Біотехнологія створення системи адресної доставки лікарських засобів на основі бактерій Escherichia coli Nissle 1917 для терапії раку

Магістерська дисертація містить 110 сторінок, 34 таблиці, 21 рисунок, перелік посилань з 76 найменувань. Мета даного дослідження – створити систему адресної доставки лікарських засобів для терапії раку на основі пробіотичної бактерії – продуцента БМН Escherichia coli Nissle 1917. Об’єкт дослідження – магнітні властивості бактерій-продуцентів БМН Escherichia coli Nissle 1917 та їх практичне застосування для адресної доставки протиракових лікарських засобів. Предмет дослідження – процес біомінералізації біогенних магнітних наночастинок (БМН) у Escherichia coli Nissle 1917 та можливість посилення магнітних властивостей E. coli Nissle 1917, як продуцента БМН, за допомогою використання хелату заліза та магнітного поля, застосування цього штаму для створення магніточутливої системи адресної доставки лікарських засобів для терапії раку. Методи дослідження: біоінформаційний аналіз, атомно-силова та магнітно-силова мікроскопія, аналіз із застосуванням системи двох постійних магнітів, аналіз та візуалізація даних у програмі «Gwyddion». У роботі було проведено біоінформаційний аналіз протеомів бактерій, що накопичуються у пухлинах, на предмет здатності до утворення БМН. Визначено продуцентів БМН серед пробіотичних бактерій і досліджено накопичення БМН у штаму E.coli Nissle 1917 методами атомно-силової та магнітно-силової мікроскопії. Досліджено вплив хелату заліза та магнітного поля на процеси біомінералізації та росту у E.coli Nissle 1917. Синтезовано ліпосоми та прикріплено їх до бактеріальних клітин у якості мікроконтейнерів для лікарських засобів.The master's thesis contains 110 pages, 34 tables, 21 figures, 76 references. The purpose of this study is to create the drug delivery system for cancer therapy based on the probiotic bacteria Escherichia coli Nissle 1917. The object of the study is magnetic properties of BMN-producing bacteria Escherichia coli Nissle 1917 and its practical application for delivery of anticancer drugs to tumor tissues. The subject of the study is the process of biomineralization of biogenic magnetic nanoparticles (BMN) in Escherichia coli Nissle 1917 and the possibility of increasing the magnetic properties of E. coli Nissle 1917, as a producer of BMN, by using iron chelate and magnetic field, and the use of this strain to create a magnetosensitive drug delivery system for cancer treatment. Research methods: bioinformatic analysis, atomic force and magnetic force microscopy, analysis using a system of two permanent magnets, analysis and data visualization with the program «Gwyddion».The bioinformatic analysis of the ability to form BMN of the bacteria that accumulate in the tumors was carried out in the work. BMN producers have been identified among probiotic bacteria and the BMN-producing strain E. coli Nissle 1917 has been investigated by atomic force and magnetic force microscopy. Influence of iron chelate and magnetic field on biomineralization and growth processes in E. coli Nissle 1917 also was investigated. Liposomes were synthesized and attached to bacterial cells as microcontainers for drugs

Extracellular vesicles and soluble factors secreted by Escherichia coli Nissle 1917 and ECOR63 protect against enteropathogenic E. coli-induced intestinal epithelial barrier dysfunction

Background: Enteric pathogens have developed mechanisms to disrupt tight junctions and increase gut permeability. Many studies have analysed the ability of live probiotics to protect intestinal epithelial cells against tight junction damage caused by bacterial pathogens. Escherichia coli Nissle 1917 (EcN) is among the probiotics that positively modulates the intestinal epithelial barrier by regulating expression and distribution of tight junction proteins. We previously reported that regulation of ZO-1, claudin-14 and claudin-2 is mediated by EcN secreted factors, either free-released or associated with outer membrane vesicles (OMVs). Factors secreted by commensal ECOR63 elicited comparable effects in intact epithelial T-84 and Caco-2 cell monolayers. Results:Here we analyse the ability of OMVs and soluble secreted factors to protect epithelial barrier function in polarized T-84 and Caco-2 cells infected with enteropathogenic Escherichia coli (EPEC). Transepithelial electrical resistance, paracellular permeability, mRNA levels and subcellular distribution of tight junction proteins were monitored in the absence or presence of EcN and ECOR63 extracellular fractions. EPEC downregulated expression of ZO-1 ZO-2, occludin and claudin-14 and altered the subcellular localization of ZO-1, occludin and F-actin cytoskeleton. OMVs and soluble factors secreted by EcN and ECOR63 counteracted EPEC- altered transepithelial resistance and paracellular permeability, preserved occludin and claudin-14 mRNA levels, retained ZO-1 and occludin at tight junctions in the cell boundaries and ameliorated F-actin disorganization. Redistribution of ZO-1 was not accompanied by changes at mRNA level. Conclusions: This study provides new insights on the role of microbiota secreted factors on the modulation of intestinal tight junctions, expanding their barrier- protective effects against pathogen-induced disruption

Substantial extracellular metabolic differences found between phylogenetically closely related probiotic and pathogenic strains of Escherichia coli

Since its first isolation a century ago, the gut inhabitant Escherichia coli strain Nissle 1917 has been shown to have probiotic activities; however, it is yet not fully elucidated which differential factors play key roles in its beneficial interactions with the host. To date, no metabolomics studies have been reported investigating the potential role of small molecules in functional strain differentiation of Nissle from its genetically close neighbors. Here, we present results of liquid chromatography coupled to high-resolution mass spectrometry characterization of extracellular metabolomes of E. coli strains as a proxy of their bioactivity potential. We found that phylogroup B2 strains exported a more diverse arsenal of metabolites than strains of other phylogroups. Zooming into the phylogroup B2 metabolome identified consistent substantial differences between metabolic output of E. coli Nissle and other strains, particularly in metabolites associated to the Argimine biosynthesis pathway. Nissle was found to release higher levels of Ornithine and Citrulline whilst depleting greater amounts of Arginine from the medium. Moreover, a novel Nissle-specific metabolite not reported before in bacteria, 5-(Carbamoylamino)-2-hydroxypentanoic acid (Citrulline/Arginic Acid related) was observed. Finally, Nissle, CFT073 and NCTC12241/ATCC25922 shared the excretion of N5-Acetylornithine, whereas other strains released N2-Acetylornithine or no N-Acetylornithine at all. Thus, we found substantial metabolic differences in phylogenetically very similar E. coli strains, an observation which suggests that it is justified to further investigate roles of small molecules as potential modulators of the gut environment by probiotic, commensal, and pathogenic strains, including E. coli Nissle 1917

The emerging therapy with probiotics in the management of inflammatory bowel disease: current status

Inflammatory Bowel Disease (IBD) comprises Ulcerative Colitis (UC) and Crohn’s Disease (CD) with unknown aetiology. Most of the drugs used to treat IBD as standard treatment produce adverse effects during long term therapy. Evidence has suggested a role of intestinal microbiota in IBD. The use of probiotics and prebiotics is the natural approach to treat IBD. The objective of this article was to review the studies on probiotics that cover the therapeutic status in Inflammatory Bowel Disease. Appraisal of published articles from peer reviewed journals, search from PubMed and Wiley Blackwell website for English language publications using defined key words according to disease type. Studies have shown that probiotic agents play an important role in IBD and these are VSL#3, Bifido-ferminted milk, Escherichia coli Nissle 1917, Saccharomyces boulardi and “BIO-THREE for inducing remission in patients with active UC, for preventing relapses in inactive UC patients and also in UC patients with ileo-anal pouch anastomosis. Lactobacillllus rhamnosus GG and Lactobacillllus johnsonii LA1 can prevent endoscopic recurrences in patients with inactive CD. Probiotic intervention study designs in IBD patients searched were RCT vs Placebo / RCT vs standard treatment . Studies - with uncontrolled design, - with prebiotics intervention and with helminths were also searched. There is a promising role of probiotics and prebiotics in chronic mucosal inflammation that occurs in Inflammatory Bowel Disease. Sufficient evidence to support the role of probiotics in CD are not available. Well designed RCT studies based on intention -to- treat analyses are required

Escherichia coli Nissle 1917 in ulcerative colitis treatment: Systematic review and meta-analysis

Background & Aims: Escherichia coli Nissle 1917 (EcN) has been recommended as a therapeutic tool for ulcerative colitis (UC) treatment. However, to date, no meta-analysis has been performed on this topic. Methods. We performed a literature search on PubMed, MEDLINE, Science Direct and EMBASE. We evaluated success rates for induction of remission, relapse rates and side effects, expressed as Intention-To-Treat. Odd ratios (OR), pooled OR and 95% confidence intervals (CI) were calculated, based on the Mantel-Haenszel method. Heterogeneity was assessed by using the χ2 and I2 statistics and, if present, a random-effects model was adopted. Results. We selected six eligible trials, with 719 patients, 390 assigned to the study group and 329 to the control group. EcN induced remission in 61.6% of cases, while in the control group (mesalazine) the remission was achieved in 69.5% of cases, with a mean difference of 7.9%. The pooled OR was 0.92 (95% CI 0.15-9.66, p=0.93). A single study showed a better performance of EcN than the placebo. A relapse of the disease occurred in 36.8% in the EcN group and in 36.1% in the control group (mesalazine), with a mean difference of 0.8%, OR=1.07, with a 95% CI of 0.70-1.64 (p=0.74). Side effects were comparable (OR=1.44, 95% CI 0.80-2.59, p=0.22). Conclusions. EcN is equivalent to mesalazine in preventing disease relapse, thus confirming current guideline recommendations. EcN seems to be as effective as controls in inducing remission and therefore, its use cannot be recommended as in one study the comparison was performed against placebo. Further studies may be helpful for this subject

Regulation of biofilm formation in Salmonella typhimurium and Escherichia coli Nissle 1917

Bacteria have the ability to grow in cell communities designated biofilms. This mode of growth is widespread and offers numerous advantages to the bacteria in terms of survival, persistence and propagation. Bacteria have developed different ways of building up a biofilm. Complex regulatory mechanisms control this sophisticated mode of growth in response to environmental conditions. This thesis focuses on the regulation of biofilm formation by the food-borne pathogen Salmonella enterica serovar Typhimurium and the probiotic strain Escherichia coli Nissle 1917. Commonly, species of the family of Enterobacteriaceae produce the biofilm extracellular matrix components cellulose and curli fimbriae at low temperature. The expression of cellulose and curli is activated by the transcriptional regulator CsgD. In this work, we demonstrated an altered pattern of biofilm regulation in E. coli Nissle 1917 (Paper I). Biogenesis of curli fimbriae was activated by CsgD at low temperature, while cellulose production at 28°C and 37°C did not require CsgD nor the di-guanylate cyclase AdrA. Cellulose production was, however, still dependent on the second messenger c-di-GMP. This regulatory pattern of cellulose and curli fimbriae production has been conserved in E. coli Nissle 1917 clonal isolates for more than 80 years implying biological significance. Production of cellulose mediated adhesion of E. coli Nissle 1917 to the gastrointestinal epithelial cell line HT-29 and to the mouse epithelium in vivo, thus possibly playing a role in colonization of the gut. A characteristic of biofilm formation is cell heterogeneity. In S. typhimurium, expression of the master regulator CsgD was bistable during biofilm development (Paper II). Bistability led to task distribution, whereby the subpopulation of cells, which expressed high amounts of CsgD, was associated with microcolony formation and the production of cellulose. CsgD expression is tightly regulated and responds to a variety of environmental conditions such as nutrient starvation and oxygen tension. Several global regulators contribute directly or indirectly to CsgD regulation. In this work, we identified novel factors involved in the complex CsgD regulation. Two lytic transglycosylases, MltE and MltC redundantly activated CsgD and rdar morphotype expression (Manuscript III). The absence of these two lytic transglycosylases could be partially compensated by the second messenger c-di-GMP. The chaperone Hfq and two Hfq dependent sRNAs, ArcZ and RyeB, also activated rdar morphotype expression by controlling the expression of CsgD (Manuscript IV). We demonstrated that ArcZ is a key regulator of biofilm formation. In addition, ArcZ played a role in the transition between sessility and motility and was involved in the timing of type 1 versus curli fimbriae surface attachment

Efficacy of Probiotic Escherichia coli Nissle 1917 in Patients with Irritable Bowel Syndrome: a Double Blind Placebo-controlled Randomized Trial

AIM: to evaluate potential improvement effect for probiotic E. coliNissle 1917 in the management of refractory IBS in an Iranian population. METHODS: a double blind placebo controlled approach has been used in the current clinical trial. 139 confirmed IBS patients were included into the study, and were given probiotic E.coli Nissle 1917 for 6 weeks. 11 items Birmingham IBS Symptom Questionnairehas been used for evaluation of changes in the symptoms every 2 weeks. CONCLUSION: probiotic therapy with E.coli Nissle 1917 was not able to induce significant improvement in the symptoms of patients with non-categorized IBS. Nevertheless, when IBS patients were recategorized to subgroups according to their main symptoms, evaluation of the efficacy of the probiotic on some individual items in the symptom list reached the significance level. Prospective clinical trials are recommended to confirm our findings. RESULTS: sixty eight subjects (49) were males. Mean±SD age of the participants was 38±13.3 years. 49(35.3) of the patients were diarrhea-predominant. The total scores showed no significant difference between the intervention vs. control group(-6.7±6.8 vs. -6.7±6.5, respectively; p=0.95); neither did any of the questionnaire items any significant alterations in the two groups. After stratification of patients based on their IBS type, diarrhea-predominant patients showed a positive response to the probiotic improving their sleep (p=0.05 and 0.03 at weeks 2 and 6, respectively). Patients with constipation-predominant IBS showed no response to the probiotic; while patients with diarrhea-constipation mixed IBS showed unfavorable response to the probiotic in the need for strain to pass a motion compared to the placebo (p=0.03 and 0.02 at weeks 4 and 6, respectively)