RESUMO
Arginase 1 (ARG1) is a cytosolic enzyme that cleaves L-arginine, the substrate of inducible nitric oxide synthase (iNOS), and thereby impairs the control of various intracellular pathogens. Herein, we investigated the role of ARG1 during infection with Salmonella enterica serovar Typhimurium (S.tm). To study the impact of ARG1 on Salmonella infections in vitro, bone marrow-derived macrophages (BMDM) from C57BL/6N wild-type, ARG1-deficient Tie2Cre+/-ARG1fl/fl and NRAMPG169 C57BL/6N mice were infected with S.tm. In wild-type BMDM, ARG1 was induced by S.tm and further upregulated by the addition of interleukin (IL)-4, whereas interferon-γ had an inhibitory effect. Deletion of ARG1 did not result in a reduction in bacterial numbers. In vivo, Arg1 mRNA was upregulated in the spleen, but not in the liver of C57BL/6N mice following intraperitoneal S.tm infection. The genetic deletion of ARG1 (Tie2Cre+/-ARG1fl/fl) or its pharmacological inhibition with CB-1158 neither affected the numbers of S.tm in spleen, liver and blood nor the expression of host response genes such as iNOS, IL-6 or tumour necrosis factor (TNF). Furthermore, ARG1 was dispensable for pathogen control irrespective of the presence or absence of the phagolysosomal natural resistance-associated macrophage protein 1 (NRAMP1). Thus, unlike the detrimental function of ARG1 seen during infections with other intraphagosomal microorganisms, ARG1 did not support bacterial survival in systemic salmonellosis, indicating differential roles of arginine metabolism for host immune response and microbe persistence depending on the type of pathogen.
Assuntos
Arginase/metabolismo , Citocinas/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Salmonelose Animal/enzimologia , Salmonella typhimurium/fisiologia , Animais , Células da Medula Óssea/microbiologia , Proteínas de Transporte de Cátions , Integrases/metabolismo , Interleucina-4/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pirrolidinas/farmacologia , Regulação para CimaRESUMO
The aim of this study was to evaluate anaemia, serum iron concentrations and δ-aminolevulinate dehydratase (ALA-D) activity in laying hens infected naturally by Salmonella Gallinarum and having severe hepatic lesions. Liver and serum samples were collected from 27 laying hens (20 infected and seven uninfected). The δ-ALA-D activity, haematocrit and serum iron concentrations were evaluated. There were significant decreases in δ-ALA-D activity, haematocrit and serum iron concentrations (P <0.01) in birds infected by S. Gallinarum when compared with uninfected birds. There was a positive correlation (P <0.001) between serum iron concentration, haematocrit (r(2) = 0.82) and δ-ALA-D activity (r(2) = 0.75). A positive correlation was also observed between δ-ALA-D activity and haematocrit (r(2) = 0.78; P <0.01). Liver samples showed moderate focal coagulative necrosis associated with infiltration of lymphoplasmacytic cells, macrophages and heterophils. The anaemia in the infected hens may be related to reduction in δ-ALA-D activity and serum iron concentrations, since both are important for haemopoiesis.
Assuntos
Anemia/veterinária , Sintase do Porfobilinogênio/metabolismo , Salmonelose Animal/complicações , Anemia/etiologia , Animais , Galinhas , Feminino , Ferro/sangue , Salmonelose Animal/enzimologia , Salmonelose Animal/patologia , Salmonella entericaRESUMO
Salmonella Gallinarum is the etiologic agent of fowl typhoid that affects chickens and turkeys causing egg production drops, infertility, lower hatchability, high mortality, and as a consequence severe economic losses to the poultry industry. The alterations in NTPDase and adenosine deaminase (ADA) activities have been demonstrated in several inflammatory conditions; however, there are no data in the literature associated with this infection. Thus, the aim of this study was to evaluate the activities of NTPDase, 5'nucleotidase, and ADA in serum and hepatic tissue of laying hens naturally infected by Salmonella Gallinarum. Liver and serum samples were collected of 27 laying hens (20 S. Gallinarum infected and 7 uninfected). NTPDase and 5'-nucleotidase activities in serum were increased (P < 0.001) in infected animals to hydrolysis of substrate ATP, ADP and AMP. In addition, it was observed decreased (P < 0.001) in ADA activity in serum of laying hens naturally infected by S. Gallinarum; as well as increased (P < 0.001) ADA activity in liver tissue of infected laying hens. Histopathological analyses revealed that S. Gallinarum caused fibrinoid necrosis in liver and spleen associated with infiltrates of heterophils, macrophages, lymphocytes, and plasma cells. Considering that NTPDase and ADA are involved in the cell-mediated immunity, this study suggests that activities of these enzymes could be important biomarkers to determine the severity of inflammatory and immune responses in salmonellosis, contributing to clarify the pathogenesis of the disease.
Assuntos
Adenosina Desaminase/imunologia , Nucleotidases/imunologia , Doenças das Aves Domésticas/enzimologia , Salmonelose Animal/enzimologia , Salmonella enterica/fisiologia , Adenosina Desaminase/genética , Animais , Galinhas/microbiologia , Feminino , Imunidade Celular , Fígado/microbiologia , Fígado/patologia , Nucleotidases/genética , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/patologia , Salmonelose Animal/imunologia , Salmonelose Animal/microbiologia , Salmonelose Animal/patologia , Baço/microbiologia , Baço/patologiaRESUMO
The pnp gene encodes polynucleotide phosphorylase, an exoribonuclease involved in RNA processing and degradation. A mutation in the pnp gene was previously identified by our group in a signature-tagged mutagenesis screen designed to search for Salmonella enterica serovar Typhimurium genes required for survival in an ex vivo swine stomach content assay. In the current study, attenuation and colonization potential of a S. Typhimurium pnp mutant in the porcine host was evaluated. Following intranasal inoculation with 10(9) cfu of either the wild-type S. Typhimurium χ4232 strain or an isogenic derivative lacking the pnp gene (n = 5/group), a significant increase (p < 0.05) in rectal temperature (fever) was observed in the pigs inoculated with wild-type S. Typhimurium compared to the pigs inoculated with the pnp mutant. Fecal shedding of the pnp mutant was significantly reduced during the 7-day study compared to the wild-type strain (p < 0.001). Tissue colonization was also significantly reduced in the pigs inoculated with the pnp mutant compared to the parental strain, including the tonsils, ileocecal lymph nodes, Peyer's Patch region of the ileum, cecum and contents of the cecum (p < 0.05). The data indicate that the pnp gene is required for S. Typhimurium virulence and gastrointestinal colonization of the natural swine host.
Assuntos
Polirribonucleotídeo Nucleotidiltransferase/genética , Salmonelose Animal/enzimologia , Salmonella typhimurium/enzimologia , Doenças dos Suínos/enzimologia , Suínos/microbiologia , Animais , Fezes/microbiologia , Trato Gastrointestinal/microbiologia , Mutação , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Salmonelose Animal/genética , Salmonelose Animal/microbiologia , Salmonella typhimurium/genética , Doenças dos Suínos/genética , Doenças dos Suínos/microbiologiaRESUMO
Caspases are either apoptotic or inflammatory. Among inflammatory caspases, caspase-1 and -11 trigger pyroptosis, a form of programmed cell death. Whereas both can be detrimental in inflammatory disease, only caspase-1 has an established protective role during infection. Here, we report that caspase-11 is required for innate immunity to cytosolic, but not vacuolar, bacteria. Although Salmonella typhimurium and Legionella pneumophila normally reside in the vacuole, specific mutants (sifA and sdhA, respectively) aberrantly enter the cytosol. These mutants triggered caspase-11, which enhanced clearance of S. typhimurium sifA in vivo. This response did not require NLRP3, NLRC4, or ASC inflammasome pathways. Burkholderia species that naturally invade the cytosol also triggered caspase-11, which protected mice from lethal challenge with B. thailandensis and B. pseudomallei. Thus, caspase-11 is critical for surviving exposure to ubiquitous environmental pathogens.
Assuntos
Caspases/metabolismo , Morte Celular , Citosol/microbiologia , Infecções por Bactérias Gram-Negativas/imunologia , Macrófagos/microbiologia , Vacúolos/microbiologia , Animais , Burkholderia/patogenicidade , Burkholderia/fisiologia , Infecções por Burkholderia/enzimologia , Infecções por Burkholderia/imunologia , Infecções por Burkholderia/metabolismo , Burkholderia pseudomallei/patogenicidade , Burkholderia pseudomallei/fisiologia , Caspases Iniciadoras , Infecções por Bactérias Gram-Negativas/enzimologia , Infecções por Bactérias Gram-Negativas/microbiologia , Imunidade Inata , Inflamassomos/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fagossomos/microbiologia , Salmonelose Animal/enzimologia , Salmonelose Animal/imunologia , Salmonelose Animal/microbiologia , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/fisiologiaRESUMO
Inflammasomes are cytosolic multiprotein complexes assembled by intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and they initiate innate immune responses to invading pathogens and danger signals by activating caspase-1 (ref. 1). Caspase-1 activation leads to the maturation and release of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18, as well as lytic inflammatory cell death known as pyroptosis. Recently, a new non-canonical inflammasome was described that activates caspase-11, a pro-inflammatory caspase required for lipopolysaccharide-induced lethality. This study also highlighted that previously generated caspase-1 knockout mice lack a functional allele of Casp11 (also known as Casp4), making them functionally Casp1 Casp11 double knockouts. Previous studies have shown that these mice are more susceptible to infections with microbial pathogens, including the bacterial pathogen Salmonella enterica serovar Typhimurium (S. typhimurium), but the individual contributions of caspase-1 and caspase-11 to this phenotype are not known. Here we show that non-canonical caspase-11 activation contributes to macrophage death during S. typhimurium infection. Toll-like receptor 4 (TLR4)-dependent and TIR-domain-containing adaptor-inducing interferon-ß (TRIF)-dependent interferon-ß production is crucial for caspase-11 activation in macrophages, but is only partially required for pro-caspase-11 expression, consistent with the existence of an interferon-inducible activator of caspase-11. Furthermore, Casp1(-/-) mice were significantly more susceptible to infection with S. typhimurium than mice lacking both pro-inflammatory caspases (Casp1(-/-) Casp11(-/-)). This phenotype was accompanied by higher bacterial counts, the formation of extracellular bacterial microcolonies in the infected tissue and a defect in neutrophil-mediated clearance. These results indicate that caspase-11-dependent cell death is detrimental to the host in the absence of caspase-1-mediated innate immunity, resulting in extracellular replication of a facultative intracellular bacterial pathogen.
Assuntos
Caspases/metabolismo , Suscetibilidade a Doenças/enzimologia , Salmonelose Animal/enzimologia , Adjuvantes Imunológicos/farmacologia , Animais , Caspases Iniciadoras , Morte Celular , Células Cultivadas , Regulação da Expressão Gênica , Inflamassomos/imunologia , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/microbiologia , Camundongos , Camundongos Knockout , Salmonelose Animal/genética , Salmonella typhimurium/fisiologia , Transdução de SinaisRESUMO
We examined antimicrobial susceptibility and efflux systems in laboratory-derived mutants of Salmonella enterica serovar Choleraesuis selected by culture on fluoroquinolone-containing plates. The mutants exhibited decreased susceptibilities to quinolones and several other antimicrobials. Mutations in the gyrA gene were not always found in the mutants. Accumulation assays revealed that intracellular enrofloxacin concentrations were significantly lower in the mutants compared with parent isolates. Increased expression of acrB mRNA can explain the decreased susceptibilities to several antimicrobials but not in the case of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Decreased susceptibility to CCCP may result from the increased expression of emrA mRNA. These results suggest that the enhancement of multiple efflux pumps is responsible for decreased susceptibilities to several antimicrobials in the laboratory-derived mutants.
Assuntos
Anti-Infecciosos/farmacologia , Fluoroquinolonas/farmacologia , Salmonelose Animal/microbiologia , Salmonella enterica/efeitos dos fármacos , Doenças dos Suínos/microbiologia , Animais , Anti-Infecciosos/uso terapêutico , DNA Girase/genética , DNA Girase/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/genética , Fluoroquinolonas/uso terapêutico , Testes de Sensibilidade Microbiana/veterinária , Mutação , Reação em Cadeia da Polimerase/veterinária , Salmonelose Animal/tratamento farmacológico , Salmonelose Animal/enzimologia , Salmonella enterica/enzimologia , Salmonella enterica/genética , Salmonella enterica/metabolismo , Análise de Sequência de DNA , Suínos , Doenças dos Suínos/tratamento farmacológicoRESUMO
pR(ST98) is a chimeric plasmid isolated from Salmonella enterica serovar Typhi (S. typhi) that mediates the functions of drug resistance and virulence. Previously, we reported that Salmonella plasmid virulence (spv) genes were present in S. typhi. In our current study, we investigated whether plasmid pR(ST98) exhibits significant cytotoxicity in macrophages. pR(ST98) was transferred into the avirulent Salmonella enterica serovar Typhimurium (S. typhimurium) strain RIA to create the transconjugant pR(ST98)/RIA. The standard S. typhimurium virulent strain SR-11, which carries a 100-kb virulence plasmid, was used as a positive control. The bacterial strains were incubated with a murine macrophage-like cell line (J774A.1) in vitro. Apoptosis of J774A.1 cells was examined by electron microscopy and flow cytometry after annexin-V/propidium iodide labeling, and the survival of Salmonella strains in J774A.1 cells was determined. Results showed that macrophages infected with strain pR(ST98)/RIA displayed greater levels of apoptosis than those infected with RIA and that pR(ST98 )may increase bacterial survival in macrophages. Further studies showed that the pR(ST98)-induced death of macrophages was associated with the loss of mitochondrial membrane potential and that pR(ST98 )may activate caspase-9 and then caspase-3. The research data indicate that the virulence of bacteria that contain the pR(ST98) plasmid is enhanced; the presence of this plasmid increases the survival of the bacterial pathogen and acts through the mitochondrial pathway to mediate macrophage apoptosis.
Assuntos
Apoptose , Macrófagos/microbiologia , Macrófagos/patologia , Plasmídeos/metabolismo , Salmonelose Animal/microbiologia , Salmonelose Animal/patologia , Salmonella typhi/genética , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Conjugação Genética/efeitos dos fármacos , Eletroforese em Gel de Ágar , Ativação Enzimática/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Citometria de Fluxo , Genes Bacterianos/genética , Macrófagos/enzimologia , Macrófagos/ultraestrutura , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Fosfatidilserinas/farmacologia , Plasmídeos/genética , Reação em Cadeia da Polimerase , Salmonelose Animal/enzimologia , Salmonella typhi/efeitos dos fármacos , Salmonella typhi/ultraestruturaRESUMO
The present study was undertaken to investigate the relationship between plasmid isolated from S. enterica serovar Typhi (pR(ST98)) and macrophage apoptosis. pR(ST98) was transferred into an attenuated S. enterica serovar Typhimurium strain RIA to create a transconjugant pRsT98/RIA. Standard S. enterica serovar Typhimurium virulence strain SR-11 was used as a positive control, and RIA as a negative one. Murine macrophage-like cell line (J774A.1) was used as an infectious cell model in vitro. In order to determine the inhibition and bactericidal effect of amikacin (AMK) to extracellular bacteria and the best optimization co-culture ratio between Salmonella and J774A.1, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of AMK to strains SR-11, pR(ST98)/RIA and RIA and multiplicity of infection (MOI) were detected first, and then J774A.1 was infected by the above three serovar Typhimurium strains. Apoptosis of J774A.1 was examined with electron microscopy and flow cytometry after annexin-V/propidium iodide labeling at 0, 1, 3, 6, 12 and 24 h. Mitochondrial membrane potential was detected by JC-1 staining method. It was demonstrated that MIC of AMK to the three strains was 10 microg/ml, MBC was 80 microg/ml, and optimal MOI was 100:1. pR(ST98)/RIA resulted in a higher apoptosis of J774A.1 than RIA, apoptotic features such as chromatin margination could be observed after 3 h, and death of J774A.1 cells was associated with the loss of mitochondrial membrane potential. These results indicated that pR(ST98) could enhance the virulence of its host bacteria, evidenced by increased macrophage apoptosis.
Assuntos
Apoptose , Macrófagos/microbiologia , Macrófagos/patologia , Salmonelose Animal/microbiologia , Salmonelose Animal/patologia , Salmonella typhi/genética , Amicacina/farmacologia , Animais , Antibacterianos/farmacologia , Caspase 3/metabolismo , Células Cultivadas , Ativação Enzimática , Citometria de Fluxo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Salmonelose Animal/enzimologia , Salmonella typhi/efeitos dos fármacos , Virulência/efeitos dos fármacosRESUMO
Signaling mechanisms of NO-mediated host defense are yet to be elucidated. In this study, we report a unique signal pathway for cytoprotection during Salmonella infection that involves heme oxygenase 1 (HO-1) induced by a nitrated cyclic nucleotide, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP). Wild-type C57BL/6 mice and C57BL/6 mice lacking inducible NO synthase (iNOS) were infected with Salmonella enterica serovar Typhimurium LT2. HO-1 was markedly up-regulated during the infection, the level being significantly higher in wild-type mice than in iNOS-deficient mice. HO-1 up-regulation was associated with 8-nitro-cGMP formation detected immunohistochemically in Salmonella-infected mouse liver and peritoneal macrophages. 8-Nitro-cGMP either exogenously added or formed endogenously induced HO-1 in cultured macrophages infected with Salmonella. HO-1 inhibition by polyethylene glycol-conjugated zinc-protoporphyrin IX impaired intracellular killing of bacteria in mouse liver and in both RAW 264 cells and peritoneal macrophages. Infection-associated apoptosis was also markedly increased in polyethylene glycol-conjugated zinc-protoporphyrin IX-treated mouse liver cells and cultured macrophages. This effect of HO-1 inhibition was further confirmed by using HO-1 short interfering RNA in peritoneal macrophages. Our results suggest that HO-1 induced by NO-mediated 8-nitro-cGMP formation contributes, via its potent cytoprotective function, to host defense during murine salmonellosis.
Assuntos
GMP Cíclico/análogos & derivados , Citoproteção/imunologia , Heme Oxigenase-1/fisiologia , Óxido Nítrico/metabolismo , Salmonelose Animal/imunologia , Salmonelose Animal/patologia , Animais , Linhagem Celular , Células Cultivadas , GMP Cíclico/metabolismo , GMP Cíclico/fisiologia , Indução Enzimática/imunologia , Inibidores Enzimáticos/farmacologia , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/biossíntese , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/fisiologia , Salmonelose Animal/enzimologia , Salmonella typhimurium/imunologia , Transdução de Sinais/imunologia , Regulação para Cima/imunologiaRESUMO
Aerobic metabolism generates biologically challenging reactive oxygen species (ROS) by the endogenous autooxidation of components of the electron transport chain (ETC). Basal levels of oxidative stress can dramatically rise upon activation of the NADPH oxidase-dependent respiratory burst. To minimize ROS toxicity, prokaryotic and eukaryotic organisms express a battery of low-molecular-weight thiol scavengers, a legion of detoxifying catalases, peroxidases, and superoxide dismutases, as well as a variety of repair systems. We present herein blockage of bacterial respiration as a novel strategy that helps the intracellular pathogen Salmonella survive extreme oxidative stress conditions. A Salmonella strain bearing mutations in complex I NADH dehydrogenases is refractory to the early NADPH oxidase-dependent antimicrobial activity of IFNgamma-activated macrophages. The ability of NADH-rich, complex I-deficient Salmonella to survive oxidative stress is associated with resistance to peroxynitrite (ONOO(-)) and hydrogen peroxide (H(2)O(2)). Inhibition of respiration with nitric oxide (NO) also triggered a protective adaptive response against oxidative stress. Expression of the NDH-II dehydrogenase decreases NADH levels, thereby abrogating resistance of NO-adapted Salmonella to H(2)O(2). NADH antagonizes the hydroxyl radical (OH(.)) generated in classical Fenton chemistry or spontaneous decomposition of peroxynitrous acid (ONOOH), while fueling AhpCF alkylhydroperoxidase. Together, these findings identify the accumulation of NADH following the NO-mediated inhibition of Salmonella's ETC as a novel antioxidant strategy. NO-dependent respiratory arrest may help mitochondria and a plethora of organisms cope with oxidative stress engendered in situations as diverse as aerobic respiration, ischemia reperfusion, and inflammation.
Assuntos
Proteínas de Bactérias/metabolismo , Macrófagos Peritoneais/enzimologia , NADPH Desidrogenase/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo , Peroxirredoxinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória , Salmonelose Animal/enzimologia , Salmonella typhimurium/enzimologia , Animais , Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Interferon gama/farmacologia , Macrófagos Peritoneais/microbiologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , NADPH Desidrogenase/genética , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo/genética , Peroxirredoxinas/genética , Ácido Peroxinitroso/farmacologia , Explosão Respiratória/genética , Salmonelose Animal/genética , Salmonella typhimurium/genéticaRESUMO
A recessive Salmonella Typhimurium susceptibility locus (immunity to Typhimurium (Ity3) was reported previously on distal mouse chromosome 1 using a cross between C57BL/6J and wild-derived MOLF/Ei mice. This quantitative trait locus is located in a genomic region spanning 84 Mb, rich in candidate genes for which a role in host resistance to Salmonella infection is either known or can be envisioned. In this study, we report the evaluation of neutrophil cytosolic factor 2 (Ncf2) as a candidate Salmonella susceptibility gene for Ity3. Ncf2 encodes p67phox, a subunit of the multiprotein enzyme complex NADPH oxidase, known to be responsible for the generation of superoxides. Congenic mice carrying the Ity3 region from MOLF/Ei, B6.MOLF-Ity/Ity3 were more susceptible to infection compared with control mice heterozygous at Ity3, B6.MOLF-Ity/Ity3(MOLF/B6), confirming the existence of a recessive Salmonella susceptibility locus on distal chromosome 1. Spleen Ncf2 expression levels were lower in infected congenic mice homozygous for the MOLF/Ei allele at Ity3 compared with mice heterozygous at Ity3. C57BL/6J and MOLF/Ei Ncf2 sequence comparisons revealed one nonconservative amino acid change (R394Q) in the functional and highly conserved Phox and Bem1 domain of the protein. Functional analysis revealed that the MOLF/Ei allele had reduced PMA- and Salmonella-induced superoxide induction as compared with their wild-type counterparts ex vivo. The R394Q substitution seems to occur on an amino acid involved in electrostatic interactions with p40phox, crucial in its activation. Moreover, a human mutation in the corresponding R395W, resulting in chronic granulatomous disease, is known to lead to reduced superoxide levels. These results support the candidacy of Ncf2 as the gene underlying Ity3.
Assuntos
Predisposição Genética para Doença , NADPH Oxidases/biossíntese , NADPH Oxidases/genética , Salmonelose Animal/enzimologia , Salmonelose Animal/genética , Salmonella typhimurium/imunologia , Animais , Proteínas de Transporte de Cátions/genética , Feminino , Marcadores Genéticos , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , NADPH Oxidases/fisiologia , Fenótipo , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Fosfoproteínas/fisiologia , Locos de Características Quantitativas , Salmonelose Animal/imunologia , Análise de Sequência de DNARESUMO
Preinoculation of susceptible 5-day-old gnotobiotic piglets with Salmonella enterica serovar Infantis strain 1326/28Phi(r) stimulates neutrophil migration into the intestine, which rapidly protects the pigs against a subsequent (normally lethal) challenge with S. enterica serovar Typhimurium strain F98. Here we show that inoculation with either 1326/28Phi(r) or F98 activated reactive oxygen species (ROS) in neutrophils via NADPH pathways in vivo and in vitro and that the survival of both Salmonella strains was increased if neutrophils were cocultured with the ROS inhibitor N-acetylcysteine (captopril). Neither F98 nor 1326/28Phi(r) significantly increased reactive nitrogen species (RNS) levels in neutrophils isolated from uninfected pigs. Our results indicate the following: (i) rapid protection of highly susceptible gnotobiotic piglets against F98-induced gastroenteritis by preinoculation with 1326/28Phi(r) is likely to be due to stimulation of ROS-producing neutrophils in the intestinal epithelium prior to challenge with the lethal strain; (ii) pathological lesions of the intestine during severe gastroenteritis are not necessarily induced by neutrophil migration per se; and (iii) if neutrophil migration into the intestine is responsible for pathology, then neither increased production of ROS or RNS (in pigs inoculated with the lethal strain) nor reduced production (in protected pigs in which pathological lesions are ameliorated by preinoculation with 1326/28Phi(r)) can account for this phenomenon.
Assuntos
Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Salmonelose Animal/prevenção & controle , Salmonella typhimurium/imunologia , Doenças dos Suínos/prevenção & controle , Animais , Vida Livre de Germes/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Intestinos/patologia , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , NADPH Oxidases/genética , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salmonelose Animal/enzimologia , Salmonelose Animal/imunologia , Salmonelose Animal/metabolismo , Suínos , Doenças dos Suínos/enzimologia , Doenças dos Suínos/imunologia , Doenças dos Suínos/metabolismo , Doenças dos Suínos/microbiologia , Fatores de TempoRESUMO
Solute carrier family 11a member 1 (Slc11a1; formerly natural resistance-associated macrophage protein 1) encodes a late endosomal/lysosomal protein/divalent cation transporter, which regulates iron homeostasis in macrophages. During macrophage activation, Slc11a1 exerts pleiotropic effects on gene regulation and function, including generation of nitric oxide (NO) via inducible NO synthase (iNOS; encoded by Nos2A) and of reactive oxygen intermediates (ROI) via the phagocyte oxidase complex. As NO and ROI have potent antimicrobial activity in macrophages, it was assumed that their activities would contribute to Slc11a1-regulated innate resistance to Salmonella enterica serovar Typhimurium and Leishmania donovani. By intercrossing mice with gene disruptions at Nos2A and Cybb (encoding gp91phox, the heavy chain subunit of cytochrome b-245 and an essential component of phagocyte NADPH oxidase) onto equivalent Slc11a1 wild-type and mutant genetic backgrounds, we demonstrate that neither iNOS nor gp91phox activity is required for Slc11a1-mediated innate resistance to either infection. Functional gp91phox and iNOS are required to control S. enterica serovar Typhimurium in non-Slc11a1-regulated phases of infection. For L. donovani, an organ-specific requirement for iNOS to clear parasites from the spleen was observed at 50 days post-infection, but neither iNOS nor gp91phox influenced late-phase infection in the liver. This contrasted with Leishmania major infection, which caused rapid lesion growth and death in iNOS knockout mice and some exacerbation of disease with gp91phox deficiency. This highlights the adaptive differences in tissue and cellular tropisms between L. donovani and L. major and the different genes and mechanisms that regulate visceral versus cutaneous forms of the disease.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Leishmaniose Visceral/enzimologia , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase/metabolismo , Fagócitos/enzimologia , Salmonelose Animal/enzimologia , Animais , Proteínas de Transporte de Cátions/genética , Modelos Animais de Doenças , Genótipo , Imunidade Inata/genética , Imunidade Inata/imunologia , Imuno-Histoquímica , Técnicas In Vitro , Leishmania donovani/patogenicidade , Leishmania major/patogenicidade , Leishmaniose Visceral/microbiologia , Fígado/microbiologia , Fígado/parasitologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Salmonelose Animal/microbiologia , Salmonella typhimurium/patogenicidade , Baço/microbiologia , Baço/parasitologiaRESUMO
Cyclooxygenase-2 (COX-2) and nitric oxide synthase 2 (NOS2) were detected and localized in 20 pigs with ulcerative colitis caused by natural infection with Salmonella typhimurium. Evidence of NOS2 activity was determined by the formation of nitrotyrosine, a reaction product of peroxynitrite, in NOS2-expressing ulcerative colons by immunohistochemistry. Transcript RNA of COX-2 and NOS2 was consistently detected in colonic tissues from the 20 pigs with ulcerative colitis by using reverse transcription-polymerase chain reaction. Immunohistochemical signals for COX-2 and NOS2 were detected in the ulcerated area of all 20 pigs. Expression of COX-2 and NOS2 was identified continuously within inflammatory intestinal lesions but was minimal in unaffected regions of the colon of S. typhimurium-infected pigs. The immunohistochemistry of serial sections of intestine indicated that the majority of colons containing numerous COX-2-positive cells also had numerous NOS2-positive cells. Localization of NOS2 and a nitrotyrosine antigen was prominent in neutrophils and macrophages in the periphery of the lesions. Simultaneous detection of COX-2 and NOS2 RNA and protein indicated functional activity of prostaglandin and NO production in vivo. This study suggested that COX-2 and NOS2 expression may play a role in the pathophysiologic processes in ulcerative colitis caused by S. typhimurium.
Assuntos
Colite Ulcerativa/enzimologia , Colite Ulcerativa/veterinária , Isoenzimas/metabolismo , Óxido Nítrico Sintase/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Salmonelose Animal/enzimologia , Salmonella typhimurium/patogenicidade , Doenças dos Suínos/enzimologia , Tirosina/análogos & derivados , Animais , Colite Ulcerativa/microbiologia , Ciclo-Oxigenase 2 , Primers do DNA/genética , Imuno-Histoquímica/veterinária , Isoenzimas/genética , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Ácido Peroxinitroso , Reação em Cadeia da Polimerase , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandinas/biossíntese , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Suínos , Doenças dos Suínos/microbiologia , Tirosina/imunologiaRESUMO
Although numerous studies have demonstrated the ability of intestinal epithelial cells to produce PGs after infection with wild-type strains of Salmonella, few studies have focused on Salmonella-induced prostanoids in mucosal lymphoid tissues. This is surprising in view of the profound effects PGs can have on the host response. To begin to address PG production at mucosal sites, mice were orally inoculated with Salmonella, and at varying times postinfection cyclooxygenase-2 (COX-2) mRNA expression and PGE(2) synthesis were investigated. COX-2 mRNA expression was highly inducible in the mesenteric lymph nodes, whereas COX-1 mRNA levels were constitutive. PGE(2) production also increased significantly in the mesenteric lymph nodes following exposure to viable Salmonella, but not after exposure to killed bacteria. This increased PGE(2) response could be blocked by treatment of mice with the selective COX-2 inhibitor, celecoxib. Treatment of mice with celecoxib during salmonellosis resulted in increased viable bacteria in the mesenteric lymph nodes by day 3 postinfection. However, celecoxib treatment prolonged the survival of lethally infected animals. In vitro studies demonstrated Salmonella-induced up-regulation of COX-2 mRNA expression and PGE(2) secretion by both macrophages and dendritic cells, which could also be blocked in the presence of celecoxib. Interestingly, exposure of these cultured APCs to viable Salmonella was a much greater stimulus for induction of PGE(2) synthesis than exposure to Salmonella-derived LPS. The present study demonstrates induction of PGE(2) synthesis in mesenteric lymph nodes, macrophages, and dendritic cells after infection with wild-type salmonella.
Assuntos
Células Dendríticas/metabolismo , Dinoprostona/biossíntese , Isoenzimas/fisiologia , Linfonodos/metabolismo , Macrófagos/metabolismo , Prostaglandina-Endoperóxido Sintases/fisiologia , Salmonelose Animal/imunologia , Salmonelose Animal/metabolismo , Administração Oral , Animais , Células da Medula Óssea/enzimologia , Células da Medula Óssea/metabolismo , Celecoxib , Células Cultivadas , Ciclo-Oxigenase 2 , Células Dendríticas/enzimologia , Dinoprostona/metabolismo , Feminino , Isoenzimas/antagonistas & inibidores , Isoenzimas/biossíntese , Isoenzimas/genética , Linfonodos/enzimologia , Macrófagos/enzimologia , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/metabolismo , Mesentério , Camundongos , Camundongos Endogâmicos BALB C , Prostaglandina-Endoperóxido Sintases/biossíntese , Prostaglandina-Endoperóxido Sintases/genética , Pirazóis , RNA Mensageiro/biossíntese , Salmonelose Animal/tratamento farmacológico , Salmonelose Animal/enzimologia , Salmonella typhimurium/imunologia , Sulfonamidas/administração & dosagemRESUMO
After bacterial infection, neutrophils dominate the cellular infiltrate. Their main function is assumed to be killing invading pathogens and resolving the inflammation they cause. Activated neutrophils are also known to release a variety of molecules, including the neutrophil serine proteinases, extracellularly. The release of these proteinases during inflammation creates a proteolytic environment where degradation of different molecules modulates the inflammatory response. Flagellin, the structural component of flagella on many bacterial species, is a virulence factor with a strong proinflammatory activity on epithelial cells and other cell types. In this study we show that both human and mouse neutrophil serine proteinases cleave flagellin from Pseudomonas aeruginosa and other bacterial species. More important, cleavage of P. aeruginosa flagellin by the neutrophil serine proteinases neutrophil elastase and cathepsin G resulted in loss of the biological activity of this virulence factor, as evidenced by the lack of innate host defense gene expression in human epithelial cells. The finding that flagellin is susceptible to cleavage by neutrophil serine proteinases suggests a novel role for these enzymes in the inflammatory response to infection. Not only can these enzymes kill bacteria, but they also degrade their virulence factors to halt the inflammatory response they trigger.
Assuntos
Flagelina/antagonistas & inibidores , Flagelina/metabolismo , Neutrófilos/enzimologia , Neutrófilos/microbiologia , Serina Endopeptidases/fisiologia , Animais , Catepsina G , Catepsinas/metabolismo , Linhagem Celular Tumoral , Cromatografia em Gel , Flagelina/isolamento & purificação , Flagelina/farmacologia , Humanos , Hidrólise , Elastase de Leucócito/metabolismo , Camundongos , Camundongos Knockout , Infecções por Pseudomonas/enzimologia , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Salmonelose Animal/enzimologia , Salmonelose Animal/imunologia , Salmonelose Animal/microbiologia , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Serina Endopeptidases/metabolismo , Especificidade por Substrato , VirulênciaRESUMO
The phagolysosomal compartment is crucial for the defense against infection with intracellular pathogens. Within this compartment, the TNF- and IFN-gamma-responsive acid sphingomyelinase (ASMase) generates the signaling molecule ceramide, resulting in the activation of proteases like cathepsin D. To investigate the possible role of ASMase as a mediator of the antibacterial effects of TNF and IFN-gamma, ASMase(-/-) mice were infected with Listeria monocytogenes. ASMase(-/-) mice showed a dramatically increased susceptibility to L. monocytogenes (LD(50) approximately 100 CFU) when compared with syngeneic wild-type mice (LD(50) approximately 10,000 CFU). In L. monocytogenes-challenged ASMase(-/-) mice, IFN-gamma serum levels as well as IL-1 beta and IL-6 secretion by macrophages were similar to those observed in wild-type C57BL/6 mice. Although macrophages and granulocytes from ASMase(-/-) mice showed intact production of reactive nitrogen intermediates and oxidative burst, ASMase(-/-) macrophages proved completely incapable of restricting the growth of L. monocytogenes in vitro. The results of this study suggest that ASMase is crucially required for the intracellular control of L. monocytogenes in macrophages and granulocytes by nonoxidative mechanisms.
Assuntos
Listeria monocytogenes/imunologia , Listeriose/genética , Listeriose/imunologia , Esfingomielina Fosfodiesterase/deficiência , Esfingomielina Fosfodiesterase/genética , Animais , Predisposição Genética para Doença , Imunidade Inata/genética , Dose Letal Mediana , Listeriose/enzimologia , Listeriose/mortalidade , Fígado/imunologia , Fígado/microbiologia , Fígado/patologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose , Oxirredução , Fagocitose/genética , Fagocitose/imunologia , Salmonelose Animal/enzimologia , Salmonelose Animal/genética , Salmonelose Animal/imunologia , Salmonella typhimurium/imunologiaRESUMO
OBJECTIVE: To evaluate hematologic and serum biochemical changes in Salmonella ser Typhimurium-infected calves. ANIMALS: 16 male 3- to 4-week-old dairy calves. PROCEDURE: 13 calves were experimentally infected with S Typhimurium (strains IR715 and CS401, which are derivatives of ATCC 14028), and 3 calves were uninfected controls. Several hematologic and serum biochemical parameters were measured. RESULTS: Hematologic changes included increases in PCV, RBC count, and hemoglobin concentration, associated with a transitory leukopenia characterized by neutropenia and lymphopenia. Biochemical findings included hypoglycemia, increased BUN, creatinine, and fibrinogen concentrations, and decreased sodium, total CO2, calcium, total protein, and albumin concentrations. Increased total bilirubin concentration associated with decreased conjugated bilirubin concentration was also observed. No significant changes in aspartate aminotransferase, gamma-glutamyltranspeptidase, alkaline phosphatase, and creatinine kinase activities were detected. CONCLUSIONS AND CLINICAL RELEVANCE: Experimental salmonellosis of calves results in marked to severe dehydration, accompanied by metabolic acidosis, hypoglycemia, and an acute inflammatory response associated with increased fibrinogen concentrations and severe neutropenia immediately after inoculation.
Assuntos
Doenças dos Bovinos/sangue , Doenças dos Bovinos/microbiologia , Salmonelose Animal/sangue , Salmonelose Animal/microbiologia , Salmonella typhimurium , Fosfatase Alcalina/sangue , Animais , Aspartato Aminotransferases/sangue , Análise Química do Sangue/veterinária , Bovinos , Doenças dos Bovinos/enzimologia , Creatina Quinase/sangue , Contagem de Eritrócitos/veterinária , Feminino , Fibrinogênio/análise , Hematócrito/veterinária , Hemoglobinas/análise , Contagem de Leucócitos/veterinária , Contagem de Linfócitos , Masculino , Salmonelose Animal/enzimologia , gama-Glutamiltransferase/sangueRESUMO
To identify host genes differentially expressed during Salmonella enterica serotype Typhimurium infection, an RNA differential display was made with total RNA extracted from ileal loops that were infected with Salmonella Typhimurium 2.5 h after infection. Down-regulated cDNA was identified in bovine Peyer's patches after infection that was highly homologous to a human plasma membrane calcium-transporting ATPase (PMCA). Differential expression of PMCA, evaluated by Northern analysis, was found to have more than a 4.6-fold decrease in expression of mRNA (size, approximately 5.1 kb). PMCA mRNA was detected by in situ hybridization exclusively within epithelial cells in the Peyer's patches. cDNA (4.4 kb) was amplified by rapid amplification of cDNA ends, cloned, and sequenced and showed a high homology to hPMCA. Bovine PMCA is down-regulated in epithelial cells of Peyer's patches after infection with Salmonella Typhimurium and, subsequently, may influence cellular calcium levels that contribute to the inflammatory processes in the pathogenesis of diarrhea.