Team:Tuebingen/einweiterer

Nissle

Introduction

Escherichia coli Nissle 1917 (EcN) is probably the most intensively investigated bacterial strain today[1]. Despite the fact that the EcN strain is widely used as a probiotic, a lot of questions remain. That is why we decided to dive deep into the characterization of EcN. Our goal was to find out more about EcN itself and to provide crucial information in order to include EcN as platform organism for other iGEM Teams and researchers.

Growth Curves

The first step in the characterization of a bacterium are growth curves on different media and under different conditions. We chose to perform half of our experiments under aerobic and the other half under anaerobic conditions. Under aerobic conditions, as a control we let EcN grow in LB medium at 37°C and pH7. We changed the temperature to 25°C, 8°C, 4°C and 0°C, showing that EcN growth is inhibited at 8°C, the temperature often used for cold shock.

Fig. 1: EcN growth in LB medium at pH7. All cultures were inoculated with an overnight culture and grown for 120 minutes at 37°C. Next, temperature was changed to 0°C, 4°C, 8°C and 25°C, while one control was kept at 37°C. Shown are mean values of triplicates.

Moreover, we evaluated the growth in the pH range from pH6 to pH1, since in our application EcN will have to pass the acidity of the stomach. Here, at pH4 we discovered an interesting recovery of EcN growth after three hours.

Fig. 2: EcN growth curve in LB at 37°C under different pH levels. Until 120min all cultures inoculated from one overnight culture grew at pH7. Then, media was changed to the respective pH and cultures were grown for 3 hours. pH5 and pH6 grew as usual, pH1,2,3 did not grow at all, while pH4 started to grow after 3 hours. Thus, media was changed again after 3 hours to pH7. pH6 and pH5 grew as usual, pH1,2,3 did not recover. Cultures grown in pH4 recovered and grew as usual. Shown are values of one culture for each pH.

Fig. 3: EcN growth in LB at 37°C with pH changed from pH7 to pH4 after two hours of growth. After 150 minutes within pH4, EcN started to grow comparable to pH7 cultures. Shown are the three cultures inoculated from the same overnight culture of EcN.

To look into inflammatory stress, we subjected EcN to up to 100µM hydrogen peroxide [2] and showed that the growth is not substantially influenced by it.

Fig. 4: EcN growth in different concentrations of Hydrogen Peroxide, infused after two hours of growth at 37°C, pH7 in LB medium. 100µM Hydrogen Peroxide with slightly inhibited growth. Shown are the means of triplicates for each concentration.

Lastly, we performed dryfreezing and recovered EcN afterwards under control conditions [3][4][5], showing that dryfreezing within skim milk protects the bacteria from substantial death.

Fig. 5: EcN growth of cultures previously subjected to a dryfreeze protocol. Shown are the mean values of triplicates of cultures grown at 37°C, pH7 in LB grown for recovery.

Anaerobic conditions were used to characterize EcN growth under the circumstances provided in the human intestines. Thus, as a control we compared EcN growth in LB medium versus mGAM medium under anaerobic conditions, showing that mGAM, a medium designed for anaerobic application, is in fact a more suitable medium. Then, we subjected the bacteria to metformin treatment, since it is one of the most common treatments of diabetes and is known to accumulate to 720 µM to 7,2 mM within the intestines due to low bioavailability. [6][7]. We showed that EcN growth is not substantially influenced by 1,3 mM of metformin into mGAM medium. Moreover, we tested for EcN’s resistance against cholic acid, since it is commonly secreted into the ileum. Here, we added 0.25 mM of cholic acid to mGAM medium, considering that the concentration of free bile salts may rise to 0.25 to 1 mM of total bile salts in the Ileum, with cholic acid only being one component [8][9]. Our results suggest that EcN growth is not greatly influenced by cholic acid.

Fig. 6: EcN growth under four anaerobic conditions compared to EcN aerobic growth. Shown are the mean values of four samples inoculated with the same overnight culture. Experiments were conducted at 37°C and pH7. LB aerobic growth as a control. Other samples are LB anaerobic growth, mGAM anaerobic growth, as well as mGAM medium supplemented with either 1.3 mM Metformin or 0.25mM Cholic Acid. Samples for RNA sequencing were taken after 270 minutes for all anaerobic conditions, since the increase of pressure within the flasks indicated a change of metabolism of EcN to fermentation processes. Aerobic LB growth samples were taken at OD600=1.0.

Finally, we tested EcN interaction with other bacteria by adding bacterial supernatant to our medium. The supernatant was sterilized and provided by Dr. Lisa Maier. We chose Bacteroides thetaiotaomicron, Prevotella copri and Ruminococcus gnavus since they are commonly found within the human microbiome [10]. Bifidobacterium adolescentis was chosen due to its probiotic nature and Clostridium difficile supernatant was used, since it is often found in the microbiome of people with chronic inflammation [10][11]. The results suggest that EcN grows under all additions, however was initially inhibited in its growth by the addition of Bacteroides spp., thus we used these samples for RNA-seq.

Fig. 7: EcN growth in mGAM medium under anaerobic conditions at 37°C, pH 7 supplemented with either 50% supernatant of different species or 50% of water as a control for depleted nutrients. All cultures were inoculated with the same overnight culture of EcN. Bacteroides spp. with strongest change in growth within the first 200 min of the experiment. Shown are mean values of four samples each. Bacteroides samples were taken for RNA sequencing after 225 minutes.

RNASeq

The transcriptome describes the set of whole RNA molecules in a population of cells and is subject to continuous changes. Understanding the complete transcriptome, the expressed genes, post-transcriptional modifications, single-nucleotide polymorphisms (SNPs) and additional properties of interest is imperative towards understanding genetic cause, disease and possible treatment strategies. The inherent complexity of the transcriptome requires precise and scaling analysis techniques. RNA sequencing (RNA-seq), also known as whole transcriptome shotgun sequencing (WTSS), is most commonly used for this purpose, today. It uses next-generation sequencing (NGS) to detect and quantify RNA in biological samples. The generated RNA-Seq read data is then analyzed according to a sample RNA-Seq work-flow as shown below.

We set ourselves the goal of understanding the transcriptomic changes that E.coli Nissle undergoes under various stress conditions to gain a deeper insight into its responses. Understanding the stress responses of E.coli Nissle could lead to the development of more stress robust strains, which not only our project would benefit from, but also scientists working on probiotic drugs in general.

We divided our experimental design into two parts. We not only wanted to investigate the effect of environmental stress on E.coli Nissle under aerobic conditions, but were also specifically interested in anaerobic conditions. Hence, our experimental design looks as follows:

The respective temperature, pH values and doses were determined by growth curves, shown above. To get the most out of RNA-Seq applied to stress factors, it is important to find the cutoff values, where E.coli Nissle is put under stress the most, but still survives.

Sequencing and library preparation was conducted on two sites. All aerobic samples were prepared and sequenced at the NGS Competence Center Tübingen (NCCT), whereas all anaerobic samples were prepared and sequenced at the European Molecular Biology Laboratory (EMBL) in Heidelberg. For more details please visit our Notebook and Attributions.

Data Analysis

Quality Control Differential Expression Analysis Pathway Analysis

Metabolic Model

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References

1. Ulrich Sonnenborn, Escherichia coli strain Nissle 1917—from bench to bedside and back: history of a special Escherichia coli strain with probiotic properties, FEMS Microbiology Letters, Volume 363, Issue 19, October 2016, fnw212,
2. Sana Ben Othman and Tomio Yabe. Use of Hydrogen Peroxide and Peroxl Radicals to induce Oxidative Stress in Neuronal Cells. Reviews in Agricultural Science, 3:40-45, 2015. Doi: 10.7831/ras.3.40
3. P. Capela, T.K.C. Hay, N.P. Shah. Effect of cryoprotectants, prebiotics and microencapsulation on survival of probiotic organisms in yoghurt and freeze-dried yoghurt. Food Research International. Volume 39, Issue 2, 2006, Pages 203-211, ISSN 0963-9969, https://doi.org/10.1016/j.foodres.2005.07.007.
4. Govender M, Choonara YE, Kumar P, du Toit LC, van Vuuren S, Pillay V. A review of the advancements in probiotic delivery: Conventional vs. non-conventional formulations for intestinal flora supplementation. AAPS PharmSciTech. 2014;15(1):29–43. doi:10.1208/s12249-013-0027-1
5. https://opsdiagnostics.com/applications/lyophilization/ecoli_lyophilization_stability.html
6. Bailey, C. J, Wilcock, C, Scarpello, J. H. B. Metformin and the intestine. Diabetologia. 2008. Volume 51, Issue 8, Pages 1552, ISSN 1432-0428, Doi: 10.1007/s00125-008-1053-5,
7. High Accumulation of Metformin in Colonic Tissue of Subjects With Diabetes or the Metabolic Syndrome. Paleari, LauraBurhenne, JürgenFoersch, SebastianParodi, AndreaGnant, MichaelScherer, DominiqueUlrich, Cornelia M.Stabuc, BorutPuntoni, MatteoCoccia, GianniPetrera, MarilenaHaefeli, Walter-EmilDeCensi, Andrea et al. Gastroenterology, Volume 154, Issue 5, 1543 - 1545
8. Martinez-Augustin O, Sanchez de Medina F. Intestinal bile acid physiology and pathophysiology. World J Gastroenterol. 2008;14(37):5630–5640. doi:10.3748/wjg.14.5630
9. Postprandial concentrations of free and conjugated bile acids down the length of the normal human small intestine. T. C. NORTHFIELD AND I. McCOLL. Gut, 1973, 14, 513-518
10. Belizário JE, Napolitano M. Human microbiomes and their roles in dysbiosis, common diseases, and novel therapeutic approaches. Front Microbiol. 2015;6:1050. Published 2015 Oct 6. doi:10.3389/fmicb.2015.01050
11. Lloyd-Price J, Abu-Ali G, Huttenhower C. The healthy human microbiome. Genome Med. 2016;8(1):51. Published 2016 Apr 27. doi:10.1186/s13073-016-0307-y

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