RESUMEN
Flagellum-mediated motility is essential to Pseudomonas aeruginosa (P. aeruginosa) virulence. Antibody against flagellin reduces motility and inhibits the spread of the bacteria from the infection site. The standard soft-agar assay to demonstrate anti-flagella motility inhibition requires long incubation times, is difficult to interpret, and requires large amounts of antibody. We have developed a time-lapse video microscopy method to analyze anti-flagellin P. aeruginosa motility inhibition that has several advantages over the soft agar assay. Antisera from mice immunized with flagellin type A or B were incubated with Green Fluorescent Protein (GFP)-expressing P. aeruginosa strain PAO1 (FlaB+) and GFP-expressing P. aeruginosa strain PAK (FlaA+). We analyzed the motion of the bacteria in video taken in ten second time intervals. An easily measurable decrease in bacterial locomotion was observed microscopically within minutes after the addition of small volumes of flagellin antiserum. From data analysis, we were able to quantify the efficacy of anti-flagellin antibodies in the test serum that decreased P. aeruginosa motility. This new video microscopy method to assess functional activity of anti-flagellin antibodies required less serum, less time, and had more robust and reproducible endpoints than the standard soft agar motility inhibition assay.
Asunto(s)
Anticuerpos Antibacterianos , Flagelos , Flagelina , Sueros Inmunes , Microscopía por Video , Pseudomonas aeruginosa , Flagelina/inmunología , Pseudomonas aeruginosa/inmunología , Animales , Sueros Inmunes/inmunología , Anticuerpos Antibacterianos/inmunología , Flagelos/inmunología , Ratones , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiologíaRESUMEN
To gain insight into sialic acid biology and sialidase/neuraminidase (NEU) expression in mature human neutrophil (PMN)s, we studied NEU activity and expression in PMNs and the HL60 promyelocytic leukemic cell line, and changes that might occur in PMNs undergoing apoptosis and HL60 cells during their differentiation into PMN-like cells. Mature human PMNs contained NEU activity and expressed NEU2, but not NEU1, the NEU1 chaperone, protective protein/cathepsin A(PPCA), NEU3, and NEU4 proteins. In proapoptotic PMNs, NEU2 protein expression increased > 30.0-fold. Granulocyte colony-stimulating factor protected against NEU2 protein upregulation, PMN surface desialylation and apoptosis. In response to 3 distinct differentiating agents, dimethylformamide, dimethylsulfoxide, and retinoic acid, total NEU activity in differentiated HL60 (dHL60) cells was dramatically reduced compared to that of nondifferentiated cells. With differentiation, NEU1 protein levels decreased > 85%, PPCA and NEU2 proteins increased > 12.0-fold, and 3.0-fold, respectively, NEU3 remained unchanged, and NEU4 increased 1.7-fold by day 3, and then returned to baseline. In dHL60 cells, lectin blotting revealed decreased α2,3-linked and increased α2,6-linked sialylation. dHL60 cells displayed increased adhesion to and migration across human bone marrow-derived endothelium and increased bacterial phagocytosis. Therefore, myeloid apoptosis and differentiation provoke changes in NEU catalytic activity and protein expression, surface sialylation, and functional responsiveness.
Asunto(s)
Ácido N-Acetilneuramínico , Neuraminidasa , Apoptosis , Diferenciación Celular , Humanos , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/metabolismo , Neutrófilos/metabolismoRESUMEN
Mammalian neuraminidases (NEUs), also known as sialidases, are enzymes that cleave off the terminal neuraminic, or sialic, acid resides from the carbohydrate moieties of glycolipids and glycoproteins. A rapidly growing body of literature indicates that in addition to their metabolic functions, NEUs also regulate the activity of their glycoprotein targets. The simple post-translational modification of NEU protein targets-removal of the highly electronegative sialic acid-affects protein folding, alters protein interactions with their ligands, and exposes or covers proteolytic sites. Through such effects, NEUs regulate the downstream processes in which their glycoprotein targets participate. A major target of desialylation by NEUs are mucins (MUCs), and such post-translational modification contributes to regulation of disease processes. In this review, we focus on the regulatory roles of NEU-modified MUCs as coordinators of disease pathogenesis in fibrotic, inflammatory, infectious, and autoimmune diseases. Special attention is placed on the most abundant and best studied NEU1, and its recently discovered important target, mucin-1 (MUC1). The role of the NEU1 - MUC1 axis in disease pathogenesis is discussed, along with regulatory contributions from other MUCs and other pathophysiologically important NEU targets.
Asunto(s)
Enfermedades del Sistema Inmune , Mucinas , Animales , Glicoproteínas/metabolismo , Mamíferos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/metabolismoRESUMEN
We previously reported that flagellin-expressing Pseudomonas aeruginosa (Pa) provokes NEU1 sialidase-mediated MUC1 ectodomain (MUC1-ED) desialylation and MUC1-ED shedding from murine lungs in vivo. Here, we asked whether Pa in the lungs of patients with ventilator-associated pneumonia might also increase MUC1-ED shedding. The levels of MUC1-ED and Pa-expressed flagellin were dramatically elevated in bronchoalveolar lavage fluid (BALF) harvested from Pa-infected patients, and each flagellin level, in turn, predicted MUC1-ED shedding in the same patient. Desialylated MUC1-ED was only detected in BALF of Pa-infected patients. Clinical Pa strains increased MUC1-ED shedding from cultured human alveolar epithelia, and FlaA and FlaB flagellin-expressing strains provoked comparable levels of MUC1-ED shedding. A flagellin-deficient isogenic mutant generated dramatically reduced MUC1-ED shedding compared with the flagellin-expressing wild-type strain, and purified FlaA and FlaB recapitulated the effect of intact bacteria. Pa:MUC1-ED complexes were detected in the supernatants of alveolar epithelia exposed to wild-type Pa, but not to the flagellin-deficient Pa strain. Finally, human recombinant MUC1-ED dose-dependently disrupted multiple flagellin-driven processes, including Pa motility, Pa biofilm formation, and Pa adhesion to human alveolar epithelia, while enhancing human neutrophil-mediated Pa phagocytosis. Therefore, shed desialylated MUC1-ED functions as a novel flagellin-targeting, Pa-responsive decoy receptor that participates in the host response to Pa at the airway epithelial surface.
Asunto(s)
Flagelina/metabolismo , Pulmón/metabolismo , Mucina-1/metabolismo , Neumonía Bacteriana/metabolismo , Neumonía Asociada al Ventilador/metabolismo , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Células A549 , Anciano , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/microbiología , Femenino , Flagelina/genética , Interacciones Huésped-Patógeno , Humanos , Pulmón/microbiología , Masculino , Persona de Mediana Edad , Mutación , Neuraminidasa/metabolismo , Neumonía Bacteriana/diagnóstico , Neumonía Bacteriana/microbiología , Neumonía Asociada al Ventilador/diagnóstico , Neumonía Asociada al Ventilador/microbiología , Infecciones por Pseudomonas/diagnóstico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidadRESUMEN
The extracellular domain (ED) of the membrane-spanning sialoglycoprotein, mucin-1 (MUC1), is an in vivo substrate for the lysosomal sialidase, neuraminidase-1 (NEU1). Engagement of the MUC1-ED by its cognate ligand, Pseudomonas aeruginosa-expressed flagellin, increases NEU1-MUC1 association and NEU1-mediated MUC1-ED desialylation to unmask cryptic binding sites for its ligand. However, the mechanism(s) through which intracellular NEU1 might physically interact with its surface-expressed MUC1-ED substrate are unclear. Using reciprocal coimmunoprecipitation and in vitro binding assays in a human airway epithelial cell system, we show here that NEU1 associates with the MUC1-cytoplasmic domain (CD) but not with the MUC1-ED. Prior pharmacologic inhibition of the NEU1 catalytic activity using the NEU1-selective sialidase inhibitor, C9-butyl amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid, did not diminish NEU1-MUC1-CD association. In addition, glutathione-S-transferase (GST) pull-down assays using the deletion mutants of the MUC1-CD mapped the NEU1-binding site to the membrane-proximal 36 aa of the MUC1-CD. In a cell-free system, we found that the purified NEU1 interacted with the immobilized GST-MUC1-CD and the purified MUC1-CD associated with the immobilized 6XHis-NEU1, indicating that the NEU1-MUC1-CD interaction was direct and independent of its chaperone protein, protective protein/cathepsin A. However, the NEU1-MUC1-CD interaction was not required for the NEU1-mediated MUC1-ED desialylation. Finally, we demonstrated that overexpression of either WT NEU1 or a catalytically dead NEU1 G68V mutant diminished the association of the established MUC1-CD binding partner, PI3K, to MUC1-CD and reduced downstream Akt kinase phosphorylation. These results indicate that NEU1 associates with the juxtamembranous region of the MUC1-CD to inhibit PI3K-Akt signaling independent of NEU1 catalytic activity.
Asunto(s)
Mucina-1/metabolismo , Neuraminidasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Células A549 , Sustitución de Aminoácidos , Células HEK293 , Humanos , Mucina-1/genética , Mutación Missense , Neuraminidasa/genética , Fosfatidilinositol 3-Quinasas/genética , Dominios Proteicos , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
Pulmonary fibrosis remains a serious biomedical problem with no cure and an urgent need for better therapies. Neuraminidases (NEUs), including NEU1, have been recently implicated in the mechanism of pulmonary fibrosis by us and others. We now have tested the ability of a broad-spectrum neuraminidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), to modulate the in vivo response to acute intratracheal bleomycin challenge as an experimental model of pulmonary fibrosis. A marked alleviation of bleomycin-induced body weight loss and notable declines in accumulation of pulmonary lymphocytes and collagen deposition were observed. Real-time polymerase chain reaction analyses of human and mouse lung tissues and primary human lung fibroblast cultures were also performed. A predominant expression and pronounced elevation in the levels of NEU1 mRNA were observed in patients with idiopathic pulmonary fibrosis and bleomycin-challenged mice compared with their corresponding controls, whereas NEU2, NEU3, and NEU4 were expressed at far lower levels. The levels of mRNA for the NEU1 chaperone, protective protein/cathepsin A (PPCA), were also elevated by bleomycin. Western blotting analyses demonstrated bleomycin-induced elevations in protein expression of both NEU1 and PPCA in mouse lungs. Two known selective NEU1 inhibitors, C9-pentyl-amide-DANA (C9-BA-DANA) and C5-hexanamido-C9-acetamido-DANA, dramatically reduced bleomycin-induced loss of body weight, accumulation of pulmonary lymphocytes, and deposition of collagen. Importantly, C9-BA-DANA was therapeutic in the chronic bleomycin exposure model with no toxic effects observed within the experimental timeframe. Moreover, in the acute bleomycin model, C9-BA-DANA attenuated NEU1-mediated desialylation and shedding of the mucin-1 ectodomain. These data indicate that NEU1-selective inhibition offers a potential therapeutic intervention for pulmonary fibrotic diseases. SIGNIFICANCE STATEMENT: Neuraminidase-1-selective therapeutic targeting in the acute and chronic bleomycin models of pulmonary fibrosis reverses pulmonary collagen deposition, accumulation of lymphocytes in the lungs, and the disease-associated loss of body weight-all without observable toxic effects. Such therapy is as efficacious as nonspecific inhibition of all neuraminidases in these models, thus indicating the central role of neuraminidase-1 as well as offering a potential innovative, specifically targeted, and safe approach to treating human patients with a severe malady: pulmonary fibrosis.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Ácido N-Acetilneuramínico/análogos & derivados , Neuraminidasa/antagonistas & inhibidores , Neumonía/tratamiento farmacológico , Fibrosis Pulmonar/tratamiento farmacológico , Animales , Bleomicina/toxicidad , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Femenino , Fibroblastos/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Mucina-1/metabolismo , Ácido N-Acetilneuramínico/farmacología , Ácido N-Acetilneuramínico/uso terapéutico , Neuraminidasa/genética , Neuraminidasa/metabolismo , Neumonía/etiología , Fibrosis Pulmonar/etiologíaRESUMEN
Magnolia bark extract administered as a dietary supplement to poultry confers a performance and health benefit, but the mechanisms are unknown. Here, a metabolomics approach was used to identify changes in intestinal metabolite levels in chickens fed an unsupplemented diet or a diet supplemented with magnolia bark extract. Total body weight gains of chickens fed magnolia bark-supplemented diets were increased 2% (from 861 to 878 g/chicken), compared with chickens fed an unsupplemented diet. Compared with unsupplemented controls, the levels of 278 intestinal biochemicals (metabolites) were altered (165 increased, 113 decreased) in chickens given the magnolia-supplemented diet. Data for biochemicals of intestinal contents of chickens fed the unsupplemented diet clustered on the left side of the PCA score plot, while those of the magnolia-supplemented diet were separated and clustered on the right side. The biochemicals included changes in the levels of amino acids, fatty acids, peptides, and nucleosides, which provided a distinctive biochemical signature unique to the magnolia-supplemented group, compared with the unsupplemented group. These results provide the foundation for future studies to identify naturally-produced biochemicals that might be used to improve poultry growth performance.
RESUMEN
Direct-fed microbials (DFMs) are dietary supplements containing live microorganisms which confer a performance and health benefit to the host, but the mechanisms are unclear. Here, a metabolomics approach was used to identify changes in intestinal metabolite levels in chickens fed an unsupplemented diet or a diet supplemented with B. subtilis strain 1781 or strain 747. Body weight gains of chickens fed the B. subtilis-supplemented diets were increased up to 5.6% in the B. subtilis 1781 group and 7.6% in the B. subtilis 747 group compared with chickens fed the unsupplemented diet. Compared with unsupplemented controls, the levels of 83 metabolites were altered (p < 0.05) (25 increased, 58 decreased) in chickens given the B. subtilis 1781-supplemented diet, while 50 were altered (p < 0.05) (12 increased, 38 decreased) with the B. subtilis 747-supplemented diet. Twenty-two metabolites were altered (p < 0.05) (18 increased, 4 decreased) in the B. subtilis 1781 vs. B. subtilis 747 groups. A random forest analysis of the B. subtilis 1781 vs. control groups gave a predictive accuracy of 87.5%, while that of the B. subtilis 747 vs. control groups was 62.5%. A random forest analysis of the B. subtilis 1781 vs. B. subtilis 747 groups gave a predictive accuracy of 75.0%. Changes in the levels of these intestinal biochemicals provided a distinctive biochemical signature unique to each B. subtilis-supplemented group, and were characterized by alterations in the levels of dipeptides (alanylleucine, glutaminylleucine, phenylalanylalanine, valylglutamine), nucleosides (N1-methyladenosine, N6-methyladenosine, guanine, 2-deoxyguanosine), fatty acids (sebacate, valerylglycine, linoleoylcholine), and carbohydrates (fructose). These results provide the foundation for future studies to identify biochemicals that might be used to improve poultry growth performance in the absence of antibiotic growth promoters.
RESUMEN
Subtherapeutic levels of dietary antibiotics increase growth performance in domestic animals, but the mechanisms are poorly understood. Here, 1-week-old broiler chickens were challenged with LPS (experiment 1), or co-infected with Eimeria maxima and Clostridium perfringens as an experimental model of necrotic enteritis (experiment 2), and fed a standard basal diet or a basal diet supplemented with virginiamycin or bacitracin methylene disalicylate. In experiment 1, LPS-challenged chickens fed the unsupplemented diet had decreased body weight gains, compared with unsupplemented controls given the PBS control. In contrast, antibiotic supplementation increased body weight gains in both the LPS-challenged and PBS groups, compared with the antibiotic-free diet. LPS-challenged chickens fed the unsupplemented diet had increased expression levels of intestinal tight junction proteins (ZO1, JAM2), MUC2 gel-forming mucin, and inflammatory cytokines (IL-1ß, IL-2, IL-6, IL-8, IL-17A) at 24 h post-challenge, compared with unsupplemented chickens given the PBS control. However, LPS-challenged chickens fed the antibiotic-supplemented diets had decreased levels of intestinal inflammatory cytokine transcripts, compared with LPS-challenged chickens given the unsupplemented basal diet. In experiment 2, E. maxima/C. perfringens-co-infected chickens fed the antibiotic-supplemented diets had increased body weight gains, decreased intestinal pathology, and greater intestinal crypt depth, compared with co-infected chickens given the unsupplemented diet. Further, similar to LPS challenge, E. maxima/C. perfringens-co-infection of chickens fed the antibiotic-supplemented diets decreased expression levels of intestinal inflammatory cytokines, compared with co-infected chickens given the unsupplemented diet. These results support the hypothesis that dietary antibiotic growth promoters might increase poultry growth, in part, through down-regulation of pathogen-induced inflammatory responses.
RESUMEN
Pseudomonas aeruginosa (Pa) expresses an adhesin, flagellin, that engages the mucin 1 (MUC1) ectodomain (ED) expressed on airway epithelia, increasing association of MUC1-ED with neuraminidase 1 (NEU1) and MUC1-ED desialylation. The MUC1-ED desialylation unmasks both cryptic binding sites for Pa and a protease recognition site, permitting its proteolytic release as a hyperadhesive decoy receptor for Pa. We found here that intranasal administration of Pa strain K (PAK) to BALB/c mice increases MUC1-ED shedding into the bronchoalveolar compartment. MUC1-ED levels increased as early as 12 h, peaked at 24-48 h with a 7.8-fold increase, and decreased by 72 h. The a-type flagellin-expressing PAK strain and the b-type flagellin-expressing PAO1 strain stimulated comparable levels of MUC1-ED shedding. A flagellin-deficient PAK mutant provoked dramatically reduced MUC1-ED shedding compared with the WT strain, and purified flagellin recapitulated the WT effect. In lung tissues, Pa increased association of NEU1 and protective protein/cathepsin A with MUC1-ED in reciprocal co-immunoprecipitation assays and stimulated MUC1-ED desialylation. NEU1-selective sialidase inhibition protected against Pa-induced MUC1-ED desialylation and shedding. In Pa-challenged mice, MUC1-ED-enriched bronchoalveolar lavage fluid (BALF) inhibited flagellin binding and Pa adhesion to human airway epithelia by up to 44% and flagellin-driven motility by >30%. Finally, Pa co-administration with recombinant human MUC1-ED dramatically diminished lung and BALF bacterial burden, proinflammatory cytokine levels, and pulmonary leukostasis and increased 5-day survival from 0% to 75%. We conclude that Pa flagellin provokes NEU1-mediated airway shedding of MUC1-ED, which functions as a decoy receptor protecting against lethal Pa lung infection.
Asunto(s)
Flagelina/metabolismo , Mucina-1/metabolismo , Neuraminidasa/metabolismo , Neumonía Bacteriana/metabolismo , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/fisiología , Animales , Femenino , Interacciones Huésped-Patógeno , Humanos , Pulmón/metabolismo , Pulmón/microbiología , Pulmón/patología , Masculino , Ratones Endogámicos BALB C , Neumonía Bacteriana/microbiología , Neumonía Bacteriana/patología , Factores Protectores , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patologíaRESUMEN
Type IV pili (T4P) are bacterial appendages composed of protein subunits, called pilins, noncovalently assembled into helical fibers. T4P are essential, in many bacterial species, for processes as diverse as twitching motility, natural competence, biofilm or microcolony formation, and host cell adhesion. The genes encoding type IV pili are found universally in the Gram-negative, aerobic, nonflagellated, and pathogenic coccobacillus Acinetobacter baumannii, but there is considerable variation in PilA, the major protein subunit, both in amino acid sequence and in glycosylation patterns. Here we report the X-ray crystal structure of PilA from AB5075, a recently characterized, highly virulent isolate, at 1.9 Å resolution and compare it to homologues from A. baumannii strains ACICU and BIDMC57, which are C-terminally glycosylated. These structural comparisons revealed that PilAAB5075 exhibits a distinctly electronegative surface chemistry. To understand the functional consequences of this change in surface electrostatics, we complemented a ΔpilA knockout strain with divergent pilA genes from ACICU, BIDMC57, and AB5075. The resulting transgenic strains showed differential twitching motility and biofilm formation while maintaining the ability to adhere to epithelial cells. PilAAB5075 and PilAACICU, although structurally similar, promote different characteristics, favoring twitching motility and biofilm formation, respectively. These results support a model in which differences in pilus electrostatics affect the equilibrium of microcolony formation, which in turn alters the balance between motility and biofilm formation in Acinetobacter.
Asunto(s)
Acinetobacter baumannii/química , Proteínas Fimbrias/química , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Sustitución de Aminoácidos , Cristalografía por Rayos X , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Glicosilación , Mutación Missense , Dominios ProteicosRESUMEN
BACKGROUND: Magnolia tree bark has been widely used in traditional Asian medicine. However, to our knowledge, no studies have been reported investigating the effects of dietary supplementation with magnolia bark extract in chickens. OBJECTIVE: We tested the hypothesis that dietary supplementation of chickens with a Magnolia officinalis bark extract would increase growth performance in uninfected and Eimeria maxima/Clostridium perfringens co-infected chickens. METHODS: A total of 168 chickens were fed from hatch either a standard diet or a diet supplemented with 0.33 mg or 0.56 mg M. officinalis bark extract/kg (M/H low or M/H high, respectively) from days 1 to 35. At day 14, half of the chickens were orally infected with E. maxima, followed by C. perfringens infection at day 18 to induce experimental avian necrotic enteritis. Daily feed intake, feed conversion ratio, body weight gain, and final body weight were measured as indicators of growth performance. Serum α1-acid glycoprotein (AGP) concentrations were measured as an indicator of systemic inflammation, and intestinal lesion scores were determined as a marker of disease progression. Transcript levels for catalase, heme oxygenase 1, and superoxide dismutase in the intestine, liver, spleen, and skeletal muscle were measured as indicators of antioxidant status. RESULTS: Growth performance increased between days 1 and 35 in uninfected and E. maxima/C. perfringens co-infected chickens fed M/H-low or M/H-high diets compared with unsupplemented controls. Gut lesion scores were decreased, whereas AGP concentrations were unchanged, in co-infected chickens fed magnolia-supplemented diets compared with unsupplemented controls. In general, transcripts for antioxidant enzymes increased in chickens fed magnolia-supplemented diets compared with unsupplemented controls, and significant interactions between dietary supplementation and co-infection were observed for all antioxidant enzyme transcript levels. CONCLUSION: Magnolia bark extract might be useful for future development of dietary strategies to improve poultry health, disease resistance, and productivity without the use of antibiotic growth promoters.
RESUMEN
Although dietary antibiotic growth promoters have long been used to increase growth performance in commercial food animal production, the biochemical details associated with these effects remain poorly defined. A metabolomics approach was used to characterize and identify the biochemical compounds present in the intestine of broiler chickens fed a standard, unsupplemented diet or a diet supplemented with the antibiotic growth promoters, virginiamycin or bacitracin methylene disalicylate. Compared with unsupplemented controls, the levels of 218 biochemicals were altered (156 increased, 62 decreased) in chickens given the virginiamycin-supplemented diet, while 119 were altered (96 increased, 23 decreased) with the bacitracin-supplemented diet. When compared between antibiotic-supplemented groups, 79 chemicals were altered (43 increased, 36 decreased) in virginiamycin- vs. bacitracin-supplemented chickens. The changes in the levels of intestinal biochemicals provided a distinctive biochemical signature unique to each antibiotic-supplemented group. These biochemical signatures were characterized by increases in the levels of metabolites of amino acids (e.g. 5-hydroxylysine, 2-aminoadipate, 5-hydroxyindoleaceate, 7-hydroxyindole sulfate), fatty acids (e.g. oleate/vaccenate, eicosapentaenoate, 16-hydroxypalmitate, stearate), nucleosides (e.g. inosine, N6-methyladenosine), and vitamins (e.g. nicotinamide). These results provide the framework for future studies to identify natural chemical compounds to improve poultry growth performance without the use of in-feed antibiotics.
Asunto(s)
Antibacterianos/metabolismo , Bacitracina/metabolismo , Pollos/crecimiento & desarrollo , Intestinos/fisiología , Metaboloma/fisiología , Salicilatos/metabolismo , Virginiamicina/metabolismo , Aminoácidos/metabolismo , Análisis de Varianza , Alimentación Animal/análisis , Animales , Antibacterianos/farmacología , Bacitracina/farmacología , Suplementos Dietéticos , Ácidos Grasos/metabolismo , Microbioma Gastrointestinal/efectos de los fármacos , Niacinamida/metabolismo , Nucleósidos/metabolismo , Salicilatos/farmacología , Virginiamicina/farmacologíaRESUMEN
MUC1 is a membrane-bound mucin expressed on the apical surfaces of most mucosal epithelial cells. In normal lung epithelia, MUC1 is a binding site for Pseudomonas aeruginosa, an opportunistic human pathogen of great clinical importance. It has now been established that MUC1 also serves an anti-inflammatory role in the airways that is initiated late in the course of a bacterial infection and is mediated through inhibition of Toll-like receptor (TLR) signaling. MUC1 expression was initially shown to interfere with TLR5 signaling in response to P. aeruginosa flagellin, but has since been extended to other TLRs. These new findings point to an immunomodulatory role for MUC1 during P. aeruginosa lung infection, particularly during the resolution phase of inflammation. This review briefly summarizes the recent characterization of MUC1's anti-inflammatory properties in both the respiratory tract and extrapulmonary tissues.
RESUMEN
Alveolar macrophages (AMs) play a critical role in the clearance of Pseudomonas aeruginosa (Pa) from the airways. However, hyper-activation of macrophages can impair bacterial clearance and contribute to morbidity and mortality. MUC1 mucin is a membrane-tethered, high molecular mass glycoprotein expressed on the apical surface of mucosal epithelial cells and some hematopoietic cells, including macrophages, where it counter-regulates inflammation. We recently reported that Pa up-regulates the expression of MUC1 in primary human AMs and THP-1 macrophages, and that increased MUC1 expression in these cells prevents hyper-activation of macrophages that appears to be important for host defense against severe pathology of Pa lung infection. The aims of this study were to elucidate the mechanism by which Pa increases MUC1 expression in macrophages. The results showed that: (a) Pa stimulation of THP-1 macrophages increased MUC1 expression both at transcriptional and protein levels in a dose-dependent manner; (b) Both Pa- and LPS-induced MUC1 expression in THP-1 cells were significantly diminished by an inhibitory peptide of TLR4; and (c) LPS-stimulated MUC1 expression was diminished at both the mRNA and protein levels by an inhibitor of the p38 mitogen-activated protein kinase, but not by inhibitors of ERK1/2, JNK, or IKK. We conclude that Pa-stimulated MUC1 expression in THP-1 macrophages is regulated mainly through the TLR4-p38 signaling pathway.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Macrófagos/microbiología , Mucina-1/genética , Infecciones por Pseudomonas/genética , Pseudomonas aeruginosa/fisiología , Regulación hacia Arriba , Línea Celular , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Mucina-1/inmunología , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/patología , Transducción de Señal , Receptor Toll-Like 4/inmunologíaRESUMEN
Avian coccidiosis is caused by multiple species of the apicomplexan protozoan, Eimeria, and is one of the most economically devastating enteric diseases for the poultry industry worldwide. Host immunity to Eimeria infection, however, is relatively species-specific. The ability to immunize chickens against different species of Eimeria using a single vaccine will have a major beneficial impact on commercial poultry production. In this paper, we describe the molecular cloning, purification, and vaccination efficacy of a novel Eimeria vaccine candidate, elongation factor-1α (EF-1α). One day-old broiler chickens were given two subcutaneous immunizations one week apart with E. coli-expressed E. tenella recombinant (r)EF-1α protein and evaluated for protection against challenge infection with E. tenella or E. maxima. rEF-1α-vaccinated chickens exhibited increased body weight gains, decreased fecal oocyst output, and greater serum anti-EF-1α antibody levels following challenge infection with either E. tenella or E. maxima compared with unimmunized controls. Vaccination with EF-1α may represent a new approach to inducing cross-protective immunity against avian coccidiosis in the field.
Asunto(s)
Pollos/parasitología , Coccidiosis/veterinaria , Eimeria tenella/inmunología , Factor 1 de Elongación Peptídica/inmunología , Enfermedades de las Aves de Corral/prevención & control , Vacunas Antiprotozoos/inmunología , Animales , Antígenos de Protozoos/inmunología , Pollos/inmunología , Coccidiosis/parasitología , Coccidiosis/prevención & control , Escherichia coli/genética , Escherichia coli/metabolismo , Masculino , Enfermedades de las Aves de Corral/parasitología , Proteínas Recombinantes/inmunología , Vacunación/veterinaria , Aumento de PesoRESUMEN
In postconfluent human pulmonary microvascular endothelial cell (HPMEC)s, NEU1 sialidase associates with and desialylates the src family kinase (SFK) substrate, CD31, and disrupts angiogenesis. We asked whether the NEU1-CD31 interaction might be SFK-driven. We found that normalized phospho-SFK (PY416) signal is increased in postconfluent HPMECs compared to subconfluent cells and prior SFK inhibition with PP2 or SU6656 completely blocked NEU1 association with and desialylation of CD31. Prior silencing of each of the four SFKs expressed in HPMECs, as well as CD31, dramatically reduced confluence-induced SFK activation. No increases in tyrosine phosphorylation of NEU1 or CD31 were detected. However, in postconfluent cells, we found increased tyrosine phosphorylation of a 120 kDa protein that was identified as p120 catenin (p120ctn). Prior silencing of c-src, fyn, or yes each reduced p120ctn phosphorylation. Prior knockdown of p120ctn prevented NEU1-CD31 association in both co-immunoprecipitation and pull-down assays. In these same assays, p120ctn associated with each of the four HPMEC-expressed SFKs as well as CD31 and NEU1. The CD31-p120ctn interaction was SFK-dependent whereas the NEU1-p120ctn interaction was not. Using purified recombinant binding partners in a cell-free system, direct protein-protein interactions between NEU1, CD31, and p120ctn were detected. Our combined data indicate that as HPMECs achieve confluence and CD31 ectodomains become homophilically engaged, multiple SFKs are activated to increase tyrosine phosphorylation of p120ctn, which in turn, functions as a cross-bridging adaptor molecule that physically couples NEU1 to CD31, permitting NEU1-mediated desialylation of CD31. These findings establish a SFK-driven, p120ctn-dependent mechanism for NEU1 recruitment to CD31.
Asunto(s)
Cateninas/genética , Neuraminidasa/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Cateninas/metabolismo , Línea Celular , Sistema Libre de Células , Células Endoteliales/metabolismo , Humanos , Pulmón/metabolismo , Microvasos/metabolismo , Ácido N-Acetilneuramínico/genética , Ácido N-Acetilneuramínico/metabolismo , Neovascularización Fisiológica/genética , Neuraminidasa/metabolismo , Fosforilación , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Unión Proteica , Mapas de Interacción de Proteínas/genética , Proteínas Proto-Oncogénicas c-fyn/genética , Proteínas Proto-Oncogénicas c-yes/genética , Transducción de Señal/genética , Familia-src Quinasas/genética , Catenina deltaRESUMEN
OBJECTIVE: The effects of vaccinating 18-day-old chicken embryos with the combination of recombinant Eimeria profilin plus Clostridium perfringens (C. perfringens) NetB proteins mixed in the Montanide IMS adjuvant on the chicken immune response to necrotic enteritis (NE) were investigated using an Eimeria maxima (E. maxima)/C. perfringens co-infection NE disease model that we previously developed. METHODS: Eighteen-day-old broiler embryos were injected with 100 µL of phosphate-buffered saline, profilin, profilin plus necrotic enteritis B-like (NetB), profilin plus NetB/Montanide adjuvant (IMS 106), and profilin plus Net-B/Montanide adjuvant (IMS 101). After post-hatch birds were challenged with our NE experimental disease model, body weights, intestinal lesions, serum antibody levels to NetB, and proinflammatory cytokine and chemokine mRNA levels in intestinal intraepithelial lymphocytes were measured. RESULTS: Chickens in ovo vaccinated with recombinant profilin plus NetB proteins/IMS106 and recombinant profilin plus NetB proteins/IMS101 showed significantly increased body weight gains and reduced gut damages compared with the profilin-only group, respectively. Greater antibody response to NetB toxin were observed in the profilin plus NetB/IMS 106, and profilin plus NetB/IMS 101 groups compared with the other three vaccine/adjuvant groups. Finally, diminished levels of transcripts encoding for proinflammatory cytokines such as lipopolysaccharide-induced tumor necrosis factor-α factor, tumor necrosis factor superfamily 15, and interleukin-8 were observed in the intestinal lymphocytes of chickens in ovo injected with profilin plus NetB toxin in combination with IMS 106, and profilin plus NetB toxin in combination with IMS 101 compared with profilin protein alone bird. CONCLUSION: These results suggest that the Montanide IMS adjuvants potentiate host immunity to experimentally-induced avian NE when administered in ovo in conjunction with the profilin and NetB proteins, and may reduce disease pathology by attenuating the expression of proinflammatory cytokines and chemokines implicated in disease pathogenesis.
RESUMEN
Acinetobacter baumannii is a Gram-negative coccobacillus found primarily in hospital settings that has recently emerged as a source of hospital-acquired infections. A. baumannii expresses a variety of virulence factors, including type IV pili, bacterial extracellular appendages often essential for attachment to host cells. Here, we report the high resolution structures of the major pilin subunit, PilA, from three Acinetobacter strains, demonstrating that A. baumannii subsets produce morphologically distinct type IV pilin glycoproteins. We examine the consequences of this heterogeneity for protein folding and assembly as well as host-cell adhesion by Acinetobacter Comparisons of genomic and structural data with pilin proteins from other species of soil gammaproteobacteria suggest that these structural differences stem from evolutionary pressure that has resulted in three distinct classes of type IVa pilins, each found in multiple species.