Establishing the phenotypic basis of adherent-invasive Escherichia coli (AIEC) pathogenicity in intestinal inflammation

2019 ◽  
Author(s):  
Hatem Kittana ◽  
João C. Gomes-Neto ◽  
Kari Heck ◽  
Jason Sughroue ◽  
Yibo Xian ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial ◽  
Disease Etiology ◽  
E Coli ◽  
Caco2 Cells ◽  
Phenotypic Group ◽  
Tnf Α

AbstractBackground & AimsAdherent-invasive Escherichia coli (AIEC) are enriched in ileal Crohn’s disease patients and implicated in disease etiology. However, AIEC pathogenesis is poorly understood, and it is unclear if the expansion of these organisms contributes to inflammatory bowel disease (IBD). Questions also remain as to what extent the various in vitro phenotypes used to classify AIEC are pathologically relevant.MethodsWe utilized a combination of in vitro phenotyping and a murine model of intestinal inflammation to systematically relate AIEC phenotypes to pathogenicity for 30 mucosa-associated human-derived E. coli strains. In vitro assays used included survival/replication in and TNF-α production by J774 macrophages as well as invasion/replication in Caco2 intestinal epithelial cells.ResultsAIEC do not form a phenotypic group that is clearly separated from non-AIEC. However, E. coli strains displaying in vitro AIEC phenotypes caused, on average, more severe intestinal inflammation. Survival/replication of strains in J774 and Caco2 cells were positively correlated with disease in vivo, while adherence to Caco2 cells and TNF-α production by J774 cells were not. Importantly, co-colonization with adherent non-AIEC strains ameliorated AIEC-mediated disease.ConclusionOur findings do not support the existence of an AIEC pathovar that can be clearly separated from commensal E. coli. However, intracellular survival/replication phenotypes do contribute to murine intestinal inflammation, suggesting that the AIEC overgrowth observed in human IBD makes a causal contribution to disease. The ability to differentiate pathologically-relevant AIEC phenotypes from those that are not provides an important foundation for developing strategies to predict, diagnose and treat human IBD through characterizing and modulating patient E. coli populations.

scholarly journals Development of Heptylmannoside-Based Glycoconjugate Antiadhesive Compounds against Adherent-Invasive Escherichia coli Bacteria Associated with Crohn's Disease

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Adeline Sivignon ◽  
Xibo Yan ◽  
Dimitri Alvarez Dorta ◽  
Richard Bonnet ◽  
Julie Bouckaert ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial ◽  
Content Type ◽  
Type 1 Pili

ABSTRACTThe ileal lesions of Crohn's disease (CD) patients are colonized by adherent-invasiveEscherichia coli(AIEC) bacteria. These bacteria adhere to mannose residues expressed by CEACAM6 on host cells in a type 1 pilus-dependent manner. In this study, we investigated different antagonists of FimH, the adhesin of type 1 pili, for their ability to block AIEC adhesion to intestinal epithelial cells (IEC). Monovalent and multivalent derivatives ofn-heptyl α-d-mannoside (HM), a nanomolar antagonist of FimH, were testedin vitroin IEC infected with the AIEC LF82 strain andin vivoby oral administration to CEACAM6-expressing mice infected with LF82 bacteria.In vitro, multivalent derivatives were more potent than the monovalent derivatives, with a gain of efficacy superior to their valencies, probably owing to their ability to form bacterial aggregates. Of note, HM and the multi-HM glycoconjugates exhibited lower efficacyin vivoin decreasing LF82 gut colonization. Interestingly, HM analogues functionalized with an isopropylamide (1A-HM) or β-cyclodextrin pharmacophore at the end of the heptyl tail (1CD-HM) exerted beneficial effectsin vivo. These two compounds strongly decreased the amount of LF82 bacteria in the feces of mice and that of bacteria associated with the gut mucosa when administered orally at a dose of 10 mg/kg of body weight after infection. Importantly, signs of colitis and intestinal inflammation induced by LF82 infection were also prevented. These results highlight the potential of the antiadhesive compounds to treat CD patients abnormally colonized by AIEC bacteria and point to an alternative to the current approach focusing on blocking proinflammatory mediators.IMPORTANCECurrent treatments for Crohn's disease (CD), including immunosuppressive agents, anti-tumor necrosis factor alpha (anti-TNF-α) and anti-integrin antibodies, focus on the symptoms but not on the cause of the disease. Adherent-invasiveEscherichia coli(AIEC) bacteria abnormally colonize the ileal mucosa of CD patients via the interaction of the mannose-specific adhesin FimH of type 1 pili with CEACAM6 mannosylated proteins expressed on the epithelial cell surface. Thus, we decided to develop an antiadhesive strategy based on synthetic FimH antagonists specifically targeting AIEC bacteria that would decrease intestinal inflammation. Heptylmannoside (HM)-based glycocompounds strongly inhibit AIEC adhesion to intestinal epithelial cellsin vitro. The antiadhesive effect of two of these compounds of relatively simple chemical structure was also observedin vivoin AIEC-infected CEACAM6-expressing mice and was associated with a reduction in the signs of colitis. These results suggest a new therapeutic approach for CD patients colonized by AIEC bacteria, based on the development of synthetic FimH antagonists.

scholarly journals Prohibitin Inhibits Tumor Necrosis Factor alpha–induced Nuclear Factor-kappa B Nuclear Translocation via the Novel Mechanism of Decreasing Importin α3 Expression

2009 ◽  
Vol 20 (20) ◽  
pp. 4412-4423 ◽  
Author(s):  
Arianne L. Theiss ◽  
Aaron K. Jenkins ◽  
Ngozi I. Okoro ◽  
Jan-Michael A. Klapproth ◽  
Didier Merlin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Inflammation ◽  
Nuclear Translocation ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Factor Alpha

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-α), a cytokine that plays a central role in the pathogenesis of IBD, on PHB expression and the effect of sustained PHB expression on TNF-α activation of nuclear factor-kappa B (NF-κB) and epithelial barrier dysfunction, two hallmarks of intestinal inflammation. We show that TNF-α 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-α–induced nuclear translocation of the NF-κB protein p65, NF-κB/DNA binding, and NF-κB–mediated transcriptional activation despite robust IκB-α phosphorylation and degradation and increased cytosolic p65. Cells overexpressing PHB were protected from TNF-α–induced increased epithelial permeability. Expression of importin α3, 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 α3 levels sustained NF-κB activation by TNF-α during PHB transfection. These results suggest that PHB inhibits NF-κB nuclear translocation via a novel mechanism involving alteration of importin α3 levels. TNF-α decreases PHB expression in intestinal epithelial cells and restoration of PHB expression in these cells can protect against the deleterious effects of TNF-α and NF-κB on barrier function.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals Biological Cost of Single and Multiple Norfloxacin Resistance Mutations in Escherichia coli Implicated in Urinary Tract Infections

2005 ◽  
Vol 49 (6) ◽  
pp. 2343-2351 ◽  
Author(s):  
Patricia Komp Lindgren ◽  
Linda L. Marcusson ◽  
Dorthe Sandvang ◽  
Niels Frimodt-Møller ◽  
Diarmaid Hughes
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Infection Model ◽  
Resistance Mutations ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Tract Infections ◽  
Subsequent Selection

ABSTRACT Resistance to fluoroquinolones in urinary tract infection (UTIs) caused by Escherichia coli is associated with multiple mutations, typically those that alter DNA gyrase and DNA topoisomerase IV and those that regulate AcrAB-TolC-mediated efflux. We asked whether a fitness cost is associated with the accumulation of these multiple mutations. Mutants of the susceptible E. coli UTI isolate Nu14 were selected through three to five successive steps with norfloxacin. Each selection was performed with the MIC of the selected strain. After each selection the MIC was measured; and the regions of gyrA, gyrB, parC, and parE, previously associated with resistance mutations, and all of marOR and acrR were sequenced. The first selection step yielded mutations in gyrA, gyrB, and marOR. Subsequent selection steps yielded mutations in gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associated mutations were identified in almost all isolates after selection steps 1 and 2 but in less than 50% of isolates after subsequent selection steps. Selected strains were competed in vitro, in urine, and in a mouse UTI infection model against the starting strain, Nu14. First-step mutations were not associated with significant fitness costs. However, the accumulation of three or more resistance-associated mutations was usually associated with a large reduction in biological fitness, both in vitro and in vivo. Interestingly, in some lineages a partial restoration of fitness was associated with the accumulation of additional mutations in late selection steps. We suggest that the relative biological costs of multiple mutations may influence the evolution of E. coli strains that develop resistance to fluoroquinolones.

scholarly journals The Escherichia coli cysG promoter belongs to the ‘extended −10’ class of bacterial promoters

1993 ◽  
Vol 296 (3) ◽  
pp. 851-857 ◽  
Author(s):  
T Belyaeva ◽  
L Griffiths ◽  
S Minchin ◽  
J Cole ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Point Mutations ◽  
Growth Conditions ◽  
Upstream Region ◽  
E Coli ◽  
Run Off ◽  
A Site ◽  
Specific Sequences

The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5′-TGN-3′ motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the ‘extended -10’ class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by cleavage at a site 41 bases downstream from the transcript start point.

scholarly journals Evaluation du potentiel antimicrobien et de la toxicité des extraits de Jatropha multifida Linn, (Euphorbiaceae)

2020 ◽  
Vol 151 ◽  
pp. 15550-15558
Author(s):  
Amégninou Agban ◽  
Yao Hoekou ◽  
Passimna Pissang ◽  
Tchadjobo Tchacondo ◽  
Komlan Batawila
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Candida Albicans ◽  
Aqueous Extract ◽  
E Coli ◽  
Toxicological Studies ◽  
Jatropha Multifida

Objectif : L’objectif de ce travail était d’évaluer in vitro l’activité antimicrobienne des extraits de feuilles et tige de Jatropha multifida sur la croissance de Candida albicans, Escherichia coli et Staphylococcus aureus, puis d’évaluer in vivo la toxicité de cette plante. Méthodologie et résultats : Les méthodes de diffusion en milieu gélosé et de microdilution en milieu liquide ont été utilisées pour évaluer l’effet antimicrobien. Une étude en subaigüe était réalisée afin d’explorer les effets toxiques de l’extrait aqueux des feuilles. Les résultats des tests antimicrobiens montrent une activité des extraits de feuilles et tige de J. multifida sur la croissance des souches utilisées avec des diamètres de zones d’inhibition allant de 8 à 25 mm et des concentrations minimales inhibitrices (CMI) variant de 0,039 mg/mL à 1,25 mg/mL à l’exception des souches de E. coli qui sont résistantes aux extraits de la tige. L’administration en subaigüe de l’extrait aqueux des feuilles de J. multifida à la dose de 600 mg/kg entraîne une perte significative de poids chez les souris. Conclusion et applications des résultats : Les extraits aqueux, éthanolique et hydroéthanolique des feuilles et tige de J. multifida possèdent d’activité antimicrobienne et pourraient être utilisés dans le traitement des Candidoses à C. albicans et des infections à S. aureus. Mais l’essai de toxicité subaigüe montre que l’extrait aqueux de la plante serait toxique. Des études toxicologiques approfondies restent donc nécessaires sur ces extraits afin de mieux élucider leur inocuité. Mots-clés : Jatropha multifida, extraits de feuilles et de tige, activités antifongique et antibactérienne, toxicité. Agban et al., J. Appl. Biosci. 2020 Evaluation du potentiel antimicrobien et de la toxicité des extraits de Jatropha multifida Linn, (Euphorbiaceae) 15551 Evaluation of antimicrobial potential and toxicity of Jatropha multifida Linn, (Euphorbiaceae) extracts ABSTRACT Objective: The objective of this study was to evaluate in vitro the antimicrobial activity of leaves and stem of Jatropha multifida extracts against Candida albicans, Escherichia coli and Staphylococcus aureus, and then to evaluate in vivo the toxicity of this plant. Methodology and Results: The agar well-diffusion and the NCCLS broth microdilution methods were used to assess the antimicrobial effect. A subacute study was carried out to explore the toxic effects of the aqueous extract of the leaves. The results of the antimicrobial tests show an activity of the extracts of leaves and stems of J. multifida on the growth of the strains used with diameters of inhibitory zones ranging from 8 to 25 mm and minimum inhibitory concentrations (MIC) varying from 0.039 mg/mL to 1.25 mg/mL exception E. coli strains which are resistant to extracts from the stem. Subacute administration of the aqueous extract of the leaves of J. multifida at a dose of 600 mg/kg leads to a significant loss of weight in the mice. Conclusion and application of findings : The aqueous, ethanolic and hydroethanolic extracts of the leaves and stem of J. multifida have antimicrobial activity and could be used in the treatment of Candidiasis and bacterial infections due respectively to C. albicans and S. aureus. But the subacute toxicity test shows that the aqueous extract of the plant would be toxic. Extensive toxicological studies therefore remain necessary on these extracts in order to better elucidate their safety. Keywords: Jatropha multifida extracts of leaves and stem, antifungal and antibacterial activities, toxicity

scholarly journals In Vitro and In Vivo Assessment of the Potential of Escherichia coli Phages to Treat Infections and Survive Gastric Conditions

2021 ◽  
Vol 9 (9) ◽  
pp. 1869
Author(s):  
Joanna Kaczorowska ◽  
Eoghan Casey ◽  
Gabriele A. Lugli ◽  
Marco Ventura ◽  
David J. Clarke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Galleria Mellonella ◽  
Enterotoxigenic Escherichia Coli ◽  
E Coli ◽  
Composite Mixture ◽  
Ph Conditions ◽  
The Stability ◽  
Higher Sensitivity

Enterotoxigenic Escherichia coli (ETEC) and Shigella ssp. infections are associated with high rates of mortality, especially in infants in developing countries. Due to increasing levels of global antibiotic resistance exhibited by many pathogenic organisms, alternative strategies to combat such infections are urgently required. In this study, we evaluated the stability of five coliphages (four Myoviridae and one Siphoviridae phage) over a range of pH conditions and in simulated gastric conditions. The Myoviridae phages were stable across the range of pH 2 to 7, while the Siphoviridae phage, JK16, exhibited higher sensitivity to low pH. A composite mixture of these five phages was tested in vivo in a Galleria mellonella model. The obtained data clearly shows potential in treating E. coli infections prophylactically.

Προβιοτικές και τεχνολογικές ιδιότητες οξυγαλακτικών βακτηρίων. In vitro και in vivo επίδραση στο ελικοβακτήριο του πυλωρού

2004 ◽  
Author(s):  
Πέτρος-Αχιλλέας Μαραγκουδάκης
Keyword(s):  
E Coli ◽  
Tnf Α ◽  
Ifn Γ

Σκοπός: Ο σκοπός της παρούσας διατριβής ήταν η εξέταση του προβιοτικού δυναμικού στελεχών οξυγαλακτικών βακτηρίων μέσω μίας σειράς in vitro δοκιμών, και της μετέπειτα εφαρμογής επιλεγμένων στελεχών σε in vivo μοντέλα ποντικών έναντι της λοίμωξης του Η. pylori και της ελκώδους κολίτιδας, αλλά και σε τεχνολογικό επίπεδο στην παρασκευή γιαουρτιού.Υλικά και μέθοδοι: Στη διατριβή αυτή μελετήθηκαν 51 στελέχη Lactobacillus του Εργαστηρίου Γαλακτοκομίας του Γεωπονικού Πανεπιστημίου Αθηνών, απομονωμένα κυρίως από γαλακτοκομικά προϊόντα και κόπρανα προβάτων, καθώς και 22 στελέχη Enterococcus και Streptococcus, απομονωμένα από κόπρανα προβάτων. Τα στελέχη αυτά εξετάστηκαν σε μία σειρά in vitro δοκιμών που αφορούν την επιβίωσή τους σε συνθήκες εξομοίωσης του ανθρώπινου γαστρεντερικού συστήματος, οι οποίες περιελάμβαναν επιβίωση σε χαμηλό pH, επιβίωση παρουσία των πρωτεολυτικών ενζύμων πεψίνη και παγκρεατίνη, και επιβίωση παρουσία χολικών αλάτων.Στην συνέχεια τα στελέχη εξετάστηκαν σε δοκιμές βασικές για την ασφάλεια χρήσης τους στον ανθρώπινο οργανισμό. "Ολα τα στελέχη δοκιμάστηκαν ως προς την αιμολυτική τους δραστικότητα, και τα στελέχη γαλακτοβακίλλων συγκεκριμένα για την ανθεκτικότητά τους έναντι επιλεγμένων αντιβιοτικών.Παράλληλα, εξετάστηκαν in vitro βασικές ιδιότητες που θεωρούνται επιθυμητές για τα προβιοτικά στελέχη, όπως υδρόλυση των χολικών αλάτων, μελέτη της υδροφοβίας και της ικανότητας πρόσδεσης των γαλακτοβακίλλων σε κύτταρα Caco-2, ικανότητά διέγερσης κυττάρων του ανθρώπινου οργανισμού (PBMCs), καθώς και μελέτη της αντιμικροβιακής δράσης έναντι παθογόνων βακτηρίων και της δυνατότητάς αναστολής της πρόσδεσης παθογόνων σε κύτταρα Caco-2.Στην συνέχεια πραγματοποιήθηκαν in vivo μελέτες σε ποντίκια, για την εξακρίβωση του προβιοτικού χαρακτήρα επιλεγμένων στελεχών. Τα στελέχη L. casei Shirota ACA-DC 6002 και L. paracasei subsp. tolerans ACA-DC 4037 χορηγήθηκαν σε ποντίκια επιμολυσμένα με Η. pylori και αξιολογήθηκε η επίδραση των γαλακτοβακίλλων αυτών στον αποικισμό του παθογόνου και στην αξιολόγηση του βαθμού και της δραστικότητας στο στομάχι. Επίσης, τα στελέχη αυτά χρησιμοποιήθηκαν και για την πρόληψη TNBS- κολίτιδας σε ποντίκια.Τέλος, επιλεγμένα στελέχη γαλακτοβακίλλων αξιολογήθηκαν για την τεχνολογική τους εφαρμογή, μελετώντας την ικανότητά οξίνϊσης του γάλακτος και τη χρήση τους ως καλλιέργειες για την παρασκευή γιαούρτηςΑποτελέσματα: Ορισμένα στελέχη βρέθηκαν να έχουν ικανοποιητική επιβίωση σε συνθήκες εξομοίωσης του ανθρώπινου πεπτικού συστήματος. Κανένα στέλεχος δεν βρέθηκε να έχει ισχυρή (β-) αιμολυτική δράση, ενώ η αντοχή των γαλακτοβακίλλων έναντι επιλεγμένων αντιβιοτικών δεν παρουσιάστηκε διαφορετική από την έμφυτη και αναμενόμενη, ανάλογα το είδος των εξεταζόμενων γαλακτοβακίλλων, ενώ αρκετά στελέχη παρουσίασαν ικανότητα υδρόλυσης χολικών αλάτων. Τα στελέχη παρουσίασαν ποικιλόμορφη πρόσδεση στους οργανικούς διαλύτες, αλλά γενικά τα περισσότερα στελέχη δεν παρουσίασαν μεγάλη υδροφοβία, ενώ αρκετά στελέχη γαλακτοβακίλλων παρουσίασαν ικανοποιητική πρόσδεση σε κύτταρα Caco-2 (>5%), με το στέλεχος L. plantarum ACA-DC 146 (25% πρόσδεση) να ξεχωρίζει. Το ίδιο στέλεχος, μαζί με το L. paracasei subsp. tolerans ACA-DC 4037 βρέθηκαν να προκαλούν την έκκριση υψηλών επιπέδων φλεγμονωδών κυτταροκινών (IL-12, TNF-α και IFN-γ) από PBMCs, ενώ αντίθετα το στέλεχος L. casei Shirota ACA-DC 6002 προκάλεσε την έκκριση της αντιφλεγμονώδους κυτταροκίνης IL-10. Παρόλο που κανένα από τα υπερκείμενα των στελεχών δεν παρεμπόδισε τα στελέχη E. coli, S. typhimurium και Η. pylori, ορισμένα στελέχη, όπως τα L. casei Shirota, L. plantarum ACA-DC 146 και L. paracasei subsp. tolerans ACA-DC 4037 προκάλεσαν την αναστολή της πρόσδεσης εντερικών παθογόνων (E. coli και S. typhimurium) σε κύτταρα Caco-2 σε επίπεδα μέχρι και 50%.In vivo, η χορήγηση του L. casei Shirota ACA-DC 6002 σε ποντίκια οδήγησε στην μείωση του βαθμού και της δραστικότητας της γαστρίτιδας αλλά και στον αποικισμό του Η. pylori. Παρόμοια αποτελέσματα, αλλά σε μικρότερη έκταση, παρατηρήθηκαν με την χορήγηση του στελέχους L. paracasei subsp. tolerans ACA-DC 4037. Παράλληλα, το στέλεχος L. casei Shirota ACA-DC 6002 δείχνει να παρέχει προστασία σε ποντίκια κατά την πρόκληση ελκώδους κολίτιδας με την χορήγηση TNBS.Σε τεχνολογικό επίπεδο, κανένα από τα εξεταζόμενα στελέχη δεν παρουσίασε υψηλή ικανότητα οξύνισης στο γάλα και κατά συνέπεια δεν ήταν δυνατόν να χρησιμοποιηθούν ως εναρκτήριες καλλιέργειες για την παρασκευή γιαούρτης. Από την άλλη, καθώς κανένα στέλεχος δεν παρεμποδίζει αλλά και δεν παρεμποδίζεται από τις παραδοσιακές εναρκτήριες καλλιέργειες γιαούρτης, ήταν δυνατή η χρήση τους ως συμπληρωματικές καλλιέργειας για την παρασκευή γιαούρτης. Στην συνέχεια επιλεγμένα στελέχη χρησιμοποιήθηκαν για το σκοπό αυτό και το στέλεχος L. paracasei subsp. tolerans ACA- DC 4037 ξεχώρισε όταν χρησιμοποιήθηκε ως συμπληρωματική καλλιέργεια για την παρασκευή γιαούρτης με εμβόλιο πηγμένου γάλακτος (1% ν/ν) στους 42°C, λόγω της υψηλής μικροβιακής, φυσικοχημικής και οργανοληπτικής ποιότητας του προϊόντος. Συμπεράσματα: Τα στελέχη L. casei Shirota ACA-DC 6002, L. plantarum ACA-DC 146 και L. paracasei subsp. tolerans ACA-DC 4037, επέδειξαν υποσχόμενο προβιοτικό δυναμικό κατά την διάρκεια εκτεταμένων in vitro και in vivo δοκιμών. Η μελέτη αυτή επιβεβαιώνει τις προβιοτικές ιδιότητες του L. casei Shirota και επιδεικνύει την πιθανή εφαρμογής του στη λοίμωξη του Η. pylori και στην ελκώδη κολίτιδα στον άνθρωπο. Επιπλέον, το στέλεχος L paracasei subsp. tolerans ACA-DC 4037 διαθέτει επίσης δυναμικό χρήσης έναντι του Η. pylori αλλά και στην παρασκευή ενός επιτυχημένου προβιοτικού γιαουρτιού. Τέλος, το στέλεχος L. plantarum ACA-DC 146 μπορεί να εξεταστεί in vivo σε μοντέλα αλλεργικών αντιδράσεων, λόγω της υψηλής αντιφλεγμονώδους επίδρασής τους σε ανθρώπινα περιφερειακά μονοπύρηνα (PBMCs.)

7 LACTOBACILLUS REUTERI SUPPRESSES PRO-INFLAMMATORY DRIVEN REACTIVE OXYGEN SPECIES IN VITRO IN HUMAN INTESTINAL EPITHELIAL CELLS AND IN VIVO IN A TNBS COLITIS MOUSE MODEL

2020 ◽  
Vol 26 (Supplement_1) ◽  
pp. S41-S41 ◽  
Author(s):  
Wenly Ruan ◽  
Melinda Engevik ◽  
Alexandra Chang-Graham ◽  
Joseph Hyser ◽  
James Versalovic
Keyword(s):  
Reactive Oxygen Species ◽  
Epithelial Cells ◽  
Lactobacillus Reuteri ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial Cells ◽  
Oxygen Species ◽  
Intestinal Epithelial ◽  
Colitis Model

Abstract Background Reactive oxygen species (ROS) play a role in maintaining intestinal epithelial homeostasis and are normally kept at low levels via antioxidant compounds. Dysregulation of ROS can lead to intestinal inflammation and contribute to inflammatory bowel disease (IBD). Select gut microbes possess the enzymatic machinery to produce antioxidants whereas others can dysregulate levels of ROS. Our model microbe, Lactobacillus reuteri (ATCC PTA 6475), has been demonstrated to reduce intestinal inflammation in mice models. It contains the genes encoding two distinct GshA-like glutamylcysteine ligases. We hypothesize that L. reuteri can secrete γ-glutamylcysteine to suppress ROS, minimize NFκB activation and regulate secretion of e pithelial cytokines. Methods & Results Conditioned media from L. reuteri was analyzed via mass spectrometry to confirm the presence of γ-glutamylcysteine. All cysteine containing products including γ-glutamylcysteine were fluorescently tagged in the conditioned media and then incubated with HT29 cell monolayers as well as human jejunal enteroid (HJE) monolayers. γ-glutamylcysteine was demonstrated to enter intestinal epithelial cells based on microscopy. Next, a Thioltracker assay was used to show increased intracellular glutathione levels by L. reuteri secreted γ-glutamylcysteine. HT29 cells and HJEs were then treated with IL-1β or hydrogen peroxide, and L. reuteri metabolites as well as γ-glutamylcysteine significantly suppressed pro-inflammatory cytokine driven ROS and IL-8 production. L. reuteri secreted products also reduced activity of NFκB as determined by a luciferase reporter assay. γ-glutamylcysteine deficient mutants were generated by targeted mutagenesis of GshA genes, and these mutant L. reuteri strains had a diminished ability to suppress IL-8 production and ROS. To further test the role of L. reuteri secreted γ-glutamylcysteine in vivo, a 2,4,6-Trinitrobenzenesulfonic acid (TNBS)- induced mouse colitis model was used. Adolescent mice were orogavaged with PBS, L. reuteri, L. reuteri GshA2 mutant, or γ-glutamylcysteine for a week after which TNBS was rectally administered to induce colitis. We demonstrate that L. reuteri and γ-glutamylcysteine can suppress histologic inflammation compared to PBS control and L. reuteri GshA2 mutant groups. Conclusions Together these data indicate that L. reuteri secretes γ-glutamylcysteine which can enter the intestinal epithelial cells and modulate epithelial cytokine production. It acts via suppression of ROS and NFκB which then decreases IL-8 production. We are able to demonstrate this in vitro in both HT 29 cells and HJEs. We now also demonstrate this in vivo in a mouse colitis model. These experiments highlight a prominent role for ROS intermediates in microbiome-mammalian cell signaling processes involved in immune responses and intestinal inflammation.

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