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2.
Cancer Sci ; 111(5): 1596-1606, 2020 May.
Artículo en Inglés | MEDLINE | ID: mdl-32198795

RESUMEN

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.


Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Células Epiteliales/metabolismo , Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , Secuencias de Aminoácidos , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Línea Celular , Membrana Celular/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/patología , Transición Epitelial-Mesenquimal , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/química , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/genética , Fosforilación , Unión Proteica , Transporte de Proteínas , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Dominios Homologos src
3.
PLoS One ; 15(3): e0230718, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32210474

RESUMEN

Chlamydia trachomatis is the most common bacterial sexually-transmitted infection and the major cause of preventable blindness worldwide. The asymptomatic nature of many infections along with uncontrolled inflammation leads to irreversible damage in the upper genital tract and the tarsal conjunctivae, with the major complications of infertility and chronic pelvic pain, and blindness, respectively. Inflammation must, therefore, be tightly regulated to avoid an unrestrained immune response. The genetic factors that regulate inflammation through Toll-like receptor (TLR) signaling pathways during C. trachomatis infection have not been fully characterized. SIGIRR (also known as IL-1R8 or TIR8) can regulate inflammation in response to various pathogens and diseases. However, nothing is known about its role during C. trachomatis infection. Expression of the pro-inflammatory chemokine, IL-8, was measured in epithelial cells infected with C. trachomatis. The effect of SIGIRR was determined by depleting SIGIRR or over-expressing SIGIRR in the epithelial cells before infection. Our results indicate that, in the absence of SIGIRR, epithelial cells induce higher levels of the pro-inflammatory chemokine, IL-8, in response to C. trachomatis infection. In addition, SIGIRR associates with MyD88 in both infected and uninfected infected cells. Collectively, our data demonstrate that SIGIRR functions as a negative regulator of the immune response to C. trachomatis infection. This finding provides insights into the immuno-pathogenesis of C. trachomatis that can be used to treat and identify individuals at risk of uncontrolled inflammation during infection.


Asunto(s)
Chlamydia trachomatis/fisiología , Receptores de Interleucina-1/metabolismo , Receptores Toll-Like/metabolismo , Chlamydia trachomatis/inmunología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Regulación de la Expresión Génica/inmunología , Silenciador del Gen , Células HeLa , Humanos , Interleucina-8/genética , Factor 88 de Diferenciación Mieloide/metabolismo , ARN Mensajero/genética , Receptores de Interleucina-1/deficiencia , Receptores de Interleucina-1/genética
4.
PLoS Pathog ; 16(3): e1008372, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32208456

RESUMEN

It is increasingly being recognised that the interplay between commensal and pathogenic bacteria can dictate the outcome of infection. Consequently, there is a need to understand how commensals interact with their human host and influence pathogen behaviour at epithelial surfaces. Neisseria meningitidis, a leading cause of sepsis and meningitis, exclusively colonises the human nasopharynx and shares this niche with several other Neisseria species, including the commensal Neisseria cinerea. Here, we demonstrate that during adhesion to human epithelial cells N. cinerea co-localises with molecules that are also recruited by the meningococcus, and show that, similar to N. meningitidis, N. cinerea forms dynamic microcolonies on the cell surface in a Type four pilus (Tfp) dependent manner. Finally, we demonstrate that N. cinerea colocalises with N. meningitidis on the epithelial cell surface, limits the size and motility of meningococcal microcolonies, and impairs the effective colonisation of epithelial cells by the pathogen. Our data establish that commensal Neisseria can mimic and affect the behaviour of a pathogen on epithelial cell surfaces.


Asunto(s)
Adhesión Bacteriana , Células Epiteliales/microbiología , Fimbrias Bacterianas/metabolismo , Neisseria cinerea/crecimiento & desarrollo , Neisseria meningitidis/crecimiento & desarrollo , Células A549 , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Neisseria cinerea/patogenicidad , Neisseria meningitidis/patogenicidad
5.
PLoS One ; 15(3): e0230667, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32208441

RESUMEN

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.


Asunto(s)
Microbiota , Glándulas Salivales/patología , Síndrome de Sjögren/patología , Acuaporinas/metabolismo , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/patogenicidad , Proteínas Bacterianas/metabolismo , Estudios de Casos y Controles , Línea Celular Tumoral , Disbiosis , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Humanos , Interferones/metabolismo , Prevotella melaninogenica/genética , Prevotella melaninogenica/aislamiento & purificación , Prevotella melaninogenica/patogenicidad , ARN Ribosómico 16S/química , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Glándulas Salivales/microbiología , Sialadenitis/complicaciones , Sialadenitis/microbiología , Sialadenitis/patología , Síndrome de Sjögren/complicaciones , Síndrome de Sjögren/microbiología
6.
PLoS One ; 15(3): e0229647, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32150574

RESUMEN

Probiotic bacteria have the ability to modulate host immune responses and have potent therapeutic functional effects against several diseases, including inflammatory diseases. However, beneficial effects of probiotics are strain specific and their interactions with host immune cells to modulate inflammatory response are largely unknown. Intestinal epithelial cells (IECs), which are the first line of defense against invading pathogens, and connects between commensals/probiotics and immune system; therefore, in this study, we used human IECs to assess the probiotic effects of three selected Lactobacillus strains in vitro. An HT-29 colonic epithelial cell and HT-29/blood mononuclear cells co-culture system were stimulated with Lactobacillus followed by Salmonella for different hours, after which the mRNA level of cytokines, ß-defensin-2 and negative regulators for TLR signaling and protein levels of ZO-1 and IκB-α were analyzed by real-time polymerase chain reaction and western blot analysis. L. brevis decreased Salmonella induced IL-6, IL-8, MCP-1 and IL-1ß levels, whereas L. pentosus suppressed IL-6 and MCP-1 in HT-29 cells. Moreover, L. brevis was able to increase the mRNA levels of A20, Tollip, SIGIRR and IRAKM, while L. pentosus reduced the levels of A20, and IRAKM in response to Salmonella. In addition, decrease in protein level of TNF-α and increase in mRNA level of IL-10 was observed in L. brevis and L. pentosus treated HT-29 cells. Lactobacillus strains were differentially modulated ZO-1 and p-IκB-α in HT-29 cells treated with Salmonella. Overall, the results of this study indicate that Lactobacillus strains attenuate Salmonella induced inflammatory responses through beneficial modulation of TLR negative regulators and the NF-κB pathway.


Asunto(s)
Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Probióticos/uso terapéutico , Salmonella/inmunología , Salmonella/patogenicidad , Técnicas de Cocultivo , Citocinas/metabolismo , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Células HT29 , Interacciones Microbiota-Huesped/inmunología , Humanos , Lactobacillus/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/microbiología , FN-kappa B/metabolismo , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/prevención & control , Transducción de Señal , Receptores Toll-Like/metabolismo , beta-Defensinas/metabolismo
7.
Life Sci ; 248: 117456, 2020 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-32097666

RESUMEN

AIMS: In this study, we will investigate the therapeutic effects of berberine (BBR) in Helicobacter pylori (H. pylori) induced chronic atrophic gastritis (CAG). Furthermore, potential mechanisms of BBR in regulating IRF8-IFN-γ signaling axis will also be investigated. MATERIALS AND METHODS: H. pylori were utilized to establish CAG model of rats. Therapeutic effects of BBR on serum supernatant indices, and histopathology of stomach were analyzed in vivo. Moreover, GES-1 cells were infected by H. pylori, and intervened with BBR in vitro. Cell viability, morphology, proliferation, and quantitative analysis were detected by high-content screening (HCS) imaging assay. To further investigate the potential mechanisms of BBR, relative mRNA, immunohistochemistry and protein expression in IRF8-IFN-γ signaling axis were measured. KEY FINDINGS: Results showed serum supernatant indices including IL-17, CXCL1, and CXCL9 were downregulated by BBR intervention, while, G-17 increased significantly. Histological injuries of gastric mucosa induced by H. pylori also were alleviated. Moreover, cell viability and morphology changes of GES-1 cells were improved by BBR intervention. In addition, proinflammatory genes and IRF8-IFN-γ signaling axis related genes, including Ifit3, Upp1, USP18, Nlrc5, were suppressed by BBR administration in vitro and in vivo. The proteins expression related to IRF8-IFN-γ signaling axis, including Ifit3, IRF1 and Ifit1 were downregulated by BBR intervention.


Asunto(s)
Antiinflamatorios/farmacología , Berberina/farmacología , Gastritis Atrófica/tratamiento farmacológico , Infecciones por Helicobacter/tratamiento farmacológico , Factores Reguladores del Interferón/genética , Interferón gamma/genética , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quimiocina CXCL1/antagonistas & inhibidores , Quimiocina CXCL1/genética , Quimiocina CXCL1/inmunología , Quimiocina CXCL9/antagonistas & inhibidores , Quimiocina CXCL9/genética , Quimiocina CXCL9/inmunología , Enfermedad Crónica , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Gastritis Atrófica/genética , Gastritis Atrófica/inmunología , Gastritis Atrófica/microbiología , Regulación de la Expresión Génica , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/crecimiento & desarrollo , Helicobacter pylori/patogenicidad , Humanos , Factores Reguladores del Interferón/antagonistas & inhibidores , Factores Reguladores del Interferón/inmunología , Interferón gamma/antagonistas & inhibidores , Interferón gamma/inmunología , Interleucina-17/agonistas , Interleucina-17/genética , Interleucina-17/inmunología , Masculino , Proteínas NLR/antagonistas & inhibidores , Proteínas NLR/genética , Proteínas NLR/inmunología , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Uridina Fosforilasa/antagonistas & inhibidores , Uridina Fosforilasa/genética , Uridina Fosforilasa/inmunología
8.
J Med Microbiol ; 69(3): 457-464, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-32100714

RESUMEN

Introduction. Helicobacter pylori is associated with gastrointestinal disease, most notably gastric cancer. Cytotoxin-associated antigen A (CagA), an important virulence factor for H. pylori pathogenicity, induces host cells to release inflammatory factors, especially interleukin-8 (IL-8). The mechanism by which C-terminal CagA induces IL-8 production has been studied extensively, but little is known about the role of the N-terminus.Aim. To investigate the effect of CagA303-456aa (a peptide in the N-terminal CagA) on IL-8 production by gastric epithelial cells.Methodology. CagA303-456aa was produced by a prokaryotic expression system and purified by Strep-tag affinity chromatography. An integrin ß1 (ITGB1)-deficient AGS cell line was constructed using the CRISPR/Cas9 technique, and NCTC 11637 cagA and/or cagL knockout mutants were constructed via homologous recombination. The levels of IL-8 production were determined by enzyme-linked immunosorbent assay (ELISA), and p38 and ERK1/2 phosphorylation were examined by Western blot.Results. CagA303-456aa induced IL-8 expression by AGS cells. IL-8 induction by CagA303-456aawas specifically inhibited by ITGB1 deficiency. Notably, CagA303-456aa activated the phosphorylation of both p38 and ERK1/2, and blocking p38 and ERK1/2 activity significantly reduced IL-8 induction by CagA303-456aa. ITGB1 deficiency also inhibited the activation of p38 phosphorylation by CagA303-456aa. Finally, experiments in CagA and/or CagL knockout bacterial lines demonstrated that extracellular CagA might induce IL-8 production by AGS cells.Conclusion. Residues 303-456 of the N-terminal region of CagA induce IL-8 production via a CagA303-456-ITGB1-p38-IL-8 pathway, and ERK1/2 is also involved in the release of IL-8. Extracellular CagA might induce IL-8 production before translocation into AGS cells.


Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Infecciones por Helicobacter/microbiología , Helicobacter pylori/inmunología , Integrina beta1/metabolismo , Interleucina-8/metabolismo , Péptidos/metabolismo , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Línea Celular Tumoral , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Femenino , Helicobacter pylori/patogenicidad , Humanos , Sistema de Señalización de MAP Quinasas , Péptidos/genética , Fosforilación , Factores de Virulencia/genética , Factores de Virulencia/metabolismo
9.
Nat Rev Gastroenterol Hepatol ; 17(5): 263-278, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32103203

RESUMEN

The human gastrointestinal tract is colonized by trillions of microorganisms that interact with the host to maintain structural and functional homeostasis. Acting as the interface between the site of the highest microbial burden in the human body and the richest immune compartment, a single layer of intestinal epithelial cells specializes in nutrient absorption, stratifies microorganisms to limit colonization of tissues and shapes the responses of the subepithelial immune cells. In this Review, we focus on the expression, regulation and functions of Toll-like receptors (TLRs) in the different intestinal epithelial lineages to analyse how epithelial recognition of bacteria participates in establishing homeostasis in the gut. In particular, we elaborate on the involvement of epithelial TLR signalling in controlling crypt dynamics, enhancing epithelial barrier integrity and promoting immune tolerance towards the gut microbiota. Furthermore, we comment on the regulatory mechanisms that fine-tune TLR-driven immune responses towards pathogens and revisit the role of TLRs in epithelial repair after injury. Finally, we discuss how dysregulation of epithelial TLRs can lead to the generation of dysbiosis, thereby increasing susceptibility to colitis and tumorigenesis.


Asunto(s)
Microbioma Gastrointestinal/inmunología , Tracto Gastrointestinal/inmunología , Mucosa Intestinal/inmunología , Receptores Toll-Like/inmunología , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Tracto Gastrointestinal/microbiología , Homeostasis , Humanos , Mucosa Intestinal/microbiología
10.
PLoS Pathog ; 16(2): e1008304, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32069333

RESUMEN

The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host. Here, we report the development and application of a human three-dimensional (3D) infection model based on tissue engineering to study host-pathogen interactions. Our intestinal 3D tissue model is built on a decellularized extracellular matrix scaffold, which is reseeded with human Caco-2 cells. Dynamic culture conditions enable the formation of a polarized mucosal epithelial barrier reminiscent of the 3D microarchitecture of the human small intestine. Infection with C. jejuni demonstrates that the 3D tissue model can reveal isolate-dependent colonization and barrier disruption phenotypes accompanied by perturbed localization of cell-cell junctions. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D model deviated from those obtained with 2D-monolayers, but recapitulated phenotypes previously observed in animal models. Moreover, we demonstrate the involvement of a small regulatory RNA pair, CJnc180/190, during infections and observe different phenotypes of CJnc180/190 mutant strains in 2D vs. 3D infection models. Hereby, the CJnc190 sRNA exerts its pathogenic influence, at least in part, via repression of PtmG, which is involved in flagellin modification. Our results suggest that the Caco-2 cell-based 3D tissue model is a valuable and biologically relevant tool between in-vitro and in-vivo infection models to study virulence of C. jejuni and other gastrointestinal pathogens.


Asunto(s)
Campylobacter jejuni/genética , Interacciones Huésped-Patógeno/fisiología , Modelos Biológicos , Células CACO-2 , Infecciones por Campylobacter/microbiología , Campylobacter jejuni/patogenicidad , Células Epiteliales/microbiología , Matriz Extracelular/fisiología , Humanos , Mucosa Intestinal/microbiología , Intestino Delgado/patología , Intestinos/microbiología , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Andamios del Tejido , Virulencia
11.
Vet Q ; 40(1): 43-50, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31939335

RESUMEN

Background: The coinfection process of Escherichia coli, an etiological agent of clinical mastitis and Mycobacterium avium subsp. paratuberculosis (MAP), a non-mastitic etiological agent in the bovine mammary gland is not fully known.Objective: Verify the ability of MAP to interfere with the invasion and translocation of E. coli in bovine mammary epithelial cell line (MAC-T).Methods: For the invasion assay, MAC-T cells were challenged with MAP K10 for 2 h and then challenged with E. coli for 10, 30 and 120 min. For the translocation assay, the trans well plates were used and the challenge sequence was repeated as previously described. The amount of E. coli in the assays was determined by counting colony forming units (CFU) in Luria-Bertani medium. Quantitative real-time PCR was used to quantify MAP in MAC-T cells. To verify the viability of the MAC-T cells, the MTT assay was performed. MAP culture supernatant was also evaluated at different percentages for E. coli growth.Results: Previous MAP infection in MAC-T cells inhibited E. coli invasion in 10, 30 and 120 min. No significant interference of MAP in the translocation of E. coli from the apical-basal direction was verified. Quantity of MAP DNA inside the MAC-T cells was statistically similar. Neither reduction in MAC-T cells viability was detected during the experiment nor MAP-released factor in the supernatant inhibited E. coli invasion.Conclusion: These findings suggest that MAP-positive cows could be more resistant to E. coli infection, but when infected, could rapidly translocate E. coli to the subepithelial region.


Asunto(s)
Traslocación Bacteriana/fisiología , Coinfección/veterinaria , Células Epiteliales/microbiología , Escherichia coli/patogenicidad , Mastitis Bovina/microbiología , Mycobacterium avium subsp. paratuberculosis/patogenicidad , Animales , Bovinos , Línea Celular , Coinfección/microbiología , Modelos Animales de Enfermedad , Infecciones por Escherichia coli/veterinaria , Femenino , Glándulas Mamarias Animales/microbiología , Leche/microbiología , Paratuberculosis
12.
Proc Natl Acad Sci U S A ; 117(5): 2645-2655, 2020 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-31964836

RESUMEN

The main risk factor for stomach cancer, the third most common cause of cancer death worldwide, is infection with Helicobacter pylori bacterial strains that inject cytotoxin-associated gene A (CagA). As the first described bacterial oncoprotein, CagA causes gastric epithelial cell transformation by promoting an epithelial-to-mesenchymal transition (EMT)-like phenotype that disrupts junctions and enhances motility and invasiveness of the infected cells. However, the mechanism by which CagA disrupts gastric epithelial cell polarity to achieve its oncogenicity is not fully understood. Here we found that the apoptosis-stimulating protein of p53 2 (ASPP2), a host tumor suppressor and an important CagA target, contributes to the survival of cagA-positive H. pylori in the lumen of infected gastric organoids. Mechanistically, the CagA-ASPP2 interaction is a key event that promotes remodeling of the partitioning-defective (PAR) polarity complex and leads to loss of cell polarity of infected cells. Blockade of cagA-positive H. pylori ASPP2 signaling by inhibitors of the EGFR (epidermal growth factor receptor) signaling pathway-identified by a high-content imaging screen-or by a CagA-binding ASPP2 peptide, prevents the loss of cell polarity and decreases the survival of H. pylori in infected organoids. These findings suggest that maintaining the host cell-polarity barrier would reduce the detrimental consequences of infection by pathogenic bacteria, such as H. pylori, that exploit the epithelial mucosal surface to colonize the host environment.


Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Bacterianas/metabolismo , Células Epiteliales/citología , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Organoides/microbiología , Antígenos Bacterianos/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Bacterianas/genética , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Helicobacter pylori/crecimiento & desarrollo , Interacciones Huésped-Patógeno , Humanos , Organoides/metabolismo , Unión Proteica , Estómago/microbiología
13.
FASEB J ; 34(1): 1783-1801, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31914584

RESUMEN

The natural product icariin (ICA) and its phosphorylated derivatives (pICA) have been shown to have outstanding anti-inflammatory and antioxidant properties. This study was to explore the protective effects of ICA and pICA on the intestinal epithelium of enterotoxigenic Escherichia coli (ETEC)-induced piglet diarrhea and its underlying mechanisms in vivo and in vitro. ETEC K88 increased pro-inflammatory cytokine expression, activated oxidative stress and inhibited antioxidant enzyme activity, induced phosphorylated p38 MAPK gene and protein expression, disrupted intestinal barrier function, and led to diarrhea in piglets. Pretreatment with ICA and pICA effectively alleviated ETEC-induced intestinal barrier dysfunction in vivo and in vitro. Pretreatment with p38 MAPK inhibitor (SB203580) significantly rescued the IPEC-J2 cells barrier function damaged by ETEC challenge. However, pretreatment with p38 MAPK activator (anisomycin) did not alleviated the IPEC-J2 cells barrier function damaged by ETEC challenge. Our data demonstrated that ICA and pICA regulate the inflammatory response and oxidative stress of intestinal epithelial cells by inhibiting the expression of p38 MAPK, thereby alleviating ETEC K88-induced disruption of intestinal barrier function and intestinal permeability. These findings provide new insights into the prevention and treatment of intestinal barrier dysfunction induced by ETEC K88.


Asunto(s)
Escherichia coli Enterotoxigénica/patogenicidad , Células Epiteliales/metabolismo , Infecciones por Escherichia coli/metabolismo , Flavonoides/metabolismo , Mucosa Intestinal/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Adhesión Bacteriana/fisiología , Línea Celular , Diarrea/metabolismo , Diarrea/microbiología , Células Epiteliales/microbiología , Infecciones por Escherichia coli/microbiología , Enfermedades Intestinales/metabolismo , Enfermedades Intestinales/microbiología , Mucosa Intestinal/microbiología , Intestinos/microbiología , Masculino , Permeabilidad , Porcinos , Enfermedades de los Porcinos/metabolismo , Enfermedades de los Porcinos/microbiología
14.
Infect Immun ; 88(2)2020 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-31712269

RESUMEN

Helicobacter pylori colonizes the stomach in about half of the world's population. H. pylori strains containing the cag pathogenicity island (cag PAI) are associated with a higher risk of gastric adenocarcinoma or peptic ulcer disease than cag PAI-negative strains. The cag PAI encodes a type IV secretion system (T4SS) that mediates delivery of the CagA effector protein as well as nonprotein bacterial constituents into gastric epithelial cells. H. pylori-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and interleukin-8 (IL-8) secretion are attributed to T4SS-dependent delivery of lipopolysaccharide metabolites and peptidoglycan into host cells, and Toll-like receptor 9 (TLR9) activation is attributed to delivery of bacterial DNA. In this study, we analyzed the bacterial energetic requirements associated with these cellular alterations. Mutant strains lacking Cagα, Cagß, or CagE (putative ATPases corresponding to VirB11, VirD4, and VirB4 in prototypical T4SSs) were capable of T4SS core complex assembly but defective in CagA translocation into host cells. Thus, the three Cag ATPases are not functionally redundant. Cagα and CagE were required for H. pylori-induced NF-κB activation, IL-8 secretion, and TLR9 activation, but Cagß was dispensable for these responses. We identified putative ATP-binding motifs (Walker-A and Walker-B) in each of the ATPases and generated mutant strains in which these motifs were altered. Each of the Walker box mutant strains exhibited properties identical to those of the corresponding deletion mutant strains. These data suggest that Cag T4SS-dependent delivery of nonprotein bacterial constituents into host cells occurs through mechanisms different from those used for recruitment and delivery of CagA into host cells.


Asunto(s)
Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células Epiteliales/microbiología , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/metabolismo , Transporte Biológico , ADN Bacteriano/metabolismo , Humanos , Interleucina-8/metabolismo , Lipopolisacáridos/metabolismo , FN-kappa B/metabolismo , Peptidoglicano/metabolismo , Receptor Toll-Like 9/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo
15.
Infect Immun ; 88(2)2020 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-31767773

RESUMEN

Aspergillus fumigatus is a ubiquitous fungal pathogen capable of causing multiple pulmonary diseases, including invasive aspergillosis, chronic necrotizing aspergillosis, fungal colonization, and allergic bronchopulmonary aspergillosis. Intact mucociliary barrier function and early airway neutrophil responses are critical for clearing fungal conidia from the host airways prior to establishing disease. Following inhalation, Aspergillus conidia deposit in the small airways, where they are likely to make their initial host encounter with epithelial cells. Challenges in airway infection models have limited the ability to explore early steps in the interactions between A. fumigatus and the human airway epithelium. Here, we use inverted air-liquid interface cultures to demonstrate that the human airway epithelium responds to apical stimulation by A. fumigatus to promote the transepithelial migration of neutrophils from the basolateral membrane surface to the apical airway surface. Promoting epithelial transmigration with Aspergillus required prolonged exposure with live resting conidia. Swollen conidia did not expedite epithelial transmigration. Using A. fumigatus strains containing deletions of genes for cell wall components, we identified that deletion of the hydrophobic rodlet layer or dihydroxynaphthalene-melanin in the conidial cell wall amplified the epithelial transmigration of neutrophils, using primary human airway epithelium. Ultimately, we show that an as-yet-unidentified nonsecreted cell wall protein is required to promote the early epithelial transmigration of human neutrophils into the airspace in response to A. fumigatus Together, these data provide critical insight into the initial epithelial host response to Aspergillus.


Asunto(s)
Aspergilosis/inmunología , Aspergillus fumigatus/inmunología , Pared Celular/inmunología , Células Epiteliales/inmunología , Neutrófilos/inmunología , Aspergilosis/microbiología , Línea Celular Tumoral , Células Epiteliales/microbiología , Humanos , Pulmón/inmunología , Pulmón/microbiología , Melaninas/inmunología , Naftoles/inmunología , Esporas Fúngicas/inmunología
16.
World J Microbiol Biotechnol ; 36(1): 10, 2019 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-31863307

RESUMEN

Aggregation and adhesion capability and survival efficacy of candidate probiotic strain Pediococcus acidilactici NCDC 252 under simulated gastric, intestinal and vaginal conditions was studied. The strain exhibited strong autoaggregation phenotype and coaggregation with other Lactic acid bacteria (LAB) and E. coli. The adhesion studies of NCDC 252 to pig's intestinal epithelial cells showed its adhesive ability. Aggregation and adhesiveness were related through cell surface proteins as removal/extraction of surface proteins resulted in altered aggregation and no adhesiveness. Cell surface proteins were analysed by SDS-PAGE and also in silico analysed from its genome. SDS-PAGE analysis of cell surface proteins of NCDC 252 revealed two potential proteins of approximately 74.3 and 53.6 kDa to be involved in host-probiotic interaction. Removal of cell surface proteins by LiCl-treatment (5 mol l-1) resulted in loss of aggregation and adhesiveness. Further survival of NCDC 252 under simulated gastrointestinal and vaginal conditions in terms of high viable counts confirmed its efficacy for its survival under gut and urogenital conditions. These observations suggest that it can be used further in functional foods, nutraceuticals and in combating urogenital infections. As NCDC 252 was able to survive in intestinal conditions, interaction of its cell surface proteins with intestinal mucins was studied in silico by docking. Highest affinity of adhesion was observed for MUC3B. In conclucion, NCDC 252, exhibited aggregation phenotype and adhesion capability. Survivability of NCDC 252 under simulated conditions and its interaction with human mucins confirms its efficacy to be used as probiotic.


Asunto(s)
Adhesión Bacteriana/fisiología , Pediococcus acidilactici/fisiología , Probióticos/metabolismo , Animales , Suplementos Dietéticos , Células Epiteliales/microbiología , Femenino , Tracto Gastrointestinal/microbiología , Humanos , Lactobacillales/fisiología , Proteínas de la Membrana , Viabilidad Microbiana , Simulación del Acoplamiento Molecular , Mucinas , Vagina/microbiología
17.
Pathog Dis ; 77(8)2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31845968

RESUMEN

The zoonotic disease Q fever caused by the intracellular bacterium Coxiella burnetii remains a global health threat due to its high infectivity, environmental stability, the debilitating nature and the long duration of treatment. Designing new and potent drugs that target previously unexplored pathways is essential to shorten treatment time and minimise antibiotic resistance. Nicotinamide adenine dinucleotide (NAD) is an essential and ubiquitous cofactor in all living organisms. NadB, an L-aspartate oxidase catalysing the first step of the prokaryotic-specific NAD de novo biosynthetic pathway, is required for C. burnetii growth and replication inside host cells. In this study, in vitro enzyme assays utilising recombinant glutathione S-transferase tagged NadB (GST-NadB) demonstrated inhibition of the L-aspartate oxidase activity of NadB by meso-tartrate. Furthermore, meso-tartrate inhibits intracellular growth and replication of C. burnetii inside host cells in a dose-dependent manner, and has no effect on the viability of mammalian cells. Unexpectedly, meso-tartrate also inhibited growth of C. burnetii in axenic medium, and further reduces replication of the nadB mutant inside host cells, suggesting it is acting more widely than simple inhibition of NadB. Overall, these results suggest that the antibacterial activity of meso-tartrate warrants further study, including investigation of its additional target(s).


Asunto(s)
Antibacterianos/farmacología , Coxiella burnetii/efectos de los fármacos , Coxiella burnetii/crecimiento & desarrollo , Inhibidores Enzimáticos/farmacología , Tartratos/farmacología , Aminoácido Oxidorreductasas/antagonistas & inhibidores , Coxiella burnetii/enzimología , Coxiella burnetii/metabolismo , Células Epiteliales/microbiología , Células HeLa , Humanos , Viabilidad Microbiana/efectos de los fármacos , NAD/metabolismo , Células THP-1
18.
Food Funct ; 10(11): 7509-7522, 2019 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-31670355

RESUMEN

Enterotoxigenic Escherichia coli (ETEC) triggers diarrhea in humans and livestock. We have previously showed that ETEC promotes intestinal epithelial cell apoptosis and increases gamma-aminobutyric acid (GABA) concentration in the jejunum, suggesting that GABA might mediate ETEC-induced apoptosis. Here, we found that GABA alleviates ETEC-induced intestinal barrier dysfunctions, including ETEC-induced apoptosis both in vivo and in vitro. Interestingly, the alleviation of GABA on ETEC-induced apoptosis largely depends on autophagy. Mechanistically, GABA attenuates ETEC-induced apoptosis via activating GABAAR signaling and the AMPK-autophagy pathway. These findings highlight that maintaining intestinal GABA concentration could alleviate intestinal ETEC infection.


Asunto(s)
Adenilato Quinasa/metabolismo , Apoptosis/efectos de los fármacos , Escherichia coli Enterotoxigénica , Células Epiteliales/microbiología , Infecciones por Escherichia coli/metabolismo , Receptores de GABA-A/metabolismo , Ácido gamma-Aminobutírico/farmacología , Adenilato Quinasa/genética , Animales , Autofagia/efectos de los fármacos , Autofagia/fisiología , Línea Celular , Células Epiteliales/efectos de los fármacos , Infecciones por Escherichia coli/patología , Mucosa Intestinal/citología , Receptores de GABA-A/genética , Porcinos
19.
BMC Complement Altern Med ; 19(1): 303, 2019 Nov 08.
Artículo en Inglés | MEDLINE | ID: mdl-31703673

RESUMEN

BACKGROUND: Candida albicans is an opportunistic pathogen that causes oral candidiasis and denture stomatitis. It has also been reported to infect oral mucositis lesions in patients who suffer from cancer affecting the head and neck and who receive chemotherapy and radiotherapy treatments. This study aimed to investigate the effects of two cinnamon bark fractions, i.e., an essential oil and an aqueous extract enriched in proanthocyanidins (Cinnulin PF®) on growth, biofilm formation, and adherence properties of C. albicans as well as on oral epithelial cells (barrier integrity, inflammatory response). METHODS: A microplate dilution assay was used to determine antifungal and anti-biofilm properties. A fluorescent assay was used to determine C. albicans adherence to oral epithelial cells. Cytotoxicity toward oral epithelial cells was assessed by determination of cell metabolic activity. Tight junction integrity of gingival keratinocytes was assessed by determination of transepithelial electrical resistance. IL-6 and IL-8 secretion by TNFα-stimulated oral epithelial cells was quantified by ELISA. RESULTS: While Cinnulin PF® did not reduce C. albicans growth, the cinnamon bark oil exhibited high antifungal activity with minimum inhibitory concentrations and minimum fungicidal concentrations in the range of 0.039 to 0.078%. The cinnamon oil was also active against a pre-formed C. albicans biofilm. Interestingly, Cinnulin PF® prevented biofilm formation by C. albicans and attenuated its adherence to oral epithelial cells. At their effective concentrations, the cinnamon oil and the Cinnulin PF® displayed no significant cytotoxicity against oral epithelial cells. In an in vitro model, both cinnamon fractions reinforced the integrity of the oral epithelial barrier. Lastly, Cinnulin PF® inhibited the secretion of interleukin-6 and interleukin-8 by oral epithelial cells stimulated with TNF-α. CONCLUSION: By their ability to attenuate growth, biofilm formation and adherence property of C. albicans, to reinforce the epithelial barrier function, and to exert anti-inflammatory properties the two cinnamon fractions (essential oil, Cinnulin PF®) investigated in the present study may be promising agents for treating oral infections involving C. albicans.


Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candidiasis Bucal/microbiología , Cinnamomum zeylanicum/química , Células Epiteliales/microbiología , Boca/microbiología , Aceites Volátiles/farmacología , Biopelículas/efectos de los fármacos , Candida albicans/crecimiento & desarrollo , Candida albicans/fisiología , Candidiasis Bucal/tratamiento farmacológico , Línea Celular , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Boca/metabolismo , Corteza de la Planta/química
20.
Int J Med Sci ; 16(10): 1320-1327, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31692996

RESUMEN

Porphyromonas gingivalis is a pivotal periodontal pathogen, and the epithelial cells serve as the first physical barrier to defend the host from bacterial attack. Within this host-bacteria interaction, P. gingivalis can modify the host immune reaction and adjust the gene expression, which is associated with periodontitis pathogenesis and developing strategies. Herein, a meta-analysis was made to get the differential gene expression profiles in epithelial cells with or without P. gingivalis infection. The network-based meta-analysis program for gene expression profiling was used. Both the gene ontology analysis and the pathway enrichment analysis of the differentially expressed genes were conducted. Our results determined that 290 genes were consistently up-regulated in P. gingivalis infected epithelial cells. 229 gene ontology biological process terms of up-regulated genes were discovered, including "negative regulation of apoptotic process" and "positive regulation of cell proliferation/migration/angiogenesis". In addition to the well-known inflammatory signaling pathways, the pathway associated with a transcriptional misregulation in cancer has also been increased. Our findings indicated that P. gingivalis benefited from the survival of epithelial cells, and got its success as a colonizer in oral epithelium. The results also suggested that infection of P. gingivalis might contribute to oral cancer through chronic inflammation. Negative regulation of the apoptotic process and transcriptional misregulation in cancer pathway are important contributors to the cellular physiology changes during infection development, which have particular relevance to the pathogenesis and progressions of periodontitis, even to the occurrence of oral cancer.


Asunto(s)
Infecciones por Bacteroidaceae/inmunología , Interacciones Huésped-Patógeno/genética , Neoplasias de la Boca/patología , Periodontitis/inmunología , Porphyromonas gingivalis/inmunología , Infecciones por Bacteroidaceae/genética , Infecciones por Bacteroidaceae/microbiología , Infecciones por Bacteroidaceae/patología , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Progresión de la Enfermedad , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Perfilación de la Expresión Génica , Ontología de Genes , Encía/citología , Encía/inmunología , Encía/microbiología , Interacciones Huésped-Patógeno/inmunología , Humanos , Mucosa Bucal/citología , Mucosa Bucal/inmunología , Mucosa Bucal/microbiología , Neoplasias de la Boca/genética , Neoplasias de la Boca/inmunología , Neoplasias de la Boca/microbiología , Periodontitis/genética , Periodontitis/microbiología , Periodontitis/patología , Porphyromonas gingivalis/aislamiento & purificación , Porphyromonas gingivalis/patogenicidad , Transducción de Señal/genética , Transducción de Señal/inmunología , Regulación hacia Arriba
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