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1.
PLoS Pathog ; 16(8): e1008766, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32857822

RESUMO

Pathogens commonly disrupt the intestinal epithelial barrier; however, how the epithelial immune system senses the loss of intestinal barrier as a danger signal to activate self-defense is unclear. Through an unbiased approach in the model nematode Caenorhabditis elegans, we found that the EGL-44/TEAD transcription factor and its transcriptional activator YAP-1/YAP (Yes-associated protein) were activated when the intestinal barrier was disrupted by infections with the pathogenic bacterium Pseudomonas aeruginosa PA14. Gene Ontology enrichment analysis of the genes containing the TEAD-binding sites revealed that "innate immune response" and "defense response to Gram-negative bacterium" were two top significantly overrepresented terms. Genetic inactivation of yap-1 and egl-44 significantly reduced the survival rate and promoted bacterial accumulation in worms after bacterial infections. Furthermore, we found that disturbance of the E-cadherin-based adherens junction triggered the nuclear translocation and activation of YAP-1/YAP in the gut of worms. Although YAP is a major downstream effector of the Hippo signaling, our study revealed that the activation of YAP-1/YAP was independent of the Hippo pathway during disruption of intestinal barrier. After screening 10 serine/threonine phosphatases, we identified that PP2A phosphatase was involved in the activation of YAP-1/YAP after intestinal barrier loss induced by bacterial infections. Additionally, our study demonstrated that the function of YAP was evolutionarily conserved in mice. Our study highlights how the intestinal epithelium recognizes the loss of the epithelial barrier as a danger signal to deploy defenses against pathogens, uncovering an immune surveillance program in the intestinal epithelium.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Permeabilidade da Membrana Celular , Células Epiteliais/imunologia , Microbioma Gastrointestinal/imunologia , Salmonelose Animal/imunologia , Salmonella typhimurium/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Camundongos , Salmonelose Animal/metabolismo , Salmonelose Animal/microbiologia , Salmonelose Animal/patologia , Transdução de Sinais
2.
Nat Cell Biol ; 22(6): 663-673, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393887

RESUMO

The linear ubiquitin chain assembly complex (LUBAC), which consists of HOIP, SHARPIN and HOIL-1L, promotes NF-κB activation and protects against cell death by generating linear ubiquitin chains. LUBAC contains two RING-IBR-RING (RBR) ubiquitin ligases (E3), and the HOIP RBR is responsible for catalysing linear ubiquitination. We found that HOIL-1L RBR plays a crucial role in regulating LUBAC. HOIL-1L RBR conjugates monoubiquitin onto all LUBAC subunits, followed by HOIP-mediated conjugation of linear chains onto monoubiquitin, and these linear chains attenuate the functions of LUBAC. The introduction of E3-defective HOIL-1L mutants into cells augmented linear ubiquitination, which protected the cells against Salmonella infection and cured dermatitis caused by reduced LUBAC levels due to SHARPIN loss. Our results reveal a regulatory mode of E3 ligases in which the accessory E3 in LUBAC downregulates the main E3 by providing preferred substrates for autolinear ubiquitination. Thus, inhibition of HOIL-1L E3 represents a promising strategy for treating severe infections or immunodeficiency.


Assuntos
Proteínas de Transporte/fisiologia , Morte Celular , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Dermatite de Contato/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Salmonelose Animal/imunologia , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitina/metabolismo , Animais , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Dermatite de Contato/metabolismo , Dermatite de Contato/patologia , Embrião de Mamíferos/imunologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Salmonella/patogenicidade , Salmonelose Animal/metabolismo , Salmonelose Animal/patologia , Índice de Gravidade de Doença , Transdução de Sinais , Ubiquitinação
3.
Am J Physiol Cell Physiol ; 318(6): C1136-C1143, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32293934

RESUMO

The transport of electrolytes and fluid by the intestinal epithelium is critical in health to maintain appropriate levels of fluidity of the intestinal contents. The transport mechanisms that underlie this physiological process are also subject to derangement in various digestive disease states, such as diarrheal illnesses. This article summarizes the 2019 Hans Ussing Lecture of the Epithelial Transport Group of the American Physiological Society and discusses some pathways by which intestinal transport is dysregulated, particularly in the setting of infection with the diarrheal pathogen, Salmonella, and in patients treated with small-molecule inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor (EGFr-TKI). The burdensome diarrhea in patients infected with Salmonella may be attributable to decreased expression of the chloride-bicarbonate exchanger downregulated in adenoma (DRA) that participates in electroneutral NaCl absorption. This outcome is possibly secondary to increased epithelial proliferation and/or decreased epithelial differentiation that occurs following infection. Conversely, the diarrheal side effects of cancer treatment with EGFr-TKI may be related to the known ability of EGFr-associated signaling to reduce calcium-dependent chloride secretion. Overall, the findings described may suggest targets for therapeutic intervention in a variety of diarrheal disease states.


Assuntos
Antiporters/metabolismo , Diarreia/metabolismo , Células Epiteliais/metabolismo , Absorção Intestinal , Mucosa Intestinal/metabolismo , Transportadores de Sulfato/metabolismo , Animais , Antineoplásicos/toxicidade , Diferenciação Celular , Proliferação de Células , Diarreia/induzido quimicamente , Diarreia/microbiologia , Diarreia/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Camundongos , Permeabilidade , Inibidores de Proteínas Quinases/toxicidade , Salmonelose Animal/metabolismo , Salmonelose Animal/microbiologia , Salmonelose Animal/patologia
4.
FASEB J ; 34(2): 2821-2839, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908018

RESUMO

Newly weaned piglets challenged with Salmonella infantis were particularly susceptible, whereas oral preadministration of Lactobacillus johnsonii L531 alleviated enteritis and promoted intestinal secretory IgA production. Salmonella infantis-induced activation of NLRC4 and NLRP3 inflammasomes and (nuclear factor kappa B) NF-κB signaling in the small intestine was also inhibited by L. johnsonii L531 pretreatment, thus limiting inflammation. An IPEC-J2 cell model of S. infantis infection yielded similar results. Salmonella infantis infection also resulted in mitochondrial damage and impaired mitophagy in the ileum and IPEC-J2 cells, as demonstrated by immunofluorescence colocalization of mitochondria with microtubule-binding protein light chain 3 (LC3) and high expression of autophagy-related proteins PTEN-induced putative kinase 1 (PINK1), sequestosome 1 (SQSTM1/p62), optineurin (OPTN), and LC3 by Western blotting analysis. However, L. johnsonii L531 pretreatment reduced both the extent of mitochondrial damage and autophagy-related protein expression. Our findings suggest that the amelioration of S. infantis-associated enteritis by L. johnsonii L531 is associated with regulation of NLRC4 and NLRP3 inflammasomes and NF-κB signaling pathway activation and suppression of mitochondrial damage. Amelioration of impaired mitophagy by L. johnsonii L531 could involve eliminating damaged mitochondria and regulating S. infantis-induced activation of the NF-κB-SQSTM1mitophagy signaling pathway in host cells to prevent the further mitochondrial damage and S. infantis dissemination.


Assuntos
Diarreia , Enterite , Lactobacillus johnsonii , Mitocôndrias/metabolismo , Mitofagia , Salmonelose Animal , Doenças dos Suínos , Animais , Diarreia/metabolismo , Diarreia/microbiologia , Diarreia/terapia , Diarreia/veterinária , Enterite/metabolismo , Enterite/microbiologia , Enterite/terapia , Enterite/veterinária , Humanos , Salmonelose Animal/metabolismo , Salmonelose Animal/microbiologia , Salmonelose Animal/terapia , Proteína Sequestossoma-1 , Transdução de Sinais , Suínos , Doenças dos Suínos/metabolismo , Doenças dos Suínos/microbiologia , Doenças dos Suínos/terapia
5.
Science ; 366(6468): 995-999, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31753999

RESUMO

The pleiotropic host resistance factor SLC11A1 (NRAMP1) defends against diverse intracellular pathogens in mammals by yet-unknown mechanisms. We compared Salmonella infection of coisogenic mice with different SLC11A1 alleles. SLC11A1 reduced Salmonella replication and triggered up-regulation of uptake systems for divalent metal cations but no other stress responses. SLC11A1 modestly diminished iron availability and acutely restricted Salmonella access to magnesium. Growth of Salmonella cells in the presence of SLC11A1 was highly heterogeneous and inversely correlated with expression of the crucial magnesium transporter gene mgtB We observed superimposable single-cell patterns in mice lacking SLC11A1 when we restricted Salmonella access to magnesium by impairing its uptake. Together, these findings identify deprivation of the main group metal magnesium as the main resistance mechanism of SLC11A1 against Salmonella.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Magnésio/metabolismo , Salmonelose Animal/microbiologia , Salmonella typhimurium/crescimento & desenvolvimento , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/genética , Resistência à Doença/genética , Aptidão Genética , Ferro/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteoma , Salmonelose Animal/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Análise de Célula Única , Baço/microbiologia
6.
PLoS Pathog ; 15(7): e1007915, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31329635

RESUMO

Expression of ABO and Lewis histo-blood group antigens by the gastrointestinal epithelium is governed by an α-1,2-fucosyltransferase enzyme encoded by the Fut2 gene. Alterations in mucin glycosylation have been associated with susceptibility to various bacterial and viral infections. Salmonella enterica serovar Typhimurium is a food-borne pathogen and a major cause of gastroenteritis. In order to determine the role of Fut2-dependent glycans in Salmonella-triggered intestinal inflammation, Fut2+/+ and Fut2-/- mice were orally infected with S. Typhimurium and bacterial colonization and intestinal inflammation were analyzed. Bacterial load in the intestine of Fut2-/- mice was significantly lower compared to Fut2+/+ mice. Analysis of histopathological changes revealed significantly lower levels of intestinal inflammation in Fut2-/- mice compared to Fut2+/+ mice and measurement of lipocalin-2 level in feces corroborated histopathological findings. Salmonella express fimbriae that assist in adherence of bacteria to host cells thereby facilitating their invasion. The std fimbrial operon of S. Typhimurium encodes the π-class Std fimbriae which bind terminal α(1,2)-fucose residues. An isogenic mutant of S. Typhimurium lacking Std fimbriae colonized Fut2+/+ and Fut2-/- mice to similar levels and resulted in similar intestinal inflammation. In vitro adhesion assays revealed that bacteria possessing Std fimbriae adhered significantly more to fucosylated cell lines or primary epithelial cells in comparison to cells lacking α(1,2)-fucose. Overall, these results indicate that Salmonella-triggered intestinal inflammation and colonization are dependent on Std-fucose interaction.


Assuntos
Fímbrias Bacterianas/metabolismo , Fucose/metabolismo , Salmonella typhimurium/patogenicidade , Animais , Aderência Bacteriana , Colite/etiologia , Colite/metabolismo , Colite/microbiologia , Feminino , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/genética , Fucosiltransferases/deficiência , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Interações entre Hospedeiro e Microrganismos , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Knockout , Óperon , Salmonelose Animal/etiologia , Salmonelose Animal/metabolismo , Salmonelose Animal/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologia
7.
Cell Rep ; 28(3): 804-818.e7, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31315056

RESUMO

Toll-like receptors (TLRs) activate innate immunity via interactions between their Toll/interleukin-1 (IL-1) receptor (TIR) domain and downstream adaptor proteins. Here we report that Salmonella Enteritidis produces a secreted protein (TcpS) that contains both a TIR domain and a coiled-coil domain. TcpS blocks MyD88- and TRIF-mediated TLR signaling, inhibits inflammatory responses, and promotes bacterial survival. Early-stage immune evasion by TcpS results in severe tissue damage in the late stage of infection and contributes to Salmonella virulence. TcpS-derived peptides inhibit nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) activation and reduce lipopolysaccharide (LPS)-elicited systemic inflammation. Therapeutic peptide administration alleviates weight loss of mice infected with H1N1 influenza. Importantly, maximal TcpS-mediated TLR inhibition requires the critical TIR-TcpS residues Y191 and I284, as well as TcpS homodimerization via its N-terminal coiled-coil domain. Our study unveils a mechanism in which TcpS suppresses innate immunity via both its homodimerization and interaction with MyD88. TcpS is also a potential therapeutic agent for inflammation-associated diseases.


Assuntos
Proteínas de Bactérias/metabolismo , Imunidade Inata , Inflamação/imunologia , Salmonelose Animal/imunologia , Salmonella enteritidis/patogenicidade , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Citocinas/metabolismo , Dimerização , Células HEK293 , Humanos , Evasão da Resposta Imune/genética , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Infecções por Orthomyxoviridae/imunologia , Domínios Proteicos/genética , Estrutura Terciária de Proteína , Salmonelose Animal/genética , Salmonelose Animal/metabolismo , Salmonella enteritidis/química , Salmonella enteritidis/genética , Salmonella enteritidis/crescimento & desenvolvimento , Receptores Toll-Like/antagonistas & inibidores , Receptores Toll-Like/metabolismo , Virulência/genética
8.
PLoS Pathog ; 15(7): e1007847, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31306468

RESUMO

Salmonella exploit host-derived nitrate for growth in the lumen of the inflamed intestine. The generation of host-derived nitrate is dependent on Nos2, which encodes inducible nitric oxide synthase (iNOS), an enzyme that catalyzes nitric oxide (NO) production. However, the cellular sources of iNOS and, therefore, NO-derived nitrate used by Salmonella for growth in the lumen of the inflamed intestine remain unidentified. Here, we show that iNOS-producing inflammatory monocytes infiltrate ceca of mice infected with Salmonella. In addition, we show that inactivation of type-three secretion system (T3SS)-1 and T3SS-2 renders Salmonella unable to induce CC- chemokine receptor-2- and CC-chemokine ligand-2-dependent inflammatory monocyte recruitment. Furthermore, we show that the severity of the pathology of Salmonella- induced colitis as well as the nitrate-dependent growth of Salmonella in the lumen of the inflamed intestine are reduced in mice that lack Ccr2 and, therefore, inflammatory monocytes in the tissues. Thus, inflammatory monocytes provide a niche for Salmonella expansion in the lumen of the inflamed intestine.


Assuntos
Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Monócitos/metabolismo , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Animais , Quimiocina CCL2/deficiência , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Feminino , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Inflamação/metabolismo , Inflamação/microbiologia , Inflamação/patologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Monócitos/patologia , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores CCR2/deficiência , Receptores CCR2/genética , Receptores CCR2/metabolismo , Salmonelose Animal/metabolismo , Salmonelose Animal/microbiologia , Salmonelose Animal/patologia , Salmonella typhimurium/genética , Sistemas de Secreção Tipo III/metabolismo
9.
Inflamm Bowel Dis ; 25(10): 1629-1643, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31066456

RESUMO

BACKGROUND: Intestinal fibrosis is a common and serious complication of Crohn's disease characterized by the accumulation of fibroblasts, deposition of extracellular matrix, and formation of scar tissue. Although many factors including cytokines and proteases contribute to the development of intestinal fibrosis, the initiating mechanisms and the complex interplay between these factors remain unclear. METHODS: Chronic infection of mice with Salmonella enterica serovar Typhimurium was used to induce intestinal fibrosis. A murine protease-specific CLIP-CHIP microarray analysis was employed to assess regulation of proteases and protease inhibitors. To confirm up- or downregulation during fibrosis, we performed quantitative real-time polymerase chain reaction (PCR) and immunohistochemical stainings in mouse tissue and tissue from patients with inflammatory bowel disease. In vitro infections were used to demonstrate a direct effect of bacterial infection in the regulation of proteases. RESULTS: Mice develop severe and persistent intestinal fibrosis upon chronic infection with Salmonella enterica serovar Typhimurium, mimicking the pathology of human disease. Microarray analyses revealed 56 up- and 40 downregulated proteases and protease inhibitors in fibrotic cecal tissue. Various matrix metalloproteases, serine proteases, cysteine proteases, and protease inhibitors were regulated in the fibrotic tissue, 22 of which were confirmed by quantitative real-time PCR. Proteases demonstrated site-specific staining patterns in intestinal fibrotic tissue from mice and in tissue from human inflammatory bowel disease patients. Finally, we show in vitro that Salmonella infection directly induces protease expression in macrophages and epithelial cells but not in fibroblasts. CONCLUSIONS: In summary, we show that chronic Salmonella infection regulates proteases and protease inhibitors during tissue fibrosis in vivo and in vitro, and therefore this model is well suited to investigating the role of proteases in intestinal fibrosis.


Assuntos
Fibrose/metabolismo , Enteropatias/metabolismo , Macrófagos/metabolismo , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/metabolismo , Salmonelose Animal/complicações , Salmonella enterica/patogenicidade , Animais , Citocinas/metabolismo , Fibrose/microbiologia , Fibrose/patologia , Perfilação da Expressão Gênica , Enteropatias/microbiologia , Enteropatias/patologia , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Peptídeo Hidrolases/genética , Salmonelose Animal/metabolismo , Salmonelose Animal/microbiologia
10.
Environ Sci Pollut Res Int ; 26(16): 16274-16278, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30977003

RESUMO

This study evaluated the effects of Bacillus subtilis, Saccharomyces boulardii, oregano, and calcium montmorillonite on the physical growth, intestinal histomorphology, and blood metabolites in Salmonella-challenged birds during the finisher phase. In this study, a total of 600 chicks (Ross 308) were randomly distributed into the following dietary treatments: basal diet with no treatment; infected with Salmonella; T1, infected + avilamycin; T2, infected + Bacillus subtilis; T3, Saccharomyces boulardii; T4, infected + oregano; T5, infected + calcium montmorillonite. Our results indicated that feed consumption, body weight gain, total body weight, and feed conversion ratio increased significantly (P < 0.01) in T1 and T2. Villus width increased significantly (P < 0.01) in T1 while the total area was significantly (P < 0.01) higher in T1 and T2 among the treatment groups. Blood protein was significantly (P < 0.01) high in T3 and T4; however, the glucose concentration was significantly (P < 0.01) high in T2, T3, and T4. The treatments increased significantly (P< 0.01) in the treatment groups compared to the negative control. Aspirate aminotransferase (AST) was significantly (P < 0.05) low in T3 compared to the positive control. In conclusion, the results indicated that supplementation of Bacillus subtilis and calcium montmorillonite improved the production performance compared to other feed additives in broiler chicks infected with Salmonella during the finisher phase.


Assuntos
Bentonita/farmacologia , Galinhas/crescimento & desenvolvimento , Intestinos/efeitos dos fármacos , Origanum , Salmonelose Animal/dietoterapia , Ração Animal/análise , Animais , Bacillus subtilis , Sangue/metabolismo , Sangue/microbiologia , Peso Corporal/efeitos dos fármacos , Cálcio/farmacologia , Galinhas/metabolismo , Galinhas/microbiologia , Intestinos/microbiologia , Intestinos/patologia , Oligossacarídeos/farmacologia , Doenças das Aves Domésticas/microbiologia , Probióticos/farmacologia , Saccharomyces boulardii , Salmonelose Animal/metabolismo , Salmonella enterica/patogenicidade , Hormônios Tireóideos/sangue
11.
Autophagy ; 15(5): 796-812, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30676840

RESUMO

Innate immune defense against intracellular pathogens, like Salmonella, relies heavily on the autophagy machinery of the host. This response is studied intensively in epithelial cells, the target of Salmonella during gastrointestinal infections. However, little is known of the role that autophagy plays in macrophages, the predominant carriers of this pathogen during systemic disease. Here we utilize a zebrafish embryo model to study the interaction of S. enterica serovar Typhimurium with the macroautophagy/autophagy machinery of macrophages in vivo. We show that phagocytosis of live but not heat-killed Salmonella triggers recruitment of the autophagy marker GFP-Lc3 in a variety of patterns labeling tight or spacious bacteria-containing compartments, also revealed by electron microscopy. Neutrophils display similar GFP-Lc3 associations, but genetic modulation of the neutrophil/macrophage balance and ablation experiments show that macrophages are critical for the defense response. Deficiency of atg5 reduces GFP-Lc3 recruitment and impairs host resistance, in contrast to atg13 deficiency, indicating that Lc3-Salmonella association at this stage is independent of the autophagy preinitiation complex and that macrophages target Salmonella by Lc3-associated phagocytosis (LAP). In agreement, GFP-Lc3 recruitment and host resistance are impaired by deficiency of Rubcn/Rubicon, known as a negative regulator of canonical autophagy and an inducer of LAP. We also found strict dependency on NADPH oxidase, another essential factor for LAP. Both Rubcn and NADPH oxidase are required to activate a Salmonella biosensor for reactive oxygen species inside infected macrophages. These results identify LAP as the major host protective autophagy-related pathway responsible for macrophage defense against Salmonella during systemic infection. Abbreviations: ATG: autophagy related gene; BECN1: Beclin 1; CFU: colony forming units; CYBA/P22PHOX: cytochrome b-245, alpha chain; CYBB/NOX2: cytochrome b-245 beta chain; dpf: days post fertilization; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein; hfp: hours post fertilization; hpi: hours post infection; IRF8: interferon regulatory factor 8; Lcp1/L-plastin: lymphocyte cytosolic protein 1; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1A/1B-light chain 3; mCherry: red fluorescent protein; mpeg1: macrophage expressed gene 1; mpx: myeloid specific peroxidase; NADPH oxidase: nicotinamide adenine dinucleotide phosphate oxidase; NCF4/P40PHOX: neutrophil cytosolic factor 4; NTR-mCherry: nitroreductase-mCherry fusion; PTU: phenylthiourea; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; RB1CC1/FIP200: RB-1 inducible coiled coin 1; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; RUBCN/RUBICON: RUN and cysteine rich domain containing BECN1-interacting protein; SCV: Salmonella-containing vacuole; S. Typhimurium/S.T: Salmonella enterica serovar Typhimurium; TEM: transmission electron microscopy; Tg: transgenic; TSA: tyramide signal amplification; ULK1/2: unc-51-like autophagy activating kinase 1/2; UVRAG: UVRAG: UV radiation resistance associated; wt: wild type.


Assuntos
Modelos Animais de Doenças , Macrófagos/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Fagocitose/genética , Salmonelose Animal , Salmonella typhimurium/imunologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Autofagia/fisiologia , Bacteriemia/genética , Bacteriemia/imunologia , Bacteriemia/microbiologia , Bacteriemia/patologia , Embrião não Mamífero , Proteínas Associadas aos Microtúbulos/genética , Fagocitose/imunologia , Espécies Reativas de Oxigênio/metabolismo , Salmonelose Animal/genética , Salmonelose Animal/imunologia , Salmonelose Animal/metabolismo , Salmonelose Animal/microbiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Peixe-Zebra/microbiologia , Proteínas de Peixe-Zebra/genética
12.
PLoS Pathog ; 14(10): e1007391, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30379938

RESUMO

Salmonella enterica serovar Typhimurium (S. Tm) is a cause of food poisoning accompanied with gut inflammation. Although mucosal inflammation is generally thought to be protective against bacterial infection, S. Tm exploits the inflammation to compete with commensal microbiota, thereby growing up to high densities in the gut lumen and colonizing the gut continuously at high levels. However, the molecular mechanisms underlying the beneficial effect of gut inflammation on S. Tm competitive growth are poorly understood. Notably, the twin-arginine translocation (Tat) system, which enables the transport of folded proteins outside bacterial cytoplasm, is well conserved among many bacterial pathogens, with Tat substrates including virulence factors and virulence-associated proteins. Here, we show that Tat and Tat-exported peptidoglycan amidase, AmiA- and AmiC-dependent cell division contributes to S. Tm competitive fitness advantage in the inflamed gut. S. Tm tatC or amiA amiC mutants feature a gut colonization defect, wherein they display a chain form of cells. The chains are attributable to a cell division defect of these mutants and occur in inflamed but not in normal gut. We demonstrate that attenuated resistance to bile acids confers the colonization defect on the S. Tm amiA amiC mutant. In particular, S. Tm cell chains are highly sensitive to bile acids as compared to single or paired cells. Furthermore, we show that growth media containing high concentrations of NaCl and sublethal concentrations of antimicrobial peptides induce the S. Tm amiA amiC mutant chain form, suggesting that gut luminal conditions such as high osmolarity and the presence of antimicrobial peptides impose AmiA- and AmiC-dependent cell division on S. Tm. Together, our data indicate that Tat and the Tat-exported amidases, AmiA and AmiC, are required for S. Tm luminal fitness in the inflamed gut, suggesting that these proteins might comprise effective targets for novel antibacterial agents against infectious diarrhea.


Assuntos
Amidoidrolases/metabolismo , Trato Gastrointestinal/microbiologia , Inflamação/microbiologia , Peptidoglicano/metabolismo , Salmonelose Animal/microbiologia , Salmonella typhimurium/fisiologia , Sistema de Translocação de Argininas Geminadas/metabolismo , Animais , Divisão Celular , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/patologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Salmonelose Animal/metabolismo , Salmonelose Animal/patologia
13.
Int J Mol Sci ; 19(10)2018 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-30360353

RESUMO

Mounting evidence has indicated that lipopolysaccharide (LPS) is implicated in neuroimmunological responses, but the body's response to subclinical doses of bacterial endotoxin remains poorly understood. The influence of a low single dose of LPS from Salmonella Enteritidis, which does not result in any clinical symptoms of intoxication (subclinical lipopolysaccharide), on selected cells and signal molecules of the neuroimmune system was tested. Five juvenile crossbred female pigs were intravenously injected with LPS from S. Enteritidis (5 µg/kg body weight (b.w.)), while five pigs from the control group received sodium chloride in the same way. Our data demonstrated that subclinical LPS from S. Enteritidis increased levels of dopamine in the brain and neuropeptides such as substance P (SP), galanin (GAL), neuropeptide Y (NPY), and active intestinal peptide (VIP) in the cervical lymph nodes with serum hyperhaptoglobinaemia and reduction of plasma CD4 and CD8 T-lymphocytes seven days after lipopolysaccharide administration. CD4 and CD8 T-lymphocytes from the cervical lymph node and serum interleukin-6 and tumour necrosis factor α showed no significant differences between the control and lipopolysaccharide groups. Subclinical lipopolysaccharide from S. Enteritidis can affect cells and signal molecules of the neuroimmune system. The presence of subclinical lipopolysaccharide from S. Enteritidis is associated with unknown prolonged consequences and may require eradication and a deeper search into the asymptomatic carrier state of Salmonella spp.


Assuntos
Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Salmonelose Animal/imunologia , Salmonella enteritidis/imunologia , Salmonella enteritidis/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Dopamina/metabolismo , Haptoglobinas/metabolismo , Interleucina-6/sangue , Neuropeptídeos/metabolismo , Córtex Pré-Frontal/imunologia , Córtex Pré-Frontal/metabolismo , Salmonelose Animal/sangue , Salmonelose Animal/metabolismo , Suínos , Fator de Necrose Tumoral alfa/sangue
14.
J Immunol ; 201(9): 2710-2720, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30249808

RESUMO

Perforin-2, the product of the MPEG1 gene, limits the spread and dissemination of bacterial pathogens in vivo. It is highly expressed in murine and human phagocytes, and macrophages lacking Perforin-2 are compromised in their ability to kill phagocytosed bacteria. In this study, we used Salmonella enterica serovar Typhimurium as a model intracellular pathogen to elucidate the mechanism of Perforin-2's bactericidal activity. In vitro Perforin-2 was found to facilitate the degradation of Ags contained within the envelope of phagocytosed bacteria. In contrast, degradation of a representative surface Ag was found to be independent of Perforin-2. Consistent with our in vitro results, a protease-sensitive, periplasmic superoxide dismutase (SodCII) contributed to the virulence of S. Typhimurium in Perforin-2 knockout but not wild-type mice. In aggregate, our studies indicate that Perforin-2 breaches the envelope of phagocytosed bacteria, facilitating the delivery of proteases and other antimicrobial effectors to sites within the bacterial cell.


Assuntos
Proteínas Citotóxicas Formadoras de Poros/imunologia , Salmonelose Animal/imunologia , Animais , Parede Celular , Camundongos , Camundongos Knockout , Fagocitose/imunologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Salmonelose Animal/metabolismo , Salmonella typhimurium
15.
Vet Res Commun ; 42(4): 265-273, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30097755

RESUMO

Pigs are an important livestock and serve as a large animal model due to physiological and anatomical similarities with humans. Thus, components of the porcine immune system such as inflammasomes need to be characterized for disease control, vaccination, and translational research purposes. Previously, we and others elucidated porcine nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family Pyrin domain containing 3 (NLRP3) inflammasome activation. However, until now, porcine NLR family caspase recruitment domain (CARD)-containing 4 (NLRC4) and absent in melanoma 2 (AIM2) inflammasomes have been not well studied. In this study, we treated well defined NLRC4 and AIM2 inflammasome triggers to porcine peripheral blood mononuclear cells (PBMCs) and murine bone-marrow derived macrophages (BMDMs) and observed interleukin (IL)-1ß maturation as a readout of inflammasome activation. NLRC4 (flagellin) and AIM2 (dsDNA) triggers led to IL-1ß secretion in both porcine PBMCs and mice macrophages. In addition, porcine and mouse NLRC4 and AIM2 inflammasomes responded differently to NLRP3 inhibitors. Bacterial inflammasome triggers, Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Escherichia coli, also induced IL-1ß secretion in porcine PBMCs. Taken together, we suggest that known triggers of NLRC4 and AIM2 inflammasomes in mice induce IL-1ß secretion in porcine PBMCs.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Proteínas Adaptadoras de Sinalização NOD/metabolismo , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Inflamassomos/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Salmonelose Animal/imunologia , Salmonelose Animal/metabolismo , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/metabolismo , Doenças dos Suínos/microbiologia
16.
Auton Neurosci ; 213: 51-59, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30005740

RESUMO

The cholinergic system plays a central role in regulating critical gastrointestinal functions, including motility, secretion, barrier and immune function. In rodent models of acute, non-infectious gastrointestinal injury, the cholinergic system functions to inhibit inflammation; however, during inflammation local expression and regulation of the cholinergic system is not well known, particularly during infectious enteritis. The objective of this study was to determine the intrinsic expression of the enteric cholinergic system in pig ileum following an acute challenge with Salmonella enterica serovar Typhimurium DT104 (S. Typhimurium). At 2 d post-challenge, a three-fold reduction in ileal acetylcholine (ACh) levels was observed in challenged animals, compared with controls. Ileal acetylcholinesterase (AChE) activity was decreased (by four-fold) while choline acetyltransferase (ChAT) expression was increased in both the ileum and mesenteric lymph nodes. Elevated ChAT found to localize preferentially to mucosa overlying lymphoid follicles of the Peyers patch in challenged pigs, with more intense labeling for ChAT in S. Typhimurium challenged pigs compared to controls. Ileal mRNA gene expression of muscarinic receptor 1 and 3 was also increased in challenged pigs, while muscarinic receptor 2 and the nicotinic receptor alpha 7 subunit gene expression were unaffected. A positive correlation was observed between ChAT protein expression in the ileum, rectal temperature, and histopathological severity in challenged animals. These data show that inflammation from S. Typhimurium challenge alters enteric cholinergic expression by down-regulating acetylcholine concentration and acetylcholine degrading enzymes while increasing acetylcholine synthesis proteins and receptors. Given the known anti-inflammatory role of the cholinergic system, the divergent expression of cholinergic genes may represent an attempt to limit tissue damage by preserving cholinergic signaling in the face of low ligand availability.


Assuntos
Íleo/metabolismo , Inflamação/veterinária , Mucosa Intestinal/metabolismo , Salmonelose Animal/metabolismo , Salmonella typhimurium , Doenças dos Suínos/metabolismo , Acetilcolina/metabolismo , Animais , Temperatura Corporal , Colina O-Acetiltransferase/metabolismo , Feminino , Regulação da Expressão Gênica , Íleo/patologia , Inflamação/metabolismo , Inflamação/patologia , Mucosa Intestinal/patologia , Linfonodos/metabolismo , Masculino , Receptores Muscarínicos/metabolismo , Salmonelose Animal/patologia , Sorogrupo , Sus scrofa , Suínos , Doenças dos Suínos/patologia
17.
BMC Vet Res ; 14(1): 180, 2018 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-29884179

RESUMO

BACKGROUND: Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) is a zoonotic pathogen responsible for severe intestinal pathology in young chickens. Natural resistance-associated macrophage protein (NRAMP) family has been shown to be associated with resistance to intracellular pathogens, including Salmonella Typhimurium. The role of NRAMP proteins in macrophage defence against microbial infection has been ascribed to changes in the metal-ion concentrations inside the bacteria-containing phagosomes. The present study was conducted to investigate tissue-specific (liver, spleen and caecum) expression kinetics of NRAMP gene family (NRAMP1 and NRAMP2) in broilers from day 0 to day 15 after Salmonella Typhimurium challenge concomitant to clinical, blood biochemical and immunological parameters survey. RESULTS: Clinical symptoms appeared 4 days post-infection (dpi) in infected birds. Symptoms like progressive weakness, anorexia, diarrhoea and lowering of the head were seen in infected birds one-week post-infection. On postmortem examination, liver showed congestion, haemorrhage and necrotic foci on the surface, while as the spleen, lungs and intestines revealed congestion and haemorrhages. Histopathological alterations were principally found in liver comprising of necrosis, reticular endothelial hyperplasia along with mononuclear cell and heterophilic infiltration. Red Blood Cell (RBC) count, Haemoglobin (Hb) and Packed Cell Volume (PCV) decreased significantly (P < 0.05) in blood while heterophil counts increased up to 7 days post-infection. Serum glucose, aspartate transaminase (AST) and alanine transaminase (ALT) enzymes concentrations increased significantly throughout the study. A gradual increase of specific humoral IgG response confirmed Salmonella infection. Meanwhile, expression of NRAMP1 and NRAMP2 genes was differentially regulated after infection in tissues such as liver, spleen and caecum known to be the target of Salmonella Typhimurium replication in the chicken. CONCLUSION: Thus the specific roles of NRAMP1 and NRAMP2 genes in Salmonella Typhimurium induced disease may be supposed from their differential expression according to tissues and timing after per os infection. However, these roles remain to be analyzed related to the severity of the disease which can be estimated by blood biochemistry and immunological parameters.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Galinhas , Doenças das Aves Domésticas/metabolismo , Salmonelose Animal/metabolismo , Salmonella typhimurium , Animais , Proteínas de Transporte de Cátions/genética , Regulação da Expressão Gênica , Doenças das Aves Domésticas/microbiologia , Salmonelose Animal/microbiologia
18.
Poult Sci ; 97(6): 2034-2043, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29514269

RESUMO

This study compared the efficacy of Pediococcus acidilactici, mannan-oligosaccharide, butyric acid, and their combination on growth performance and intestinal health in broiler chickens challenged with S. Typhimurium. Ross 308 male broilers (n = 420) were randomly assigned to one of the 6 treatments, resulting in 5 replicate pens of 14 chicks per treatment. The treatments included a negative control [(NC), no additive, not challenged]; positive control [(PC), no additive, but challenged with S. Typhimurium at d 3 posthatch], and 4 groups whereby birds were challenged with S. Typhimurium at d 3 posthatch and fed diets supplemented with either probiotic [0.1 g/kg Pediococcus acidilactici (PA)], prebiotic [2 g/kg mannan-oligosaccharides (MOS)], organic acid [0.5 g/kg butyric acid (BA)], or a combination of the 3 additives (MA). The S. Typhimurium challenge decreased feed intake, body weight gain and increased feed conversion ratio and reduced jejunum villus height (VH) and VH to crypt depth (CD) ratio (P < 0.05). Birds on the MA treatment exhibited similar performance to birds on the NC treatment (P > 0.05) and had a lower population of Salmonella in the ceca compared with birds on the PC treatment, at d 14 and 21 post-challenge (P < 0.05). The lowest heterophil to lymphocyte ratio was observed in birds on the MA and NC treatments (P < 0.05). Birds fed diets supplemented with MA or PA had greater VH and VH: CD ratio than birds on the PC treatment at d 7, 14 and 21 d post-challenge (P < 0.05). Suppressed amylase and protease activity was observed as a result of the S. Typhimurium challenge; the enzyme levels were restored in birds fed the additive-supplemented diets, when compared to the birds on the PC treatment, particularly at d 21 post-challenge (P < 0.05). These results indicate that dietary supplementation with a combination of PA, BA, and MOS in broiler chickens could be used as an effective tool for controlling S. Typhimurium and promoting growth performance.


Assuntos
Ácido Butírico/metabolismo , Galinhas/fisiologia , Mananas/metabolismo , Oligossacarídeos/metabolismo , Pediococcus acidilactici/química , Doenças das Aves Domésticas/metabolismo , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Animais , Ácido Butírico/administração & dosagem , Galinhas/crescimento & desenvolvimento , Dieta/veterinária , Suplementos Nutricionais/análise , Mananas/administração & dosagem , Oligossacarídeos/administração & dosagem , Probióticos/administração & dosagem , Distribuição Aleatória , Salmonelose Animal/metabolismo , Salmonella typhimurium/fisiologia
19.
Infect Immun ; 86(5)2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29483291

RESUMO

Salmonella enterica elicits intestinal inflammation to gain access to nutrients. One of these nutrients is fructose-asparagine (F-Asn). The availability of F-Asn to Salmonella during infection is dependent upon Salmonella pathogenicity islands 1 and 2, which in turn are required to provoke inflammation. Here, we determined that F-Asn is present in mouse chow at approximately 400 pmol/mg (dry weight). F-Asn is also present in the intestinal tract of germfree mice at 2,700 pmol/mg (dry weight) and in the intestinal tract of conventional mice at 9 to 28 pmol/mg. These findings suggest that the mouse intestinal microbiota consumes F-Asn. We utilized heavy-labeled precursors of F-Asn to monitor its formation in the intestine, in the presence or absence of inflammation, and none was observed. Finally, we determined that some members of the class Clostridia encode F-Asn utilization pathways and that they are eliminated from highly inflamed Salmonella-infected mice. Collectively, our studies identify the source of F-Asn as the diet and that Salmonella-mediated inflammation is required to eliminate competitors and allow the pathogen nearly exclusive access to this nutrient.


Assuntos
Asparagina/metabolismo , Frutose/metabolismo , Microbioma Gastrointestinal/imunologia , Inflamação/metabolismo , Salmonelose Animal/imunologia , Salmonelose Animal/metabolismo , Salmonella enterica/imunologia , Salmonella enterica/metabolismo , Animais , Inflamação/imunologia , Inflamação/patologia , Salmonelose Animal/patologia , Salmonella enterica/patogenicidade
20.
Biol Trace Elem Res ; 185(2): 497-508, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29478228

RESUMO

Salmonella and the host battle for iron (Fe), due to its importance for fundamental cellular processes. To investigate Fe redistribution of Salmonella-infected hens and the effects of high dietary Fe on it, Salmonella-free hens were randomly assigned to 1 of 4 treatments in 2 (two dietary Fe level) × 2 (Salmonella-inoculation or -noninoculation) factorial assignment. After feeding a basal diet supplemented with 60 (adequate, control) or 300 mg Fe/kg (high-Fe) for 4 weeks, 59-week-old Salmonella-free hens were orally inoculated with 5 × 107 colony-forming units of Salmonella Typhimurium (infection) or PBS (vehicle). Blood, spleen, and liver samples (n = 8) were collected at 14 days post-inoculation to determine Fe concentration and Fe transporters expression. Salmonella infection decreased (P < 0.05) hematocrit, serum Fe concentration, and splenic Fe concentration regardless of high-Fe or control hens, whereas increased (P < 0.05) Fe centration in the livers of high-Fe-treated hens. High dietary Fe increased hematocrit and serum Fe concentration, but did not affect (P = 0.11) splenic Fe concentration in Salmonella-infected hens. Salmonella infection did not influence (P = 0.31) liver Fe centration in control hens, but increased (P = 0.04) it in high-Fe-treated hens. High dietary Fe decreased (P < 0.01) the mRNA abundance of divalent metal transporter 1 and transferrin receptor, but increased (P < 0.02) ferroportin-1 (FPN1) mRNA and protein in the spleens and the livers regardless of Salmonella-infected or vehicle hens. Salmonella infection increased (P < 0.02) FPN1 mRNA and protein expression in the spleens, but did not influence its expression in the livers. These results suggested Salmonella infection and high dietary Fe differently influence the Fe distribution in the spleen and the liver of Salmonella-infected hens.


Assuntos
Ferro na Dieta/farmacologia , Ferro/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Salmonelose Animal/metabolismo , Baço/efeitos dos fármacos , Baço/metabolismo , Animais , Galinhas , Feminino , Ferro/sangue , Ferro/farmacocinética , Ferro na Dieta/administração & dosagem , Ferro na Dieta/sangue , Ferro na Dieta/farmacocinética , Oviposição/efeitos dos fármacos , Distribuição Aleatória , Salmonelose Animal/sangue , Distribuição Tecidual/efeitos dos fármacos
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