RESUMEN
In spite of huge endeavors in cell line engineering to produce glycoproteins with desired and uniform glycoforms, it is still not possible in vivo. Alternatively, in vitro glycoengineering can be used for the modification of glycans. However, in vitro glycoengineering relies on expensive nucleotide sugars, such as uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) which serves as GlcNAc donor for the synthesis of various glycans. In this work, we present a systematic study for the cell-free de novo synthesis and regeneration of UDP-GlcNAc from polyphosphate, UMP and GlcNAc by a cascade of five enzymes (N-acetylhexosamine kinase (NahK), Glc-1P uridyltransferase (GalU), uridine monophosphate kinase (URA6), polyphosphate kinase (PPK3), and inorganic diphosphatase (PmPpA). All enzymes were expressed in E. coli BL21 Gold (DE3) and purified using immobilized metal affinity chromatography (IMAC). Results from one-pot experiments demonstrate the successful production of UDP-GlcNAc with a yield approaching 100%. The highest volumetric productivity of the cascade was about 0.81â¯g L-1 â¯h-1 of UDP-GlcNAc. A simple model based on mass action kinetics was sufficient to capture the dynamic behavior of the multienzyme pathway. Moreover, a design equation based on metabolic control analysis was established to investigate the effect of enzyme concentration on the UDP-GlcNAc flux and to demonstrate that the flux of UDP-GlcNAc can be controlled by means of the enzyme concentrations. The effect of temperature on the UDP-GlcNAc flux followed an Arrhenius equation and the optimal co-factor concentration (Mg2+) for high UDP-GlcNAc synthesis rates depended on the working temperature. In conclusion, the study covers the entire engineering process of a multienzyme cascade, i.e. pathway design, enzyme expression, enzyme purification, reaction kinetics and investigation of the influence of basic parameters (temperature, co-factor concentration, enzyme concentration) on the synthesis rate. Thus, the study lays the foundation for future cascade optimization, preparative scale UDP-GlcNAc synthesis and for in situ coupling of the network with UDP-GlcNAc transferases to efficiently regenerate UDP-GlcNAc. Hence, this study provides a further step towards cost-effective in vitro glycoengineering of antibodies and other glycosylated proteins.
Asunto(s)
Sistema Libre de Células/metabolismo , Enzimas/metabolismo , Uridina Difosfato N-Acetilglucosamina/biosíntesis , Vías Biosintéticas , Enzimas/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Cinética , Nucleósido-Fosfato Quinasa/genética , Nucleósido-Fosfato Quinasa/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , TemperaturaRESUMEN
Intestinal epithelial cells form a barrier that is critical in protecting the host from the hostile luminal environment. Previously, we showed that lysophosphatidic acid (LPA) receptor 1 regulates proliferation of intestinal epithelial cells, such that the absence of LPA1 mitigates the epithelial wound healing process. This study provides evidence that LPA1 is important for the maintenance of epithelial barrier integrity. The epithelial permeability, determined by fluorescently labeled dextran flux and transepithelial resistance, is increased in the intestine of mice with global deletion of Lpar1, Lpar1-/- (Lpa1-/-). Serum liposaccharide level and bacteria loads in the intestinal mucosa and peripheral organs were elevated in Lpa1-/- mice. Decreased claudin-4, caudin-7, and E-cadherin expression in Lpa1-/- mice further suggested defective apical junction integrity in these mice. Regulation of LPA1 expression in Caco-2 cells modulated epithelial permeability and the expression levels of junctional proteins. The increased epithelial permeability in Lpa1-/- mice correlated with increased susceptibility to an experimental model of colitis. This resulted in more severe inflammation and increased mortality compared with control mice. Treatment of Caco-2 cells with tumor necrosis factor-α and interferon-γ significantly increased paracellular permeability, which was blocked by cotreatment with LPA, but not LPA1 knockdown cells. Similarly, orally given LPA blocked tumor necrosis factor-mediated intestinal barrier defect in mice. LPA1 plays a significant role in maintenance of epithelial barrier in the intestine via regulation of apical junction integrity.
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Colitis/fisiopatología , Mucosa Intestinal/metabolismo , Receptores del Ácido Lisofosfatídico/fisiología , Animales , Carga Bacteriana , Células CACO-2 , Colitis/genética , Colitis/microbiología , Susceptibilidad a Enfermedades , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , Absorción Intestinal/fisiología , Mucosa Intestinal/microbiología , Masculino , Ratones Noqueados , Permeabilidad , Receptores del Ácido Lisofosfatídico/deficiencia , Receptores del Ácido Lisofosfatídico/genéticaRESUMEN
Glycosylation of proteins is a key function of the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell-cell adhesion, blood-group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein-based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose-1-phosphate-guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA), and 1-domain polyphosphate kinase 2 (1D-Ppk2) expressed in E. coli for the cell-free production and regeneration of GDP-mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP-mannose is produced at various conditions, that is pH 7-8, temperature 25-35°C and co-factor concentrations of 5-20 mM MgCl2 . The maximum reaction rate of GDP-mannose achieved was 2.7 µM/min at 30°C and 10 mM MgCl2 producing 566 nmol GDP-mannose after a reaction time of 240 min. With respect to the initial GDP concentration (0.8 mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane-deleted Alg1 (ALG1ΔTM), the first mannosyltransferase in the ER-associated lipid-linked oligosaccharide (LLO) assembly. Thereby, in a one-pot reaction, phytanyl-PP-(GlcNAc)2 -Man1 was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl-PP-(GlcNAc)2 -Man1 can serve as a substrate for the synthesis of LLO for the cell-free in vitro glycosylation of proteins. A high-performance anion exchange chromatography method with UV and conductivity detection (HPAEC-UV/CD) assay was optimized and validated to determine the enzyme kinetics. The established kinetic model enabled the optimization of the GDP-mannose regenerating cascade and can further be used to study coupling of the GDP-mannose cascade with glycosyltransferases. Overall, the study envisages a first step towards the development of a platform for the cell-free production of LLOs as precursors for in vitro glycoengineering of proteins.
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Enzimas/metabolismo , Escherichia coli/genética , Guanosina Difosfato Manosa/metabolismo , Lipopolisacáridos/metabolismo , Proteínas Recombinantes/metabolismo , Coenzimas/metabolismo , Enzimas/genética , Enzimas/aislamiento & purificación , Escherichia coli/metabolismo , Expresión Génica , Concentración de Iones de Hidrógeno , Cloruro de Magnesio/metabolismo , Manosa/metabolismo , Polifosfatos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , TemperaturaRESUMEN
Backgrounds: Recent studies have identified the role of serologic markers in characterizing disease phenotype, location, complications, and severity among Northern Europeans (NE) with Crohn's disease (CD). However, very little is known about the role of serology in CD among African Americans (AA). Our study explored the relationship between serology and disease phenotype in AA with CD, while controlling for genetic ancestry. Methods: AAs with CD were enrolled as participants through multicenter collaborative efforts. Serological levels of IgA anti-Saccharomyces cervisiae antibody (ASCA), IgG ASCA, E. coli outermembrane porin C, anti-CBir1, and ANCA were measured using enzyme-linked immunosorbent assays. Genotyping was performed using Illumina immunochip technology; an admixture rate was calculated for each subject. Multiple imputation by chained equations was performed to account for data missing at random. Logistic regression was used to calculate adjusted odds ratio (OR) for associations between serological markers and both complicated disease and disease requiring surgery. Results: A total of 358 patients were included in the analysis. The majority of our patients had inflammatory, noncomplicated disease (58.4%), perianal disease (55.7%), and documented colonic inflammation (86.8%). On multivariable analysis, both IgG ASCA and OmpC were associated with complicated disease (OR, 2.67; 95% CI, 1.67-4.28; OR, 2.23; 95% CI, 1.41-3.53, respectively) and disease requiring surgery (OR, 2.51; 95% CI, 1.49-4.22; OR, 3.57; 95% CI, 2.12-6.00). NE admixture to the African genome did not have any associations or interactions in relation to clinical outcome. Conclusions: Our study comprises the largest cohort of AAs with CD. The utility of serological markers for the prognosis of CD in NE applies equally to AA populations.
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Anticuerpos Anticitoplasma de Neutrófilos/sangre , Anticuerpos Antibacterianos/sangre , Anticuerpos Antifúngicos/sangre , Biomarcadores/sangre , Enfermedad de Crohn/sangre , Inmunoglobulina A/inmunología , Complicaciones Posoperatorias , Adolescente , Adulto , Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antifúngicos/inmunología , Niño , Estudios de Cohortes , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/cirugía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Adulto JovenRESUMEN
BACKGROUND & AIMS: The inflammatory bowel diseases (IBD) ulcerative colitis (UC) and Crohn's disease (CD) cause significant morbidity and are increasing in prevalence among all populations, including African Americans. More than 200 susceptibility loci have been identified in populations of predominantly European ancestry, but few loci have been associated with IBD in other ethnicities. METHODS: We performed 2 high-density, genome-wide scans comprising 2345 cases of African Americans with IBD (1646 with CD, 583 with UC, and 116 inflammatory bowel disease unclassified) and 5002 individuals without IBD (controls, identified from the Health Retirement Study and Kaiser Permanente database). Single-nucleotide polymorphisms (SNPs) associated at P < 5.0 × 10-8 in meta-analysis with a nominal evidence (P < .05) in each scan were considered to have genome-wide significance. RESULTS: We detected SNPs at HLA-DRB1, and African-specific SNPs at ZNF649 and LSAMP, with associations of genome-wide significance for UC. We detected SNPs at USP25 with associations of genome-wide significance for IBD. No associations of genome-wide significance were detected for CD. In addition, 9 genes previously associated with IBD contained SNPs with significant evidence for replication (P < 1.6 × 10-6): ADCY3, CXCR6, HLA-DRB1 to HLA-DQA1 (genome-wide significance on conditioning), IL12B,PTGER4, and TNC for IBD; IL23R, PTGER4, and SNX20 (in strong linkage disequilibrium with NOD2) for CD; and KCNQ2 (near TNFRSF6B) for UC. Several of these genes, such as TNC (near TNFSF15), CXCR6, and genes associated with IBD at the HLA locus, contained SNPs with unique association patterns with African-specific alleles. CONCLUSIONS: We performed a genome-wide association study of African Americans with IBD and identified loci associated with UC in only this population; we also replicated IBD, CD, and UC loci identified in European populations. The detection of variants associated with IBD risk in only people of African descent demonstrates the importance of studying the genetics of IBD and other complex diseases in populations beyond those of European ancestry.
Asunto(s)
Negro o Afroamericano/genética , Moléculas de Adhesión Celular Neuronal/genética , Colitis Ulcerosa/genética , Enfermedad de Crohn/genética , Predisposición Genética a la Enfermedad/genética , Cadenas HLA-DRB1/genética , Proteínas Represoras/genética , Ubiquitina Tiolesterasa/genética , Adenilil Ciclasas/genética , Estudios de Casos y Controles , Proteínas Ligadas a GPI/genética , Estudio de Asociación del Genoma Completo , Técnicas de Genotipaje , Cadenas alfa de HLA-DQ/genética , Humanos , Subunidad p40 de la Interleucina-12/genética , Canal de Potasio KCNQ2/genética , Polimorfismo de Nucleótido Simple , Receptores CXCR6 , Receptores de Quimiocina/genética , Receptores de Interleucina/genética , Subtipo EP4 de Receptores de Prostaglandina E/genética , Receptores Virales/genética , Nexinas de Clasificación/genética , Tenascina/genética , Población Blanca/genéticaRESUMEN
BACKGROUND: Epidemiological and genetic studies suggest a role for enteric flora in the pathogenesis of Crohn's disease (CD). Crohn's disease-associated Escherichia coli (CDEC) is characterized by their ability to invade epithelial cells and survive and induce high concentration of TNF-α from infected macrophages. However, the molecular mechanisms of CDEC survival in infected macrophages are not completely understood. METHODS: Intracellular survival of CDEC strain LF82 isolated from inflamed ileum tissue, 13I isolated from inflamed colonic tissue, and control E. coli strains were tested in the murine macrophage cell line, J774A.1 by Gentamicin protection assay. Modulation of intracellular cell signaling pathways by the E. coli strains were assessed by western blot analysis and confocal microscopy. RESULTS: 13I demonstrated increased survival in macrophages with 2.6-fold higher intracellular bacteria compared with LF82, yet both strains induced comparable levels of TNF-α. LF82 and 13I differentially modulated key mitogen-activated protein kinase pathways during the acute phase of infection; LF82 activated all 3 mitogen-activated protein kinase pathways, whereas 13I activated ERK1/2 pathway but not p38 and JNK pathways. Both 13I and LF82 suppressed nuclear translocation of NFκB compared with noninvasive E. coli strains during the acute phase of infection. However, unlike noninvasive E. coli strains, 13I and LF82 infection resulted in chronic activation of NFκB during the later phase of infection. CONCLUSIONS: Our results showed that CDEC survive in macrophages by initially suppressing NFκB activation. However, persistence of bacterial within macrophages induces chronic activation of NFκB, which correlates with increased TNF-α secretion from infected macrophages.
Asunto(s)
Apoptosis , Enfermedad de Crohn/microbiología , Células Epiteliales/microbiología , Infecciones por Escherichia coli/microbiología , Escherichia coli/patogenicidad , Macrófagos/microbiología , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adhesión Bacteriana , Western Blotting , Células Cultivadas , Escherichia coli/crecimiento & desarrollo , Escherichia coli/aislamiento & purificación , Técnica del Anticuerpo Fluorescente , Humanos , Microscopía Confocal , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de SeñalRESUMEN
Factors implicated in the pathophysiology of ulcerative colitis (UC) are an abnormal immune response, defect in intestinal epithelial barrier function, and gut microbiota. Currently, it is unclear whether specific bacterial strains are responsible for the induction of intestinal inflammation, but increased bacterial tissue invasion has been described in affected UC patients. Further, a quantitative and qualitative microbial imbalance in UC, defined as dysbiosis, has been characterized by an increase in Rhodococcus spp., Shigella spp., and Escherichia spp., but a decrease in certain Bacteroides spp.. More specifically, Campylobacter spp., Enterobacteriae, and enterohepatic Helicobacter were more prevalent in tissue sample from UC patients subjected to molecular detection methods, but not controls. In addition, serologic testing identified Fusobacterim varium as a potential contributor to the intestinal inflammation in UC. Interestingly, in-situ hybridization studies have shown anti-inflammatory Lactobacillus spp. and Pediococcus spp. were absent in samples from subjects affected by UC. Therefore, dysbiosis is a factor in the pathogenesis of UC.
RESUMEN
BACKGROUND: Metastasis, the spread and growth of tumor cells to distant organ sites, represents the most devastating attribute and plays a major role in the morbidity and mortality of cancer. Inflammation is crucial for malignant tumor transformation and survival. Thus, blocking inflammation is expected to serve as an effective cancer treatment. Among anti-inflammation therapies, chemokine modulation is now beginning to emerge from the pipeline. CXC chemokine receptor-4 (CXCR4) and its ligand stromal cell-derived factor-1 (CXCL12) interaction and the resulting cell signaling cascade have emerged as highly relevant targets since they play pleiotropic roles in metastatic progression. The unique function of CXCR4 is to promote the homing of tumor cells to their microenvironment at the distant organ sites. METHODOLOGY/PRINCIPAL FINDINGS: We describe the actions of N,N'-(1,4-phenylenebis(methylene))dipyrimidin-2-amine (designated MSX-122), a novel small molecule and partial CXCR4 antagonist with properties quite unlike that of any other reported CXCR4 antagonists, which was prepared in a single chemical step using a reductive amination reaction. Its specificity toward CXCR4 was tested in a binding affinity assay and a ligand competition assay using (18)F-labeled MSX-122. The potency of the compound was determined in two functional assays, Matrigel invasion assay and cAMP modulation. The therapeutic potential of MSX-122 was evaluated in three different murine models for inflammation including an experimental colitis, carrageenan induced paw edema, and bleomycin induced lung fibrosis and three different animal models for metastasis including breast cancer micrometastasis in lung, head and neck cancer metastasis in lung, and uveal melanoma micrometastasis in liver in which CXCR4 was reported to play crucial roles. CONCLUSIONS/SIGNIFICANCE: We developed a novel small molecule, MSX-122, that is a partial CXCR4 antagonist without mobilizing stem cells, which can be safer for long-term blockade of metastasis than other reported CXCR4 antagonists.
Asunto(s)
Antiinflamatorios/farmacología , Antineoplásicos/farmacología , Pirimidinas/farmacología , Receptores CXCR4/antagonistas & inhibidores , Animales , Antiinflamatorios/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/patología , Simulación por Computador , Diseño de Fármacos , Edema/inducido químicamente , Edema/tratamiento farmacológico , Femenino , Fibrosis/inducido químicamente , Fibrosis/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/patología , Humanos , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/secundario , Pulmón/efectos de los fármacos , Pulmón/patología , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Masculino , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Modelos Moleculares , Unión Proteica , Pirimidinas/uso terapéutico , Receptores CXCR4/metabolismo , Neoplasias de la Úvea/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
It has become increasingly clear that inflammatory bowel disease (IBD) develops on the background of genetic defects in the host, conveying an increased susceptibility to an environmental antigen or antigens. The environmental factor implicated in the pathophysiology of gut inflammation, which is undergoing increased scrutiny, is the intestinal flora. The intestinal flora as a whole and specific bacteria and their products have been found to trigger cytokine expression in various cell types. Consistently, multiple bacterial strains were found to induce tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8) in macrophage and epithelial cell systems, respectively, in particular in Crohn's disease. Interestingly, various cell types from patients with IBD display an increased susceptibility to specific bacterial products, including flagellin, pili, and lipopolysaccharides. It remains to be determined whether additional effector proteins regulate cytokine expression and the aberrant mucosal immune response in IBD.
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Bacterias/patogenicidad , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/microbiología , Animales , Humanos , Enfermedades Inflamatorias del Intestino/metabolismoAsunto(s)
Neoplasias del Conducto Colédoco/diagnóstico , Conducto Colédoco/patología , Tumores de Células Gigantes/diagnóstico , Colangiopancreatografia Retrógrada Endoscópica , Conducto Colédoco/cirugía , Neoplasias del Conducto Colédoco/cirugía , Tumores de Células Gigantes/cirugía , Humanos , Masculino , Persona de Mediana Edad , Radiografía Abdominal , TomografíaRESUMEN
Lymphostatin/EHEC factor for adherence-1 is a novel large toxin represented in various Gram negative bacteria, highly associated with the development of infectious diarrhea and hemolytic uremic syndrome. In vitro and in vivo experiments identified lymphostatin/EFA-1 as a toxin with a central role in the pathogenesis of Gram negative bacteria, responsible for bacterial adhesion, intestinal colonization, immunosuppression, and disruption of gut epithelial barrier function.
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Toxinas Bacterianas/metabolismo , Escherichia coli Enterohemorrágica/patogenicidad , Proteínas de Escherichia coli/metabolismo , Adhesión Bacteriana , Diarrea/microbiología , Escherichia coli Enterohemorrágica/metabolismo , Síndrome Hemolítico-Urémico/microbiología , Humanos , Terapia de Inmunosupresión , Intestinos/microbiologíaRESUMEN
Expression of prohibitin 1 (PHB), a multifunctional protein in the cell, is decreased during inflammatory bowel disease (IBD). Little is known regarding the regulation and role of PHB during intestinal inflammation. We examined the effect of tumor necrosis factor alpha (TNF-alpha), a cytokine that plays a central role in the pathogenesis of IBD, on PHB expression and the effect of sustained PHB expression on TNF-alpha activation of nuclear factor-kappa B (NF-kappaB) and epithelial barrier dysfunction, two hallmarks of intestinal inflammation. We show that TNF-alpha decreased PHB protein and mRNA abundance in intestinal epithelial cells in vitro and in colon mucosa in vivo. Sustained expression of prohibitin in intestinal epithelial cells in vitro and in vivo (prohibitin transgenic mice, PHB TG) resulted in a marked decrease in TNF-alpha-induced nuclear translocation of the NF-kappaB protein p65, NF-kappaB/DNA binding, and NF-kappaB-mediated transcriptional activation despite robust IkappaB-alpha phosphorylation and degradation and increased cytosolic p65. Cells overexpressing PHB were protected from TNF-alpha-induced increased epithelial permeability. Expression of importin alpha3, a protein involved in p50/p65 nuclear import, was decreased in cells overexpressing PHB and in colon mucosa of PHB TG mice. Restoration of importin alpha3 levels sustained NF-kappaB activation by TNF-alpha during PHB transfection. These results suggest that PHB inhibits NF-kappaB nuclear translocation via a novel mechanism involving alteration of importin alpha3 levels. TNF-alpha decreases PHB expression in intestinal epithelial cells and restoration of PHB expression in these cells can protect against the deleterious effects of TNF-alpha and NF-kappaB on barrier function.
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Proteínas Represoras/fisiología , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , alfa Carioferinas/fisiología , Adenocarcinoma/patología , Animales , Colon/citología , Neoplasias del Colon/patología , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/patología , Genes Reporteros , Humanos , Mucosa Intestinal/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Prohibitinas , Transporte de Proteínas/fisiología , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Proteínas Represoras/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Lymphocyte inhibitory factor A (lifA) in Citrobacter rodentium encodes the large toxin lymphostatin, which contains two enzymatic motifs associated with bacterial pathogenesis, a glucosyltransferase and a protease. Our aim was to determine the effects of each lymphostatin motif on intestinal epithelial-barrier function. In-frame mutations of C. rodentium lifA glucosyltransferase (CrGlM21) and protease (CrPrM5) were generated by homologous recombination. Infection of both model intestinal epithelial monolayers and mice with C. rodentium wild type resulted in compromised epithelial barrier function and mislocalization of key intercellular junction proteins in the tight junction and adherens junction. In contrast, CrGlM21 was impaired in its ability to reduce barrier function and influenced the tight junction proteins ZO-1 and occludin. CrPrM5 demonstrated decreased effects on the adherens junction proteins beta-catenin and E-cadherin. Analysis of the mechanisms revealed that C. rodentium wild type differentially influenced Rho GTPase activation, suppressed Cdc42 activation, and induced Rho GTPase activation. CrGlM21 lost its suppressive effects on Cdc42 activation, whereas CrPrM5 was unable to activate Rho signaling. Rescue experiments using constitutively active Cdc42 or C3 exotoxin to inhibit Rho GTPase supported a role of Rho GTPases in the epithelial barrier compromise induced by C. rodentium. Taken together, our results suggest that lymphostatin is a bacterial virulence factor that contributes to the disruption of intestinal epithelial-barrier function via the modulation of Rho GTPase activities.
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Mucosa Intestinal/inmunología , Factores de Virulencia/inmunología , Proteínas de Unión al GTP rho/inmunología , Uniones Adherentes/metabolismo , Uniones Adherentes/patología , Animales , Citrobacter rodentium , Infecciones por Enterobacteriaceae/enzimología , Infecciones por Enterobacteriaceae/inmunología , Activación Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Glucosiltransferasas/genética , Glucosiltransferasas/inmunología , Mucosa Intestinal/enzimología , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Mutación , Péptido Hidrolasas/genética , Péptido Hidrolasas/inmunología , Factores de Virulencia/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
BACKGROUND & AIMS: Krüppel-like factor 5 (KLF5) is a transcription factor that is highly expressed in proliferating crypt cells of the intestinal epithelium. KLF5 has a pro-proliferative effect in vitro and is induced by mitogenic and stress stimuli. To determine whether KLF5 is involved in mediating proliferative responses to intestinal stressors in vivo, we examined its function in a mouse model of transmissible murine colonic hyperplasia triggered by colonization of the mouse colon by the bacteria Citrobacter rodentium. METHODS: Heterozygous Klf5 knockout (Klf5(+/-)) mice were generated from embryonic stem cells carrying an insertional disruption of the Klf5 gene. Klf5(+/-) mice or wild-type (WT) littermates were infected with C rodentium by oral gavage. At various time points postinfection, mice were killed and distal colons were harvested. Colonic crypt heights were determined morphometrically from sections stained with H&E. Frozen tissues were stained by immunofluorescence using antibodies against Klf5 and the proliferation marker, Ki67, to determine Klf5 expression and numbers of proliferating cells per crypt. RESULTS: Infection of WT mice with C rodentium resulted in a 2-fold increase in colonic crypt heights at 14 days postinfection and was accompanied by a 1.7-fold increase in Klf5 expression. Infection of Klf5(+/-) mice showed an attenuated induction of Klf5 expression, and hyperproliferative responses to C rodentium were reduced in the Klf5(+/-) animals as compared with WT littermates. CONCLUSION: Our study shows that Klf5 is a key mediator of crypt cell proliferation in the colon in response to pathogenic bacterial infection.
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Citrobacter rodentium/aislamiento & purificación , Colitis/metabolismo , Colon/patología , Infecciones por Enterobacteriaceae/metabolismo , Factores de Transcripción de Tipo Kruppel/fisiología , Animales , Western Blotting , Proliferación Celular , Citrobacter rodentium/patogenicidad , Colitis/genética , Colitis/microbiología , Colon/metabolismo , Colon/microbiología , Modelos Animales de Enfermedad , Infecciones por Enterobacteriaceae/genética , Infecciones por Enterobacteriaceae/microbiología , Expresión Génica , Genotipo , Hiperplasia/etiología , Hiperplasia/genética , Hiperplasia/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaAsunto(s)
Enfermedades del Ano/diagnóstico , Enfermedad de Crohn/diagnóstico , Mycobacterium tuberculosis/aislamiento & purificación , Enfermedades del Recto/diagnóstico , Tuberculosis/diagnóstico , Adulto , Antituberculosos/uso terapéutico , Enfermedades del Ano/microbiología , Enfermedades del Ano/fisiopatología , Biopsia , Diagnóstico Diferencial , Humanos , Pulmón/diagnóstico por imagen , Pulmón/patología , Masculino , Radiografía , Enfermedades del Recto/fisiopatología , Prueba de Tuberculina , Tuberculosis/microbiología , Tuberculosis/fisiopatologíaRESUMEN
Gut barrier dysfunction may occur in short bowel syndrome (SBS). We hypothesized that systemic exposure to flagellin and lipopolysaccharide (LPS) in SBS might regulate specific immune responses. We analyzed serial serum samples obtained from parenteral nutrition (PN)-dependent patients with SBS versus non-SBS control serum. Serum from 23 adult SBS patients was obtained at baseline and 4, 8, 12, 16, 20, and 24 wk in a trial of modified diet with or without growth hormone. Control serum was obtained from 48 healthy adults and 37 adults requiring PN during critical illness. Serum flagellin was detected by an ELISA recognizing an array of gram-negative flagellins, and LPS was detected by limulus assay. Serum flagellin- and LPS-specific immunoglobulin levels (IgM, IgA, and IgG) were determined by ELISA. Serum flagellin and LPS were undetectable in control subjects. In contrast, serum flagellin, LPS, or both were detected in 14 SBS patients (61%) during one or more time points [flagellin alone, 5/23 (22%); LPS alone, 6/23 (26%); or flagellin + LPS, 3/23 (13%)]. Flagellin-specific serum IgM, IgA, and IgG levels were markedly increased in SBS patients compared with both control populations and remained elevated during the 6-mo study period. LPS-specific IgA was significantly higher in SBS patients compared with healthy controls; LPS-specific IgM, IgA, and IgG levels each decreased over time in association with PN weaning. We conclude that adults with PN-dependent SBS are systemically exposed to flagellin and LPS, presumably from the gut lumen. This likely regulates innate and adaptive immune responses to these specific bacterial products.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Flagelina/sangre , Flagelina/inmunología , Lipopolisacáridos/sangre , Lipopolisacáridos/inmunología , Síndrome del Intestino Corto/inmunología , Adulto , Anciano , Especificidad de Anticuerpos , Femenino , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Persona de Mediana Edad , Regulación hacia ArribaRESUMEN
Crohn's disease (CD) and ulcerative colitis (UC) are idiopathic inflammatory conditions of the gut. Our goal was to investigate if invasive Escherichia coli strains were present in patients with inflammatory bowel disease (IBD). Bacterial strains were isolated from biopsy material obtained from normal controls, and patients with a clinical diagnosis of CD and UC. Invasive bacteria were characterized by gentamicin protection assay and biochemical profiling (Api-20E). Strains were characterized by induction of cytokine expression in epithelial and macrophage cell cultures, measurement of epithelial barrier function, and confocal microscopy. Of all invasive bacterial strains in CD 98.9% were identified as E. coli as opposed to 42.1% in UC and 2.1% in normal controls. Epithelial invasion in vitro was significantly higher for CD-associated E. coli (8.4%, +/-5.5 of initial inoculum (I/O)) in comparison to UC (2.5%, +/-0.4 I/O), but highest for strains from inflamed CD tissue (11.3%, +/-4.3 I/O). Both, CD and UC E. coli strains induced high mean TNF-alpha expression in macrophage cell lines (2604.8 pg/10(5) cells, +/-447.4; 2,402.6 pg/10(5) cells, +/-476.3, respectively), but concentrations were significantly higher for isolates from inflamed CD tissue (3071.3 pg/10(5) cells, +/-226.0). Invasive E. coli from IBD tissue induced similar concentrations of interleukin (IL)-8 in epithelial cell cultures, but strains from inflamed CD tissue induced significantly less epithelial IL-8 (674.1 pg/10(5) cells, +/-58.0 vs 920.5 pg/10(5) cells, +/-94.6). IBD-associated E. coli strains significantly decreased transepithelial resistance, induced disorganization of F-actin and displacement of ZO-1, and E-cadherin from the apical junctional complex (AJC). In comparison to normal controls and UC, E. coli are more prevalent in CD, are highly invasive, and do not encode for known effector proteins. E. coli strains from IBD patients regulate cytokine expression and epithelial barrier function, two pathological features of IBD.
Asunto(s)
Colitis Ulcerosa/microbiología , Enfermedad de Crohn/microbiología , Citocinas/metabolismo , Escherichia coli/patogenicidad , Mucosa Intestinal/microbiología , Células CACO-2 , Estudios de Casos y Controles , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/metabolismo , Escherichia coli/genética , Humanos , Mucosa Intestinal/metabolismo , PermeabilidadRESUMEN
Sensitive biomarkers for intestinal absorptive function would be clinically useful in short bowel syndrome (SBS). Citrulline (Cit) is a product of the metabolism of glutamine (Gln) and derived amino acids by enterocytes. Cit is produced almost exclusively by the gut, which is also a major site of Gln metabolism. The goals of this study were to examine whether plasma Cit and Gln concentrations are biomarkers of residual small intestinal length and nutrient absorptive functions in adult SBS patients followed prospectively. We studied 24 stable adults with severe SBS receiving chronic parenteral nutrition (PN) in a double-blind, randomized trial of individualized dietary modification +/- recombinant human growth hormone (GH). During a baseline week, intestinal absorption studies (% absorption of fluid, kcal, nitrogen, fat, carbohydrate, sodium, phosphorus, and magnesium) were performed and concomitant plasma Cit and Gln concentrations determined. Individualized dietary modification and treatment with subcutaneous injection of placebo (n = 9) or GH (0.1 mg/kg daily x 21 days, then 3 times/week; n = 15) were then begun. PN weaning was initiated after week 4 and continued as tolerated for 24 weeks. Repeat plasma amino acid determination and nutrient absorption studies were performed at weeks 4 and 12. Residual small bowel length at baseline was positively correlated with baseline plasma Cit (r = 0.467; p = .028). However, no significant correlations between absolute Cit or Gln concentrations and the percent absorption of nutrient substrates at any time point were observed. Similarly, no correlation between the change in Cit or GLN concentration and the change in % nutrient absorption was observed (baseline vs weeks 4 and 12, respectively). By weeks 12 and 24, 7 and 13 subjects were weaned completely from PN, respectively. However, baseline plasma Cit or Gln did not predict PN weaning at these time points. We concluded that plasma Cit (but not Gln) concentrations appeared to be an indicator of small intestinal length in adult SBS. However, neither plasma Cit nor Gln was a biomarker for intestinal absorptive function in this cohort of patients with SBS.