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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.
PLoS One ; 15(8): e0237886, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32810191

RESUMO

Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) causes gastroenteritis in many countries. However, in Brazil there are few studies that have conducted a virulence characterization of this serovar. The aim of this study was to evaluate the virulence potential of S. Typhimurium strains isolated in Brazil. Forty S. Typhimurium strains isolated from humans (n = 20) and food (n = 20) from Brazil were studied regarding their invasion and survival in human epithelial cells (Caco-2) and macrophages (U937). Their virulence potential was determined using the Galleria mellonella larvae model combined with the analysis of virulence genes by whole genome sequencing (WGS). A total of 67.5% of the S. Typhimurium studied (32.5% isolated from humans and 35% isolated from food) invaded Caco-2 epithelial cells at levels similar to or greater than the S. Typhimurium SL1344 prototype strain. In addition, 37.5% of the studied strains (25% isolated from humans and 12.5% isolated from food) survived in U937 human macrophages at levels similar to or greater than SL1344. S. Typhimurium strains isolated from humans (40%) and food (25%) showed high or intermediate virulence in G. mellonella larvae after seven days exposure. Approximately, 153 virulence genes of chromosomal and plasmidial origin were detected in the strains studied. In conclusion, the ability of the S. Typhimurium to invade Caco-2 epithelial cells was strain dependent and was not related to the source or the year of isolation. However, S. Typhimurium strains isolated from humans showed greater survival rates in U937 human macrophages, and presented higher proportion of isolates with a virulent profile in G. mellonella in comparison to strains isolated from food suggesting that this difference may be related to the higher frequency of human isolates which contained plasmid genes, such as spvABCDR operon, pefABCD operon, rck and mig-5.


Assuntos
Microbiologia de Alimentos , Salmonella typhimurium/genética , Salmonella typhimurium/isolamento & purificação , Animais , Brasil , Células CACO-2 , Sobrevivência Celular , Células Epiteliais/microbiologia , Genes Bacterianos , Genótipo , Humanos , Macrófagos/microbiologia , Mariposas/microbiologia , Fenótipo , Plasmídeos/genética , Salmonella typhimurium/patogenicidade , Células U937 , Virulência/genética
3.
PLoS One ; 15(7): e0234993, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645014

RESUMO

The main functions of the choroid plexus (CP) are the production of cerebral spinal fluid (CSF), the formation of the blood-CSF barrier, and regulation of immune response. This barrier allows for the exchange of specific nutrients, waste, and peripheral immune cells between the blood stream and CSF. Borrelia burgdorferi (Bb), the causative bacteria of Lyme disease, is associated with neurological complications including meningitis-indeed, Bb has been isolated from the CSF of patients. While it is accepted that B. burgdorferi can enter the central nervous system (CNS) of patients, it is unknown how the bacteria crosses this barrier and how the pathogenesis of the disease leads to the observed symptoms in patients. We hypothesize that during infection Borrelia burgdorferi will induce an immune response conducive to the chemotaxis of immune cells and subsequently lead to a pro-inflammatory state with the CNS parenchyma. Primary human choroid plexus epithelial cells were grown in culture and infected with B. burgdorferi strain B31 MI-16 for 48 hours. RNA was isolated and used for RNA sequencing and RT-qPCR validation. Secreted proteins in the supernatant were analyzed via ELISA. Transcriptome analysis based on RNA sequencing determined a total of 160 upregulated genes and 98 downregulated genes. Pathway and biological process analysis determined a significant upregulation in immune and inflammatory genes specifically in chemokine and interferon related pathways. Further analysis revealed downregulation in genes related to cell to cell junctions including tight and adherens junctions. These results were validated via RT-qPCR. Protein analysis of secreted factors showed an increase in inflammatory chemokines, corresponding to our transcriptome analysis. These data further demonstrate the role of the CP in the modulation of the immune response in a disease state and give insight into the mechanisms by which Borrelia burgdorferi may disseminate into, and act upon, the CNS. Future experiments aim to detail the impact of B. burgdorferi on the blood-CSF-barrier (BCSFB) integrity and inflammatory response within animal models.


Assuntos
Borrelia burgdorferi/patogenicidade , Plexo Corióideo/patologia , Células Epiteliais/patologia , Doença de Lyme/microbiologia , Barreira Hematoencefálica , Borrelia burgdorferi/imunologia , Células Cultivadas , Plexo Corióideo/imunologia , Plexo Corióideo/microbiologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Inflamação/metabolismo , Doença de Lyme/imunologia , Doença de Lyme/patologia , Proteínas/análise , RNA/análise
4.
Postepy Biochem ; 66(2): 151-159, 2020 06 27.
Artigo em Polonês | MEDLINE | ID: mdl-32700509

RESUMO

The epithelial tissues have continuous contact with external environment, including pathogenic microorganisms. Endogenous antimicrobial proteins and peptides produced by epithelial cells play a key role in controlling microbial burden and composition, either directly, or by engaging immune cells. These include active derivatives of multifunctional protein chemerin, which is equipped with both antimicrobial and chemotactic function. Given an increasing number of infections caused by antibiotic-insensitive microorganisms, such as methicillin- resistant S. aureus (MRSA), it is important to fully understand how these epithelia-associated microorganisms are controlled at barrier sites, including skin and oral cavity. Chemerin-derived synthetic peptide 4 (p4) covering central Val66-Pro85 chemerin sequence exhibits broad range of antimicrobial activity against skin- and oral cavity- associated bacteria, including MRSA strains, suggesting its therapeutic potential for bacteria-mediated barrier organs pathologies. In this article we present the overview of protective functions of chemerin and chemerin-derived peptides in the epithelial tissues.


Assuntos
Antibacterianos/metabolismo , Bactérias/metabolismo , Quimiocinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Farmacorresistência Bacteriana , Células Epiteliais/citologia , Humanos , Staphylococcus aureus Resistente à Meticilina/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(29): 17249-17259, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32641516

RESUMO

Control of infections caused by carbapenem-resistant Klebsiella pneumoniae continues to be challenging. The success of this pathogen is favored by its ability to acquire antimicrobial resistance and to spread and persist in both the environment and in humans. The emergence of clinically important clones, such as sequence types 11, 15, 101, and 258, has been reported worldwide. However, the mechanisms promoting the dissemination of such high-risk clones are unknown. Unraveling the factors that play a role in the pathobiology and epidemicity of K. pneumoniae is therefore important for managing infections. To address this issue, we studied a carbapenem-resistant ST-15 K. pneumoniae isolate (Kp3380) that displayed a remarkable adherent phenotype with abundant pilus-like structures. Genome sequencing enabled us to identify a chaperone-usher pili system (Kpi) in Kp3380. Analysis of a large K. pneumoniae population from 32 European countries showed that the Kpi system is associated with the ST-15 clone. Phylogenetic analysis of the operon revealed that Kpi belongs to the little-characterized γ2-fimbrial clade. We demonstrate that Kpi contributes positively to the ability of K. pneumoniae to form biofilms and adhere to different host tissues. Moreover, the in vivo intestinal colonizing capacity of the Kpi-defective mutant was significantly reduced, as was its ability to infect Galleria mellonella The findings provide information about the pathobiology and epidemicity of Kpi+ K. pneumoniae and indicate that the presence of Kpi may explain the success of the ST-15 clone. Disrupting bacterial adherence to the intestinal surface could potentially target gastrointestinal colonization.


Assuntos
Fímbrias Bacterianas/genética , Klebsiella pneumoniae/genética , Chaperonas Moleculares/genética , Células A549 , Animais , Antibacterianos , Aderência Bacteriana/efeitos dos fármacos , Aderência Bacteriana/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Carbapenêmicos/farmacologia , Linhagem Celular , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/genética , Células Epiteliais/microbiologia , Europa (Continente) , Feminino , Deleção de Genes , Genes Bacterianos/genética , Humanos , Infecções por Klebsiella , Klebsiella pneumoniae/citologia , Klebsiella pneumoniae/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Tipagem de Sequências Multilocus , Óperon , Filogenia
6.
Nat Commun ; 11(1): 2803, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499566

RESUMO

Host-associated reservoirs account for the majority of recurrent and oftentimes recalcitrant infections. Previous studies established that uropathogenic E. coli - the primary cause of urinary tract infections (UTIs) - can adhere to vaginal epithelial cells preceding UTI. Here, we demonstrate that diverse urinary E. coli isolates not only adhere to, but also invade vaginal cells. Intracellular colonization of the vaginal epithelium is detected in acute and chronic murine UTI models indicating the ability of E. coli to reside in the vagina following UTI. Conversely, in a vaginal colonization model, E. coli are detected inside vaginal cells and the urinary tract, indicating that vaginal colonization can seed the bladder. More critically, bacteria are identified inside vaginal cells from clinical samples from women with a history of recurrent UTI. These findings suggest that E. coli can establish a vaginal intracellular reservoir, where it may reside safely from extracellular stressors prior to causing an ascending infection.


Assuntos
Células Epiteliais/microbiologia , Escherichia coli Uropatogênica/patogenicidade , Vagina/microbiologia , Animais , Aderência Bacteriana , Infecções por Escherichia coli/microbiologia , Feminino , Camundongos , Camundongos Endogâmicos C3H , Microscopia de Fluorescência , Fagocitose , Bexiga Urinária/microbiologia , Sistema Urinário/microbiologia , Infecções Urinárias/microbiologia , Vagina/citologia
7.
Eur J Clin Microbiol Infect Dis ; 39(10): 1821-1830, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32557327

RESUMO

Helicobacter pylori (H. pylori) infection is associated with some gastric diseases, such as gastritis, peptic ulcer, and gastric cancer. CagA and VacA are known virulence factors of H. pylori, which play a vital role in severe clinical outcomes. Additionally, the expression of outer membrane proteins (OMPs) helps H. pylori attach to gastric epithelial cells at the primary stage and increases the virulence of H. pylori. In this review, we have summarized the paralogs of H. pylori OMPs, their genomic loci, and the different receptors of OMPs identified so far. We focused on five OMPs, BabA (HopS), SabA (HopP), OipA (HopH), HopQ, and HopZ, and one family of OMPs: Hom. We highlight the coexpression of OMPs with other virulence factors and their relationship with clinical outcomes. In conclusion, OMPs are closely related to the pathogenic processes of adhesion, colonization, persistent infection, and severe clinical consequences. They are potential targets for the prevention and treatment of H. pylori-related diseases.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Fatores de Virulência/metabolismo , Células Epiteliais/microbiologia , Expressão Gênica , Humanos , Fatores de Virulência/genética
8.
PLoS One ; 15(5): e0233052, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32413095

RESUMO

Severe influenza virus (IV) infections still represent a major challenge to public health. To combat IV infections, vaccines and antiviral compounds are available. However, vaccine efficacies vary with very limited to no protection against newly emerging zoonotic IV introductions. In addition, the development of resistant virus variants against currently available antivirals can be rapidly detected, in consequence demanding the design of novel antiviral strategies. Virus supportive cellular signaling cascades, such as the NF-κB pathway, have been identified to be promising antiviral targets against IV in in vitro and in vivo studies and clinical trials. While administration of NF-κB pathway inhibiting agents, such as LASAG results in decreased IV replication, it remained unclear whether blocking of NF-κB might sensitize cells to secondary bacterial infections, which often come along with viral infections. Thus, we examined IV and Staphylococcus aureus growth during LASAG treatment. Interestingly, our data reveal that the presence of LASAG during superinfection still leads to reduced IV titers. Furthermore, the inhibition of the NF-κB pathway resulted in decreased intracellular Staphylococcus aureus loads within epithelial cells, indicating a dependency on the pathway for bacterial uptake. Unfortunately, so far it is not entirely clear if this phenomenon might be a drawback in bacterial clearance during infection.


Assuntos
Antivirais/efeitos adversos , Aspirina/análogos & derivados , Infecções Bacterianas/etiologia , Glicina/efeitos adversos , Influenza Humana/tratamento farmacológico , Lisina/análogos & derivados , NF-kappa B/antagonistas & inibidores , Células A549 , Aspirina/efeitos adversos , Combinação de Medicamentos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Técnicas de Silenciamento de Genes , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/complicações , Influenza Humana/virologia , Lisina/efeitos adversos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Infecções Estafilocócicas/etiologia , Superinfecção/etiologia , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/genética , Replicação Viral/efeitos dos fármacos
9.
J Vis Exp ; (159)2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32449738

RESUMO

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.


Assuntos
Células Epiteliais/microbiologia , Células Epiteliais/patologia , Pulmão/patologia , Infecções por Pseudomonas/etiologia , Fumar/efeitos adversos , Carga Bacteriana/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Gentamicinas/farmacologia , Humanos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia
10.
Nat Commun ; 11(1): 2471, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32424203

RESUMO

Gut microbes are linked to host metabolism, but specific mechanisms remain to be uncovered. Ceramides, a type of sphingolipid (SL), have been implicated in the development of a range of metabolic disorders from insulin resistance (IR) to hepatic steatosis. SLs are obtained from the diet and generated by de novo synthesis in mammalian tissues. Another potential, but unexplored, source of mammalian SLs is production by Bacteroidetes, the dominant phylum of the gut microbiome. Genomes of Bacteroides spp. and their relatives encode serine palmitoyltransfease (SPT), allowing them to produce SLs. Here, we explore the contribution of SL-production by gut Bacteroides to host SL homeostasis. In human cell culture, bacterial SLs are processed by host SL-metabolic pathways. In mouse models, Bacteroides-derived lipids transfer to host epithelial tissue and the hepatic portal vein. Administration of B. thetaiotaomicron to mice, but not an SPT-deficient strain, reduces de novo SL production and increases liver ceramides. These results indicate that gut-derived bacterial SLs affect host lipid metabolism.


Assuntos
Bacteroides/fisiologia , Ceramidas/metabolismo , Microbioma Gastrointestinal , Redes e Vias Metabólicas , Esfingolipídeos/metabolismo , Animais , Células CACO-2 , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Vida Livre de Germes , Humanos , Resistência à Insulina , Mucosa Intestinal/microbiologia , Fígado/metabolismo , Redes e Vias Metabólicas/genética , Camundongos , Mutação/genética , Serina C-Palmitoiltransferase/deficiência , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo
12.
PLoS One ; 15(5): e0228606, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32392246

RESUMO

Bordetella pertussis, the causative agent of whopping cough, produces an adenylate cyclase toxin (CyaA) that plays a key role in the host colonization by targeting innate immune cells which express CD11b/CD18, the cellular receptor of CyaA. CyaA is also able to invade non-phagocytic cells, via a unique entry pathway consisting in a direct translocation of its catalytic domain across the cytoplasmic membrane of the cells. Within the cells, CyaA is activated by calmodulin to produce high levels of cyclic adenosine monophosphate (cAMP) and alter cellular physiology. In this study, we explored the effects of CyaA toxin on the cellular and molecular structure remodeling of A549 alveolar epithelial cells. Using classical imaging techniques, biochemical and functional tests, as well as advanced cell mechanics method, we quantify the structural and functional consequences of the massive increase of intracellular cyclic AMP induced by the toxin: cell shape rounding associated to adhesion weakening process, actin structure remodeling for the cortical and dense components, increase in cytoskeleton stiffness, and inhibition of migration and repair. We also show that, at low concentrations (0.5 nM), CyaA could significantly impair the migration and wound healing capacities of the intoxicated alveolar epithelial cells. As such concentrations might be reached locally during B. pertussis infection, our results suggest that the CyaA, beyond its major role in disabling innate immune cells, might also contribute to the local alteration of the epithelial barrier of the respiratory tract, a hallmark of pertussis.


Assuntos
Toxina Adenilato Ciclase/genética , Bordetella pertussis/enzimologia , Imunidade Inata/genética , Coqueluche/genética , Toxina Adenilato Ciclase/metabolismo , Bordetella pertussis/patogenicidade , Calmodulina/metabolismo , Membrana Celular/metabolismo , AMP Cíclico/genética , Células Epiteliais/microbiologia , Humanos , Sistema Respiratório/metabolismo , Sistema Respiratório/microbiologia , Sistema Respiratório/patologia , Coqueluche/microbiologia , Coqueluche/patologia
13.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 42(2): 228-235, 2020 Apr 28.
Artigo em Chinês | MEDLINE | ID: mdl-32385030

RESUMO

Objective To investigate the mechanism of long non-coding RNA plasmacytoma variant translocation 1 (PVT1) in gastric cancer caused by helicobacter pylori (HP) infection. Methods The expression of PVT1 was detected by quantitative real-time polymerase chain reaction in HP-infected normal gastric epithelial cells GES-1. Gastric cancer cell line SGC-7901 was transfected with PVT1 small interfering RNA and co-cultured with HP,and then the inflammatory cytokines such as tumor necrosis factor-α (TNF-α),interleukin (IL) -1ß,IL-6 and IL-8 were detected. After PVT1 was knocked down,the effects of PVT1 on the proliferation and migration of gastric cancer cells were examined by cell scratch assay. RNA-pulldown combined with mass spectrometry was used to detect the protein binding to PVT1,and the result of mass spectrometry was verified by RNA-pulldown combined with Western blot. Results In HP-infected normal gastric epithelial cells GES-1,quantitative real-time polymerase chain reaction showed that PVT1 was significantly up-regulated (t=7.160,P=0.019). PVT1 was knocked down in gastric cancer cells,and then infected with HP. The expressions of inflammatory factors including TNF-α (t=3.899,P=0.011),IL-1ß (t=14.610,P=0.000),and IL-8 (t=6.557,P=0.001) were significantly inhibited. Although PVT1 knockdown had no significant effect on the proliferation ability of gastric cancer cells,it inhibited the migration of cells. PVT1 might interact with RPS8 protein. Conclusion PVT1 may act as a pro-inflammatory factor and regulate gastric cancer caused by HP infection.


Assuntos
Movimento Celular , Células Epiteliais/citologia , Células Epiteliais/microbiologia , Infecções por Helicobacter/patologia , RNA Longo não Codificante/genética , Linhagem Celular Tumoral , Citocinas/metabolismo , Técnicas de Silenciamento de Genes , Helicobacter pylori , Humanos , Inflamação
14.
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
15.
Nat Microbiol ; 5(7): 929-942, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32284563

RESUMO

Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene that converts cholesterol to the oxysterol 25-hydroxycholesterol (25HC). Circulating 25HC modulates essential immunological processes including antiviral immunity, inflammasome activation and antibody class switching; and dysregulation of CH25H may contribute to chronic inflammatory disease and cancer. Although 25HC is a potent regulator of cholesterol storage, uptake, efflux and biosynthesis, how these metabolic activities reprogram the immunological state of target cells remains poorly understood. Here, we used recently designed toxin-based biosensors that discriminate between distinct pools of plasma membrane cholesterol to elucidate how 25HC prevents Listeria monocytogenes from traversing the plasma membrane of infected host cells. The 25HC-mediated activation of acyl-CoA:cholesterol acyltransferase (ACAT) triggered rapid internalization of a biochemically defined fraction of cholesterol, termed 'accessible' cholesterol, from the plasma membrane while having little effect on cholesterol in complexes with sphingomyelin. We show that evolutionarily distinct bacterial species, L. monocytogenes and Shigella flexneri, exploit the accessible pool of cholesterol for infection and that acute mobilization of this pool by oxysterols confers immunity to these pathogens. The significance of this signal-mediated membrane remodelling pathway probably extends beyond host defence systems, as several other biologically active oxysterols also mobilize accessible cholesterol through an ACAT-dependent mechanism.


Assuntos
Bactérias/imunologia , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Membrana Celular/metabolismo , Colesterol/metabolismo , Imunidade Inata/efeitos dos fármacos , Oxisteróis/farmacologia , Infecções Bacterianas/tratamento farmacológico , Colesterol/química , Citocinas/metabolismo , Células Epiteliais/microbiologia , Humanos , Interferons/metabolismo , Listeria/efeitos dos fármacos , Listeria/imunologia , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Oxisteróis/química , Oxisteróis/metabolismo , Shigella/efeitos dos fármacos , Shigella/imunologia , Esterol O-Aciltransferase/metabolismo , Relação Estrutura-Atividade
16.
PLoS Pathog ; 16(4): e1008498, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282854

RESUMO

We investigated the role of the inflammasome effector caspases-1 and -11 during Salmonella enterica serovar Typhimurium infection of murine intestinal epithelial cells (IECs). Salmonella burdens were significantly greater in the intestines of caspase-1/11 deficient (Casp1/11-/-), Casp1-/- and Casp11-/- mice, as compared to wildtype mice. To determine if this reflected IEC-intrinsic inflammasomes, enteroid monolayers were derived and infected with Salmonella. Casp11-/- and wildtype monolayers responded similarly, whereas Casp1-/- and Casp1/11-/- monolayers carried significantly increased intracellular burdens, concomitant with marked decreases in IEC shedding and death. Pretreatment with IFN-γ to mimic inflammation increased caspase-11 levels and IEC death, and reduced Salmonella burdens in Casp1-/- monolayers, while high intracellular burdens and limited cell shedding persisted in Casp1/11-/- monolayers. Thus caspase-1 regulates inflammasome responses in IECs at baseline, while proinflammatory activation of IECs reveals a compensatory role for caspase-11. These results demonstrate the importance of IEC-intrinsic canonical and non-canonical inflammasomes in host defense against Salmonella.


Assuntos
Caspase 1/imunologia , Caspases Iniciadoras/imunologia , Inflamassomos/imunologia , Intestinos/enzimologia , Intestinos/imunologia , Infecções por Salmonella/enzimologia , Salmonella typhimurium/imunologia , Animais , Células Epiteliais/enzimologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Feminino , Imunidade nas Mucosas , Inflamassomos/metabolismo , Interferon gama/imunologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Intestinos/microbiologia , Lipopolissacarídeos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Salmonella/imunologia , Salmonella typhimurium/patogenicidade
17.
Nat Commun ; 11(1): 1892, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32312961

RESUMO

Streptococcus pneumoniae serotype 1 is the predominant cause of invasive pneumococcal disease in sub-Saharan Africa, but the mechanism behind its increased invasiveness is not well understood. Here, we use mouse models of lung infection to identify virulence factors associated with severe bacteraemic pneumonia during serotype-1 (ST217) infection. We use BALB/c mice, which are highly resistant to pneumococcal pneumonia when infected with other serotypes. However, we observe 100% mortality and high levels of bacteraemia within 24 hours when BALB/c mice are intranasally infected with ST217. Serotype 1 produces large quantities of pneumolysin, which is rapidly released due to high levels of bacterial autolysis. This leads to substantial levels of cellular cytotoxicity and breakdown of tight junctions between cells, allowing a route for rapid bacterial dissemination from the respiratory tract into the blood. Thus, our results offer an explanation for the increased invasiveness of serotype 1.


Assuntos
Autólise , Proteínas de Bactérias/metabolismo , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/patologia , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/patogenicidade , Estreptolisinas/metabolismo , Células A549 , Animais , Bacteriemia/microbiologia , Toxinas Bacterianas , Sobrevivência Celular , Modelos Animais de Doenças , Células Epiteliais/microbiologia , Feminino , Humanos , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Nasofaringe/microbiologia , Sorogrupo , Virulência , Fatores de Virulência
18.
PLoS Pathog ; 16(4): e1008446, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282860

RESUMO

Microfold (M) cell host-pathogen interaction studies would benefit from the visual analysis of dynamic cellular and microbial interplays. We adapted a human in vitro M cell model to physiological bacterial infections, expression of fluorescent localization reporters and long-term three-dimensional time-lapse microscopy. This approach allows following key steps of M cell infection dynamics at subcellular resolution, from the apical onset to basolateral epithelial dissemination. We focused on the intracellular pathogen Shigella flexneri, classically reported to transcytose through M cells to initiate bacillary dysentery in humans, while eliciting poorly protective immune responses. Our workflow was critical to reveal that S. flexneri develops a bimodal lifestyle within M cells leading to rapid transcytosis or delayed vacuolar rupture, followed by direct actin motility-based propagation to neighboring enterocytes. Moreover, we show that Listeria monocytogenes, another intracellular pathogen sharing a tropism for M cells, disseminates in a similar manner and evades M cell transcytosis completely. We established that actin-based M cell-to-enterocyte spread is the major dissemination pathway for both pathogens and avoids their exposure to basolateral compartments in our system. Our results challenge the notion that intracellular pathogens are readily transcytosed by M cells to inductive immune compartments in vivo, providing a potential mechanism for their ability to evade adaptive immunity.


Assuntos
Disenteria Bacilar/microbiologia , Enterócitos/microbiologia , Células Epiteliais/microbiologia , Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Shigella flexneri/fisiologia , Células CACO-2 , Humanos , Listeria monocytogenes/genética , Shigella flexneri/genética
19.
Cancer Sci ; 111(5): 1596-1606, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32198795

RESUMO

Chronic infection with Helicobacter pylori cagA-positive strains is causally associated with the development of gastric diseases, most notably gastric cancer. The cagA-encoded CagA protein, which is injected into gastric epithelial cells by bacterial type IV secretion, undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) segments (EPIYA-A, EPIYA-B, EPIYA-C, and EPIYA-D), which are present in various numbers and combinations in its C-terminal polymorphic region, thereby enabling CagA to promiscuously interact with SH2 domain-containing host cell proteins, including the prooncogenic SH2 domain-containing protein tyrosine phosphatase 2 (SHP2). Perturbation of host protein functions by aberrant complex formation with CagA has been considered to contribute to the development of gastric cancer. Here we show that SHIP2, an SH2 domain-containing phosphatidylinositol 5'-phosphatase, is a hitherto undiscovered CagA-binding host protein. Similar to SHP2, SHIP2 binds to the Western CagA-specific EPIYA-C segment or East Asian CagA-specific EPIYA-D segment through the SH2 domain in a tyrosine phosphorylation-dependent manner. In contrast to the case of SHP2, however, SHIP2 binds more strongly to EPIYA-C than to EPIYA-D. Interaction with CagA tethers SHIP2 to the plasma membrane, where it mediates production of phosphatidylinositol 3,4-diphosphate [PI(3,4)P2 ]. The CagA-SHIP2 interaction also potentiates the morphogenetic activity of CagA, which is caused by CagA-deregulated SHP2. This study indicates that initially delivered CagA interacts with SHIP2 and thereby strengthens H. pylori-host cell attachment by altering membrane phosphatidylinositol compositions, which potentiates subsequent delivery of CagA that binds to and thereby deregulates the prooncogenic phosphatase SHP2.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Células Epiteliais/metabolismo , Mucosa Gástrica/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Motivos de Aminoácidos , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Linhagem Celular , Membrana Celular/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/química , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosforilação , Ligação Proteica , Transporte Proteico , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Domínios de Homologia de src
20.
PLoS One ; 15(3): e0230667, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32208441

RESUMO

Key events in the pathogenesis of SjÓ§gren syndrome (SS) include the change of salivary gland epithelial cells into antigen-presenting cell-like phenotypes and focal lymphocytic sialadenitis (FLS). However, what triggers these features in SS is unknown. Dysbiosis of the gut and oral microbiomes is a potential environmental factor in SS, but its connection to the etiopathogenesis of SS remains unclear. This study aimed to characterize the oral microbiota in SS and to investigate its potential role in the pathogenesis of SS. Oral bacterial communities were collected by whole mouthwash from control subjects (14 without oral dryness and 11 with dryness) and primary SS patients (8 without oral dryness and 17 with dryness) and were analyzed by pyrosequencing. The SS oral microbiota was characterized by an increased bacterial load and Shannon diversity. Through comparisons of control and SS in combined samples and then separately in non-dry and dry conditions, SS-associated taxa independent of dryness were identified. Three SS-associated species and 2 control species were selected and used to challenge human submandibular gland tumor (HSG) cells. Among the selected SS-associated bacterial species, Prevotella melaninogenica uniquely upregulated the expression of MHC molecules, CD80, and IFNλ in HSG cells. Concomitantly, P. melaninogenica efficiently invaded HSG cells. Sections of labial salivary gland (LSG) biopsies from 8 non-SS subjects and 15 SS patients were subjected to in situ hybridization using universal and P. melaninogenica-specific probes. Ductal cells and the areas of infiltration were heavily infected with bacteria in the LSGs with FLS. Collectively, dysbiotic oral microbiota may initiate the deregulation of SGECs and the IFN signature through bacterial invasion into ductal cells. These findings may provide new insights into the etiopathogenesis of SS.


Assuntos
Microbiota , Glândulas Salivares/patologia , Síndrome de Sjogren/patologia , Aquaporinas/metabolismo , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/patogenicidade , Proteínas de Bactérias/metabolismo , Estudos de Casos e Controles , Linhagem Celular Tumoral , Disbiose , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Interferons/metabolismo , Prevotella melaninogenica/genética , Prevotella melaninogenica/isolamento & purificação , Prevotella melaninogenica/patogenicidade , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Glândulas Salivares/microbiologia , Sialadenite/complicações , Sialadenite/microbiologia , Sialadenite/patologia , Síndrome de Sjogren/complicações , Síndrome de Sjogren/microbiologia
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