assuntos
Bacteriemia/genética , Antígeno CD11a/deficiência , Predisposição Genética para Doença , Mutação com Perda de Função , Infecções por Salmonella/genética , Salmonella typhimurium/crescimento & desenvolvimento , Animais , Bacteriemia/imunologia , Bacteriemia/patologia , Carga Bacteriana , Medula Óssea/patologia , Modelos Animais de Doenças , Genes , Subpopulações de Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Salmonella/imunologia , Infecções por Salmonella/patologia , Sorogrupo , Baço/patologia , Análise de Sobrevida , Timo/patologiaRESUMO
Salmonella presents a global public health concern. Central to Salmonella pathogenicity is an ability to subvert host defences through strategically targeting host proteins implicated in restricting infection. Therefore, to gain insight into the host-pathogen interactions governing Salmonella infection, we performed an in vivo genome-wide mutagenesis screen to uncover key host defence proteins. This revealed an uncharacterized role of CYRI (FAM49B) in conferring host resistance to Salmonella infection. We show that CYRI binds to the small GTPase RAC1 through a conserved domain present in CYFIP proteins, which are known RAC1 effectors that stimulate actin polymerization. However, unlike CYFIP proteins, CYRI negatively regulates RAC1 signalling, thereby attenuating processes such as macropinocytosis, phagocytosis and cell migration. This enables CYRI to counteract Salmonella at various stages of infection, including bacterial entry into non-phagocytic and phagocytic cells as well as phagocyte-mediated bacterial dissemination. Intriguingly, to dampen its effects, the bacterial effector SopE, a RAC1 activator, selectively targets CYRI following infection. Together, this outlines an intricate host-pathogen signalling interplay that is crucial for determining bacterial fate. Notably, our study also outlines a role for CYRI in restricting infection mediated by Mycobacterium tuberculosis and Listeria monocytogenes. This provides evidence implicating CYRI cellular functions in host defence beyond Salmonella infection.
Assuntos
Infecções Bacterianas/prevenção & controle , Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Infecções Bacterianas/metabolismo , Infecções Bacterianas/microbiologia , Carga Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citoesqueleto/genética , Resistência à Doença/genética , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Listeria monocytogenes/metabolismo , Listeria monocytogenes/fisiologia , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Proteínas Mitocondriais/genética , Mutação , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/fisiologia , Fagocitose , Ligação Proteica , Salmonella typhimurium/metabolismo , Salmonella typhimurium/fisiologia , Análise de SobrevidaRESUMO
Retention of iron in tissue macrophages via upregulation of hepcidin (HAMP) and downregulation of the iron exporter ferroportin (FPN) is thought to participate in the establishment of anemia of inflammation after infection. However, an upregulation of FPN has been proposed to limit macrophages iron access to intracellular pathogens. Therefore, we studied the iron homeostasis and in particular the regulation of FPN after infection with Salmonella enterica serovar Typhimurium in mice presenting tissue macrophages with high iron (AcB61), basal iron (A/J and wild-type mice), or low iron (Hamp knock out, Hamp-/-) levels. The presence of iron in AcB61 macrophages due to extravascular hemolysis and strong erythrophagocytosis activity favored the proliferation of Salmonella in the spleen and liver with a concomitant decrease of FPN protein expression. Despite systemic iron overload, no or slight increase in Salmonella burden was observed in Hamp-/- mice compared to controls. Importantly, FPN expression at both mRNA and protein levels was strongly decreased during Salmonella infection in Hamp-/- mice. The repression of Fpn mRNA was also observed in Salmonella-infected cultured macrophages. In addition, the downregulation of FPN was associated with decreased iron stores in both the liver and spleen in infected mice. Our findings show that during Salmonella infection, FPN is repressed through an iron and hepcidin-independent mechanism. Such regulation likely provides the cellular iron indispensable for the growth of Salmonella inside the macrophages.
RESUMO
Coxsackievirus type B3 (CVB3) is a cardiotropic enterovirus. Infection causes cardiomyocyte necrosis and myocardial inflammation. The damaged tissue that results is replaced with fibrotic or calcified tissue, which can lead to permanently altered cardiac function. The extent of pathogenesis among individuals exposed to CVB3 is dictated by a combination of host genetics, viral virulence, and the environment. Here, we aimed to identify genes that modulate cardiopathology following CVB3 infection. 129S1 mice infected with CVB3 developed increased cardiac pathology compared to 129X1 substrain mice despite no difference in viral burden. Linkage analysis identified a major locus on chromosome 7 (LOD: 8.307, P<0.0001) that controlled the severity of cardiac calcification and necrosis following infection. Sub-phenotyping and genetic complementation assays identified Abcc6 as the underlying gene. Microarray expression profiling identified genotype-dependent regulation of genes associated with mitochondria. Electron microscopy examination showed elevated deposition of hydroxyapatite-like material in the mitochondrial matrices of infected Abcc6 knockout (Abcc6-/-) mice but not in wildtype littermates. Cyclosporine A (CsA) inhibits mitochondrial permeability transition pore opening by inhibiting cyclophilin D (CypD). Treatment of Abcc6 -/- mice with CsA reduced cardiac necrosis and calcification by more than half. Furthermore, CsA had no effect on the CVB3-induced phenotype of doubly deficient CypD-/-Abcc6-/- mice. Altogether, our work demonstrates that mutations in Abcc6 render mice more susceptible to cardiac calcification following CVB3 infection. Moreover, we implicate CypD in the control of cardiac necrosis and calcification in Abcc6-deficient mice, whereby CypD inhibition is required for cardioprotection.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Calcinose/tratamento farmacológico , Infecções por Coxsackievirus/tratamento farmacológico , Ciclosporina/farmacologia , Enterovirus Humano B/efeitos dos fármacos , Inflamação/tratamento farmacológico , Miocardite/tratamento farmacológico , Animais , Calcinose/patologia , Calcinose/virologia , Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/virologia , Peptidil-Prolil Isomerase F , Ciclofilinas/metabolismo , Feminino , Imunossupressores/farmacologia , Inflamação/patologia , Inflamação/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Miocardite/patologia , Miocardite/virologia , NecroseRESUMO
Salmonella enterica is a ubiquitous Gram-negative intracellular bacterium that continues to pose a global challenge to human health. The etiology of Salmonella pathogenesis is complex and controlled by pathogen, environmental, and host genetic factors. In fact, patients immunodeficient in genes in the IL-12, IL-23/IFN-γ pathway are predisposed to invasive nontyphoidal Salmonella infection. Using a forward genomics approach by N-ethyl-N-nitrosourea (ENU) germline mutagenesis in mice, we identified the Ity14 (Immunity to Typhimurium locus 14) pedigree exhibiting increased susceptibility following in vivo Salmonella challenge. A DNA-binding domain mutation (p.G418_E445) in Stat4 (Signal Transducer and Activator of Transcription Factor 4) was the causative mutation. STAT4 signals downstream of IL-12 to mediate transcriptional regulation of inflammatory immune responses. In mutant Ity14 mice, the increased splenic and hepatic bacterial load resulted from an intrinsic defect in innate cell function, IFN-γ-mediated immunity, and disorganized granuloma formation. We further show that NK and NKT cells play an important role in mediating control of Salmonella in Stat4(Ity14/Ity14) mice. Stat4(Ity14/Ity14) mice had increased expression of genes involved in cell-cell interactions and communication, as well as increased CD11b expression on a subset of splenic myeloid dendritic cells, resulting in compromised recruitment of inflammatory cells to the spleen during Salmonella infection. Stat4(Ity14/Ity14) presented upregulated compensatory mechanisms, although inefficient and ultimately Stat4(Ity14/Ity14) mice develop fatal bacteremia. The following study further elucidates the pathophysiological impact of STAT4 during Salmonella infection.
Assuntos
Regulação da Expressão Gênica , Predisposição Genética para Doença , Interferon gama/imunologia , Mutação , Fator de Transcrição STAT4/genética , Salmonelose Animal/genética , Salmonelose Animal/imunologia , Transcrição Gênica , Animais , Carga Bacteriana , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Proteínas de Transporte de Cátions/genética , Análise por Conglomerados , Análise Mutacional de DNA , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Loci Gênicos , Imunidade Inata/genética , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Fígado/imunologia , Fígado/metabolismo , Fígado/microbiologia , Camundongos , Mutação/efeitos dos fármacos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Compostos de Nitrosoureia/toxicidade , Linhagem , Salmonelose Animal/microbiologia , Salmonelose Animal/mortalidade , Salmonella typhimurium/imunologia , Baço/imunologia , Baço/metabolismo , Baço/microbiologia , TranscriptomaRESUMO
Citrobacter rodentium is a natural mouse pathogen widely used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections in humans. While C. rodentium causes self-limiting colitis in most inbred mouse strains, it induces fatal diarrhoea in susceptible strains. The physiological pathways as well as the genetic determinants leading to susceptibility have remained largely uncharacterized. Here we use a forward genetic approach to identify the R-spondin2 gene as a major determinant of susceptibility to C. rodentium infection. Robust induction of R-spondin2 expression during infection in susceptible mouse strains causes a potent Wnt-mediated proliferative response of colonic crypt cells, leading to the generation of an immature and poorly differentiated colonic epithelium with deficiencies in ion-transport components. Our data demonstrate a previously unknown role of R-spondins and Wnt signalling in susceptibility to infectious diarrhoea and identify R-spondin2 as a key molecular link between infection and intestinal homoeostasis.
Assuntos
Citrobacter rodentium/fisiologia , Diarreia/metabolismo , Diarreia/microbiologia , Suscetibilidade a Doenças/microbiologia , Infecções por Enterobacteriaceae/metabolismo , Transdução de Sinais , Trombospondinas/metabolismo , Animais , Diferenciação Celular , Mapeamento Cromossômico , Clonagem Molecular , Colo/microbiologia , Colo/patologia , Diarreia/patologia , Suscetibilidade a Doenças/metabolismo , Suscetibilidade a Doenças/patologia , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/patologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Estudos de Associação Genética , Loci Gênicos/genética , Humanos , Hiperplasia , Camundongos , Camundongos Endogâmicos , Microvilosidades/microbiologia , Microvilosidades/patologia , Modelos Biológicos , Células Estromais/metabolismo , Células Estromais/microbiologia , Células Estromais/patologia , Análise de Sobrevida , Via de Sinalização WntRESUMO
Salmonella, a ubiquitous Gram-negative intracellular bacterium, is a food borne pathogen that infects a broad range of hosts. Infection with Salmonella Typhimurium in mice is a broadly recognized experimental model resembling typhoid fever in humans. Using a N-ethyl-N-nitrosurea (ENU) mutagenesis recessive screen, we report the identification of Ity16 (Immunity to Typhimurium locus 16), a locus responsible for increased susceptibility to infection. The position of Ity16 was refined on chromosome 8 and a nonsense mutation was identified in the ankyrin 1 (Ank1) gene. ANK1 plays an important role in the formation and stabilization of the red cell cytoskeleton. The Ank1(Ity16/Ity16) mutation causes severe hemolytic anemia in uninfected mice resulting in splenomegaly, hyperbilirubinemia, jaundice, extramedullary erythropoiesis and iron overload in liver and kidneys. Ank1(Ity16/Ity16) mutant mice demonstrated low levels of hepcidin (Hamp) expression and significant increases in the expression of the growth differentiation factor 15 (Gdf15), erythropoietin (Epo) and heme oxygenase 1 (Hmox1) exacerbating extramedullary erythropoiesis, tissue iron deposition and splenomegaly. As the infection progresses in Ank1(Ity16/Ity16), the anemia worsens and bacterial load were high in liver and kidneys compared to wild type mice. Heterozygous Ank1(+/Ity16) mice were also more susceptible to Salmonella infection although to a lesser extent than Ank1(Ity16/Ity16) and they did not inherently present anemia and splenomegaly. During infection, iron accumulated in the kidneys of Ank1(+/Ity16) mice where bacterial loads were high compared to littermate controls. The critical role of HAMP in the host response to Salmonella infection was validated by showing increased susceptibility to infection in Hamp-deficient mice and significant survival benefits in Ank1(+/Ity16) heterozygous mice treated with HAMP peptide. This study illustrates that the regulation of Hamp and iron balance are crucial in the host response to Salmonella infection in Ank1 mutants.
Assuntos
Anemia Hemolítica/genética , Anquirinas/genética , Peptídeos Catiônicos Antimicrobianos/genética , Códon sem Sentido/efeitos dos fármacos , Etilnitrosoureia/toxicidade , Sobrecarga de Ferro/genética , Infecções por Salmonella/genética , Anemia Hemolítica/metabolismo , Anemia Hemolítica/microbiologia , Anemia Hemolítica/mortalidade , Animais , Anquirinas/metabolismo , Peptídeos Catiônicos Antimicrobianos/deficiência , Eritrócitos/metabolismo , Eritrócitos/patologia , Eritropoetina/genética , Eritropoetina/metabolismo , Expressão Gênica , Predisposição Genética para Doença , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Hepcidinas , Heterozigoto , Homozigoto , Ferro/metabolismo , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/microbiologia , Sobrecarga de Ferro/mortalidade , Fígado/metabolismo , Fígado/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Infecções por Salmonella/mortalidade , Salmonella typhimurium/fisiologia , Análise de SobrevidaRESUMO
The immune response is essential for survival by destroying microorganisms and pre-cancerous cells. However, inflammation, one aspect of this response, can result in short- and long-term deleterious side-effects. Mclk1âº/â» mutant mice can be long-lived despite displaying a hair-trigger inflammatory response and chronically activated macrophages as a result of high mitochondrial ROS generation. Here we ask whether this phenotype is beneficial or simply tolerated. We used models of infection by Salmonella serovars and found that Mclk1âº/â» mutants mount a stronger immune response, control bacterial proliferation better, and are resistant to cell and tissue damage resulting from the response, including fibrosis and types of oxidative damage that are considered to be biomarkers of aging. Moreover, these same types of tissue damage were found to be low in untreated 23 months-old mutants. We also examined the initiation of tumour growth after transplantation of mouse LLC1 carcinoma cells into Mclk1âº/â» mutants, as well as during spontaneous tumorigenesis in Mclk1âº/â»Trp53âº/â» double mutants. Tumour latency was increased by the Mclk1âº/â» genotype in both models. Furthermore, we used the transplantation model to show that splenic CD8⺠T lymphocytes from Mclk1âº/â» graft recipients show enhanced cytotoxicity against LLC1 cells in vitro. Mclk1âº/â» mutants thus display an association of an enhanced immune response with partial protection from age-dependent processes and from pathologies similar to those that are found with increased frequency during the aging process. This suggests that the immune phenotype of these mutants might contribute to their longevity. We discuss how these findings suggest a broader view of how the immune response might impact the aging process.
Assuntos
Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Mutação , Neoplasias/genética , Neoplasias/imunologia , Envelhecimento/genética , Envelhecimento/imunologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Feminino , Fibrose/genética , Fibrose/imunologia , Heterozigoto , Humanos , Masculino , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Mitocondriais/imunologia , Oxigenases de Função Mista , Neoplasias/mortalidade , Estresse Oxidativo , Infecções por Salmonella/genética , Infecções por Salmonella/imunologia , Infecções por Salmonella/patologia , Salmonella enteritidis/imunologia , Salmonella typhimurium/imunologiaRESUMO
Mitochondrial reactive oxygen species (ROS) are believed to stabilize hypoxia-inducible factor (HIF)-1alpha, a transcriptional regulator of the immune response. Mclk1 encodes a mitochondrial protein that is necessary for ubiquinone biosynthesis. Heterozygote Mclk1(+/-) mutant mice are long-lived despite increased mitochondrial ROS and decreased energy metabolism. In this study, Mclk1(+/-) mutant mice in the C57BL/6J background displayed increased basal and induced expression of HIF-1alpha in liver and macrophages in association with elevated expression of inflammatory cytokines, in particular TNF-alpha. Mutant macrophages showed increased classical and decreased alternative activation, and mutant mice were hypersensitive to LPS. Consistent with these observations in vivo, knock-down of Mclk1 in murine RAW264.7 macrophage-like cells induced increased mitochondrial ROS as well as elevated expression of HIF-1alpha and secretion of TNF-alpha. We used an antioxidant peptide targeted to mitochondria to show that altered ROS metabolism is necessary for the enhanced expression of HIF-1alpha, which, in turn, is necessary for increased TNF-alpha secretion. These findings provide in vivo evidence for the action of mitochondrial ROS on HIF-1alpha activity and demonstrate that changes in mitochondrial function within physiologically tolerable limits modulate the immune response. Our results further suggest that altered immune function through a limited increase in HIF-1alpha expression can positively impact animal longevity.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Imunidade , Proteínas de Membrana/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular , Citocinas/biossíntese , Metabolismo Energético , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Fígado/metabolismo , Longevidade , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/imunologia , Oxigenases de Função Mista , Mutação , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The perpetual evolution of drug-resistant microbes, the overwhelming burden of acquired immune suppression due to HIV, the emergence or re-emergence of various pathogens (West Nile virus, pandemic influenza, Creutzfeld-Jacob disease), and increased fears of bioterrorism has drawn a great deal of new attention to infectious diseases. The pathogenesis of infection is characterized by complex interactions of potentially virulent microorganisms with host genetic and acquired factors. Chemical mutagenesis of the mouse genome provides a robust method to unravel this challenging problem. To deepen our understanding of the natural host response to pathogens, our team and others are interrogating the mouse genome to define genes that are crucial to the defense against infectious diseases (pathogen recognition, viral defense, bacterial defense, prion infection). In this review we highlight the current progress of these efforts and propose a toolbox for other groups that are interested in this endeavor.
Assuntos
Doenças Transmissíveis Emergentes/prevenção & controle , Mutagênese/efeitos dos fármacos , Animais , Doenças Transmissíveis Emergentes/diagnóstico , Doenças Transmissíveis Emergentes/genética , Etilnitrosoureia/farmacologia , Programas de Rastreamento , Camundongos , LinhagemRESUMO
The chronic myeloid leukemia syndrome of the BXH-2 mouse strain (Mus musculus) is caused by a recessive mutation (R294C) in the transcriptional regulator Icsbp1/IRF-8. In trans activation assays using an IL-12p40 gene reporter construct introduced in RAW 264.7 mouse macrophages, we show that the Icsbp1(C294) isoform behaves as a partial loss-of-function. The Icsbp1(C294) hypomorph allele appears to have a threshold effect on IL-12 production, with pleiotropic consequences on resistance to different types of infections in vivo. Despite the presence of a resistance Nramp1(G169) allele, BXH-2 mice (Icsbp1(C294)) show impaired control of Mycobacterium bovis (bacille Calmette-Guérin) multiplication both early and late during infection, with uncontrolled replication linked to inability to form granulomas in infected liver and spleen. Studies in informative (BXH-2 x BALB/cJ)F(2) mice show that homozygosity for Icsbp1(C294) causes susceptibility to Salmonella enterica serovar Typhimurium to a level comparable to that seen for mice lacking functional Nramp1 or TLR4. Finally, impaired Icsbp1(C294) function is associated with the following: 1) increased replication of the Plasmodium chabaudi AS malarial parasite during the first burst of blood parasitemia, and 2) recurring waves of high blood parasitemia late during infection. These results show that Icsbp1 is required for orchestrating early innate responses and also long-term immune protection against unrelated intracellular pathogens.
Assuntos
Imunidade Inata , Fatores Reguladores de Interferon/imunologia , Macrófagos/imunologia , Malária/imunologia , Mycobacterium bovis/imunologia , Infecções por Salmonella/imunologia , Tuberculose/imunologia , Substituição de Aminoácidos , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/imunologia , Linhagem Celular , Genes Recessivos , Imunidade Inata/genética , Fatores Reguladores de Interferon/genética , Subunidade p40 da Interleucina-12/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Macrófagos/microbiologia , Macrófagos/parasitologia , Malária/genética , Malária/veterinária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Mutação de Sentido Incorreto , Parasitemia/genética , Parasitemia/imunologia , Plasmodium chabaudi/imunologia , Infecções por Salmonella/genética , Salmonella typhimurium/imunologia , Síndrome , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Tuberculose/genética , Tuberculose/veterináriaRESUMO
Toll-like receptor (TLR)4 is critical for endotoxin recognition and cellular responses. Using Tlr4 transgenic mice, we investigated the influence of Tlr4 gene dosage on acute respiratory response to endotoxin. Transgenic mice expressing three, six, or 30 copies of Tlr4, control, and Tlr4-deficient mice received intranasal administration of lipopolysaccharide (LPS; 10 ug), and the airway response was analyzed by plethysmography, lung histology, cell recruitment, cytokine and chemokine secretion and protein leakage into the bronchoalveolar space. We demonstrate that overexpression of Tlr4 augmented a LPS-induced bronchoconstrictive effect, as well as tumor necrosis factor and CXC chemokine ligand 1 (keratinocyte-derived chemokine) production. Neutrophil recruitment, microvascular and alveolar epithelial injury with protein leak in the airways, and damage of the lung microarchitecture were Tlr4 gene dose-dependently increased. Therefore, the TLR4 expression level determines the extent of acute pulmonary response to inhaled endotoxin, and TLR4 may thus be a valuable target for immunointervention in acute lung inflammation as a result of endotoxins.
Assuntos
Dosagem de Genes/imunologia , Lipopolissacarídeos/toxicidade , Pneumonia/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Doença Aguda , Administração Intranasal , Animais , Citocinas/biossíntese , Citocinas/imunologia , Relação Dose-Resposta Imunológica , Regulação da Expressão Gênica/imunologia , Lipopolissacarídeos/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neutrófilos/imunologia , Pneumonia/genética , Pneumonia/patologia , Receptor 4 Toll-Like/deficiênciaRESUMO
The extreme susceptibility to infection with Salmonella Typhimurium of wild-derived MOLF/Ei mice has been linked to one genomic region on Chromosome 1 (Ity3). A member of the Toll-like receptors family, Tlr5, located on distal Chromosome 1, was previously shown to be a candidate gene for Ity3 based on expression studies and sequencing analysis. The candidacy of Tlr5 as a Salmonella-susceptibility gene was evaluated functionally by comparing Tlr5 C57BL/6J and MOLF/Ei alleles in vitro and in vivo. In vitro studies showed that the MOLF/Ei Tlr5 allele is more transcriptionally active when the gene is removed from its natural genomic environment. This observation was supported by in vivo studies in B6.MOLF-Ity3 congenic mice that showed that mice homozygous for the MOLF/Ei allele at Ity3, including Tlr5, had an increased response to flagellin as measured by IL-6 and CXCL-1 secretion in the serum compared with parental MOLF/Ei mice. Despite the fact that both MOLF/Ei and B6.MOLF-Ity/Ity3 mice are more susceptible to Salmonella Typhimurium infection than B6.MOLF-Ity mice, they exhibit a different phenotype with respect to Tlr5 expression and Tlr5 signaling, supporting the prediction that Tlr5 is not primarily involved in the disease phenotype underlying the Ity3 locus in MOLF/Ei mice.
Assuntos
Predisposição Genética para Doença/genética , Salmonelose Animal/etiologia , Salmonella typhimurium , Receptor 5 Toll-Like/genética , Animais , Animais Congênicos , Quimiocina CXCL1 , Quimiocinas CXC/biossíntese , Clonagem Molecular , Flagelina/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Interleucina-6/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos MutantesRESUMO
BXH-2 mice develop a fatal myeloid leukemia by a two-step mutagenic process. First, a BXH-2-specific recessive mutation causes a myeloproliferative syndrome. Second, retroviral insertions alter oncogenes or tumor suppressors, resulting in clonal expansion of leukemic cells. We have identified a recessive locus on chromosome 8 (Myls) that is responsible for myeloproliferation in BXH-2. This Myls interval has been narrowed down to 2 Mb and found to contain several positional candidates, including the interferon consensus sequence-binding protein 1 gene (Icsbp, also known as interferon regulatory factor 8 [IRF8]). We show that BXH-2 mice carry a mutation (915 C to T) resulting in an arginine-to-cysteine substitution at position 294 within the predicted IRF association domain of the protein. Although expression of Icsbp1 mRNA transcripts is normal in BXH-2 splenocytes, these cells are unable to produce interleukin 12 and interferon-gamma in response to activating stimuli, confirming that R294C behaves as a loss-of-function mutation. Myeloproliferation in BXH-2 mice is concomitant to increased susceptibility to Mycobacterium bovis (BCG) despite the presence of resistance alleles at the Nramp1 locus. These results suggest a two-step model for chronic myeloid leukemia in BXH-2, in which inactivation of Icsbp1 predisposes to myeloproliferation and immunodeficiency. This event is required for retroviral replication, and subsequent insertional mutagenesis that causes leukemia in BXH-2 mice.
Assuntos
Substituição de Aminoácidos/genética , Predisposição Genética para Doença , Leucemia Mieloide/genética , Mycobacterium bovis , Mutação Puntual , Proteínas Repressoras/genética , Tuberculose/veterinária , Animais , Arginina/genética , Cromossomos de Mamíferos/genética , Cisteína/genética , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/virologia , Fatores Reguladores de Interferon , Interferon gama/biossíntese , Interleucina-12/biossíntese , Leucemia Mieloide/fisiopatologia , Leucemia Mieloide/virologia , Camundongos , Mutagênese Insercional/genética , Mutagênese Insercional/fisiologia , Locos de Características Quantitativas/genética , Locos de Características Quantitativas/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Repressoras/fisiologia , Retroviridae/fisiologia , Baço/citologia , Baço/fisiopatologia , Tuberculose/genética , Tuberculose/virologia , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
The biosynthesis of methionine in bacteria requires the mobilization of sulfur from Cys by the formation and degradation of cystathionine. Cystathionine beta-lyase, encoded by metC in bacteria and STR3 in Schizosaccharomyces pombe, catalyzes the breakdown of cystathionine to homocysteine, the penultimate step in methionine biosynthesis. This enzyme has been suggested to be the target for pyridinamine antimicrobial agents. We have demonstrated, by using purified enzymes from bacteria and yeast, that cystathionine beta-lyase is not the likely target of these agents. Nonetheless, an insertional inactivation of metC in Salmonella enterica serovar Typhimurium resulted in the attenuation of virulence in a mouse model of systemic infection. This result confirms a previous chemical validation of the Met biosynthetic pathway as a target for the development of antibacterial agents and demonstrates that cystathionine beta-lyase is important for bacterial virulence.
Assuntos
Liases/metabolismo , Salmonella typhimurium/patogenicidade , Animais , Antibacterianos/farmacologia , Cistationina/metabolismo , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Liases/antagonistas & inibidores , Liases/genética , Metionina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pirimidinas/farmacologia , Salmonelose Animal/microbiologia , Salmonelose Animal/mortalidade , Salmonella typhimurium/enzimologia , Salmonella typhimurium/genética , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , VirulênciaRESUMO
P2X(7) is a subtype of ATP-gated channels that is highly expressed in astrocytes, microglia, and other immune cells. Activation of P2X(7) purinoceptors by ATP or 3'-O-(4-benzoyl)-benzoyl ATP (BzATP) induces the formation of cytolytic pores and provokes release of interleukin-1beta from immune cells. We investigated the actions of other endogenous nucleotides on recombinant and microglial P2X(7) receptors using electrophysiology, fluorescence imaging, and interleukin-1beta release measurement. We found that initial application of ADP or AMP to Xenopus oocytes expressing P2X(7) receptors was ineffective. However, when ADP and AMP, but not UTP or adenosine, were applied after a brief exposure to ATP or BzATP, they activated P2X(7) receptors in a dose-dependent manner. Moreover, responses to ADP and AMP were also elicited after exposure to low concentrations of ATP and were recorded several minutes after removal of ATP from the extracellular medium. Whole-cell recordings from mouse microglial cells showed that significant responses to ADP and AMP were elicited only after ATP application. YO-PRO-1 dye uptake imaging revealed that, unlike ATP, prolonged application of ADP or AMP did not cause an opening of large cytolytic pores in mouse microglial cells. Finally, ADP and AMP stimulated the release of interleukin-1beta from ATP-primed mouse and human microglial cells. We conclude that selective sensitization of P2X(7) receptors to ADP and AMP requires priming with ATP. This novel property of P2X(7) leads to activation by ATP metabolites and proinflammatory cytokine release from microglia without cytotoxicity.
Assuntos
Difosfato de Adenosina/farmacologia , Monofosfato de Adenosina/farmacologia , Interleucina-1/biossíntese , Microglia/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Comunicação Autócrina/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Hexoquinase/metabolismo , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Microglia/citologia , Microglia/efeitos dos fármacos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Transfecção , Xenopus laevisRESUMO
Studies of mouse models of endotoxemia and sepsis with gram-negative bacteria have shown that the host response is genetically controlled. Mice infected with the gram-negative bacterium Salmonella enterica serovar Typhimurium exhibit marked genetic differences in disease manifestation, and the wild-derived strain Mus musculus molossinus MOLF/Ei is extremely susceptible to S. enterica serovar Typhimurium. The kinetics of bacterial proliferation within the liver and the spleen and histological examination of tissue sections have suggested that MOLF/Ei mice do not succumb to infection because of overwhelming bacterial growth in the reticuloendothelial organs or massive tissue necrosis, as observed in other Salmonella-susceptible strains. MOLF/Ei mice respond normally to lipopolysaccharide (LPS) in vivo and in vitro, as determined by the production of tumor necrosis factor alpha and spleen cell mitogenesis. However, they have a unique cytokine profile in response to infection compared to that observed for other Salmonella-susceptible mice. There was increased expression of mRNA of the interleukin-1 alpha (IL-1 alpha) and IL-1 beta genes as the infection in the spleens and livers of MOLF/Ei mice progressed. Despite the fact that MOLF/Ei mice have the ability to respond to LPS and the fact that there are significant increases in IL-1 alpha and IL-1 beta mRNA, Nos2 in the spleen is not upregulated and nitrite production by spleen cells is reduced. At the central level, the inflammatory response is characterized by strong upregulation of the inhibitory factor kappa B alpha and Toll-like receptor 2 genes, two genes known to be regulated by LPS and IL-1 in the brain. The high levels of IL-1 expression in the spleens and livers of MOLF/Ei mice may have important implications for the activation of peripheral and central innate immune mechanisms.