IDENTIFICATION OF E.COLI NISSLE 1917 PROTEINS BY USING 2-D GEL ELECTROPHORESIS UNDER THE INFLUENCE OF COCOS NUCIFERA SAP AND WINE

Received on 20 January, 2014; received in revised form, 06 March, 2014; accepted, 05 April, 2014; published 01 July, 2014

IDENTIFICATION OF E.COLI NISSLE 1917 PROTEINS BY USING 2-D GEL ELECTROPHORESIS UNDER THE INFLUENCE OF COCOS NUCIFERA SAP AND WINE

K. Chandrasekhar, S. Sreevani and J. Pramoda Kumari*

Dept. of Microbiology, S.V.University, Tirupati, Andhra Pradesh, India

E. coli Nissle, protein, 2-D electrophoresis, up and down regulation

INTRODUCTION: Cocos nucifera sap is commonly called as cocoti sap. Sap is a juice collected from the cocoti palm plants. After fermentation, cocoti sap is converted into cocoti wine. It is a sweetish, milky white, effervescent alcoholic beverage with evolved CO₂ bubbles as mildly alcoholic beverage similar to beer. This alcohol gives sour smell and sulphur like odor may also be present. It acts as toxicants. Many scientists have analyzed the chemical and microbial properties of cocoti sap ^{1, 2, 3}. Escherichia coli Nissle 1917 is one of the probiotic present in gut flora of our body and served as a model organism for countless biochemical, biological, and biotechnological studies, since the completion of the E. coli genome-sequencing project.

E. coli Nissle 1917 has demonstrated to reduce intestinal inflammatory bowel disease (IBD) ^{4, 5}.

Proteomics is regarded as a powerful approach as far as biochemical research is concerned because it directly studies the key functional components of biochemical systems, namely proteins ⁶.

Proteomics provides a powerful tool for analysing the various molecular mechanisms employed by plants, animals, insects, and microorganisms.

In most cases protein samples are compared in healthy versus infected (or) treated⁷.Proteomics that aims at the determination of proteins constituents and their isoforms in a given sample⁸.

2-D clean-up kit facilitates the preparation of low conductivity samples suitable for isoelectric focusing (IEF) and 2-D gel electrophoresis. Additionally, the kit concentrates proteins from samples that are too dilute, allowing for higher protein loads that can improve spot detection.

2-D gel electrophoresis is derived from 1-D SDS-PAGE, and expands the number of proteins resolved on an electrophoresis gel by separating the proteins based on their native charge and molecular mass ^{9, 10}. 2-D electrophoresis technique captured detailed information about protein expression, complex formation, isoforms, and post translational modifications (PMF) ¹¹. 2-D electrophoresis is regarded as a powerful technique, because it can be used to separate and resolved complex protein mixtures into thousands of individual compounds ¹². This technique has been widely used and successfully applied in a variety of biological systems.

Although a comparison of protein expression profiles from regular 2-D gel electrophoresis can be carried out with the assistance of various software programs ¹³. It typically requires some computerized justification of 2-D gel images so that two images can be superimposed and composed ¹⁴. After separation, proteins in 2-D gels were visualized by staining, with colloidal Coomassie blue stain. In this manner, proteins can be quantified and spot patterns in multiple gels can be matched and compared. Statistical analysis can be performed on group of spots in gel, and variations, differences, and similarities can be evaluated ¹⁵.

The proteins are finally visualized by radiolabeling or detected with a variety of staining methods such as silver, coomassie blue or fluorescent stains. Adapted image capture devices are used to generate digital images that can be analyzed with 2-D gel softwaresuch as ImageMaster 2-D platinum.

Down regulation is the process by which a cell decreases the quantity of a cellular component, such as RNA or protein, in response to an external variable. An increase of a cellular component is called up-regulation. Cells can increase and decrease their sensitivity to cells by regulating the number of their receptors. Receptors are proteins and are manufactured by the cell itself, so a cell can increase and decrease the amount of receptors within its plasma membrane. If a cell increases the number of receptors then we call it up-regulation and if the cell decreases the number of receptors we call it down regulation.

Down regulation is when a cell decreases its sensitivity to a hormone by decreasing the amount of available receptors.

Up regulation is used by cells to increase their sensitivity to a specific hormone. Up regulation occurs when a cell produces more receptors, the cell decreases its degradation of receptors or by activating already present receptors. Cells typically up regulate when the concentration of a hormone is very little.

If there is a lower concentration of a hormone in the blood stream and the cell increases the number of receptors, it increases the chances of interacting with that hormone. Hormones themselves can also cause cells to up regulate. Genomic techniques, including microarrays allow genomic profiling of cells under stress ¹⁶.

T-test is used to test differences in means between two groups. The t- test can be used even if sample sizes are very small, as long as the variables within each group are normally distributed and the variation of scores within the two groups is equal. With the t-test, the test statistic used to generate p-values has a student’s distribution with n-1 degrees of freedom ¹⁷.

MATERIALS AND METHODS:

Sample collection: Fresh cocoti palm sap samples were collected from coconut palm trees in a sterilized reagent bottles in Tirupati rural, A.P, INDIA. Samples were transported immediately to the laboratory for analysis, some of the sap was separated, allowed for fermentation to make palm wine (fermented palm sap is known as palm wine) at room temperature 25-28^oC. These samples were filtered by using vacuum pump nitrocellulose membrane filters to separate the microorganisms present in the sample (Filter pore size is 0.02μ).

Culture collection: Eschericia coli Nissle 1917was obtained from culture collection center of Ardeypharm GmbH, Herdecke, Germany and the culture was maintained on Luria-Bertani (LB) Medium. LB medium is the most commonly used for E. coli culture.

Protein extraction and Quantification: The MIC of the two samples, i.e., cocoti sap and wine was determined by micro dilution method. The proteins of three samples (control, sap and wine treated) of E. coli Nissle 1917 were extracted by using Trizol protein extraction kit method through sonication and centrifugation process. Quantify the protein concentration by using BCA kit method and all samples were adjusted to 400 mg.

2-D gel electrophoresis: Collected protein sample is purified because 2-D gel electrophoresis is very sensitive to salts and detergents. Sample contains some amount of salts and detergents. In this process protein samples were separated by two independent properties i.e. isoelectric point and based on molecular weight. 4 – 7 pH gradient strips were used; the strip length is 24cms.

2-DE protein sample was loaded into the strip holder, placed the strip and covered the strip by using covering gel, allowed for IEF with the help of electrodes, according to the optimal conditions. Remove the strip from the strip holder after the IEF step was completed. Then the sample treated for rehydration with the help of equilibrium buffer Iodoacetamide (IAA). Put the strip into the casting plate, set the instrument and loaded with running buffer allowed for second dimension.

After completion of the second dimension, the gel was separated from the plate and allowed for staining. Colloidal coomassie brilliant blue stain was used.

Image scanning and analysis: All destained gels were digitized using gel scanner (Typhon Variable Mode imager), and allowed for gel analysis by using Image master 2-D platinum 6.0 software programme. It quantifies the protein spots, and showed the variation between the control and treated gel samples, the spot size indicates, up-regulation and down-regulation of the protein.

The expressed protein spots were separated by using spot cutter and these spots can be analyzed by MS- for protein identification.

RESULTS AND DISCUSSION:

FIG. 1: REPRESENTATIVE 2-D GEL ELECTROPHORESIS IMAGE OF CONTROL E.COLI NISSLE 1917, COVERING PIRANGE OF 4 TO 7. THE LOCATIONS OF THE SPOTS ARE MARKED ON THE GEL

FIG- 2: REPRESENTATIVE 2-D GEL ELECTROPHORESIS IMAGE OF COCOTI SAP TREATED E.COLI NISSLE 1917, COVERINGPI RANGE OF 4 TO 7. THE LOCATIONS OF THE SPOTS ARE MARKED ON THE GEL.

FIG- 3: REPRESENTATIVE 2-D GEL ELECTROPHORESIS IMAGE OF COCOTI WINE TREATED E.COLI NISSLE1917 COVERINGPIRANGE OF 4 TO 7. THE LOCATIONS OF THE SPOTS ARE MARKED ON THE GEL.

In the present study, we analyzed control, cocoti sap and wine treated protein samples by using 2-D gel electrophoresis. In gel images, it shows the variations in all the three samples i.e. control, cocoti sap and cocoti wine treated samples. Based on the protein regulation, we were noticed 15 differentially expressed spots in cocoti sap treated sample when compared to control sample (Fig. 1 & 2). In the case of wine treated sample, totally 16 differently expressed spots were noticed. Specifically, we were identified one newly expressed protein spot i.e. 3n₁ (Fig. 3) spot which was not present in both control and cocoti sap treated samples. The protein spots sizes were increased in up-regulation and spots size were decreased in down regulation, when compared to the control. Based on this, the protein spots were separated from the gel. Over these ten samples shows ten up-regulation and five protein spots shows down-regulation. The 3 - D gel images of all the differentially expressed proteins were generated and presented in the following images (Fig. 4 & 5).

FIG. 4: UP- REGULATION OF PROTEIN EXPRESSION 3-D IMAGES

FIG. 5: DOWN REGULATION OF PROTEIN 3-D IMAGES

TABLE 1: UP REGULATION VALUES OF PROTEINS UNDER EXPOSER OF SAP AND WINE TREATMENT

Up regulation    Total numbers-10                      Mean- 18.38038

Variance- 2.19323                     Standard deviation- 1.48096

TABLE 2: DOWN REGULATION VALUES OF PROTEINS UNDER EXPOSER OF SAP AND WINE TREATMENT

Down regulation           Total numbers- 5                      Mean-26.5019

Variance-5.8786                       S.D- 2.4246

t- Calculated value: 6.8687       t- Critical value:  2.4469

P- value: 0.00047

RESULT: There is no significant difference between up and down regulation in the samples. Sonull hypothesis is rejected.

DISCUSSION:  Transferred the data square root to Sin^-1√p. After performing Sin^-1√p transformation independent samples t- test was conducted. There was no significant difference in up-regulation (Mean-18.38038, Standard deviation- 1.48096) (Table 1) and down regulation (Mean- 26.5019, Standard deviation- 2.4246) (Table 2). t- Calculated value is 6.8687, and t- critical value is 2.4469.  p – Value is 0.00047.

Where t- calculated value is higher than t- critical value, so null-hypothesis is rejected.

CONCLUSION: In the present study, the proteins were separated from cocoti sap and wine treated gel samples. Both cocoti sap and wine treated samples shows up and down regulations and also a new protein was evaluated under wine stress.

The results indicating that the sap and wine can cause stress on probiotic E.coli Nissle 1917. Further, the spots are undergoing for MALDI-TOF-MS analysis and the differentially expressed proteins are to be noticed. This will be useful for drug designing in pharmaceutical industries for the treatment against sap and wine caused by the intestinal disorders.

REFERENCES:

1. Borse B.B, Rao L.J.M, Ramalakshmi K, Raghavan B: Chemical composition of volatiles from coconut sap (neera) and effect of processing. Food chemistry 2007; 101(3): 877-880.
2. Olawale, Adetunji Kola, AkintobiAkinbiyiOlubiyi, David OluwoleMoses: Evaluation of Microbial Quality and Alcoholic Improvement of Natural and Fermented RaphiaPalmwine (“Ogoro”).New York Science Journal 2010;3.
3. Kalaiyarasi K, Sangeetha K, &Rajarajan S: A Compaeative study on the microbial flora of the fresh sap from cut inflorescence and fermented sap (toddy) of BorrassusFlabelliferLinn (Palmyrah tree) and of Cocos nucifera Linn (coconut tree) to identify the microbial fermenters. International Journal of Research in Pure and Applied Microbiology. 2013; 3(3): 43-47.
4. Mazmanian S.K, Round J.L, and Kasper D.L: A microbial symbiosis factor prevents intestinal inflammatory disease. Nature2008; 453: 620-625.
5. Kane MD S.V, Horst MD S,  Sandborn  MD W.J, Becker MD B, et al :Natalizumab for Moderate to Severe Crohn’s Disease in Clinical Practice: The Mayo Clinic Rochester Experience. Inflammatory Bowel Diseases. 2012; 18(12).
6. Freed J.K, Smith J.R, Li P, & Greene A.S: Isolation of Signal transduction complex using biotin and cross linking methodologies. Proteomics 2007; 7(14): 2371 – 2374.
7. Kim S: Neuroproteomics: expression profiling of the brain’s proteomes in health and disease. Neurochemical Research 2004; 29:1317-1331.
8. Pin E, Fredolini C, Petricoin 3rd EF: The role of proteomics in prostate cancer research: biomarker discovery and validation.  Clinical Biochemistry 2013; 46:524–38.
9. Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, and Weiss W: The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis2000;21: 1037–1053.
10. Hanash S. M: Biomedical applications of two-dimensional electrophoresis using immobilized pH gradients: current status. Electrophoresis 2000;21: 1202–1209.
11. Rabilloud T, Chevallet M, Luche S, Lelong C: Two-dimensional gel electrophoresis in proteomics: past, present and future.  Journal of Proteomics 2010; 73(11):2064–2077.
12. Görg A, Weiss W, & Dunn M.J: Current two dimensional electrophoresis technology for proteomics. Proteomics 2004; 4(12): 3665–3685.
13. Morris J.S: Evaluating the performance of new approaches to spot quantification and differential expression in 2-dimensional gel electrophoresis studies. Journal of Proteome Research 2010; 9: 595–604.
14. Righetti P.G, Castagna A, Antonucci F, Piubelli C, Cecconi D, Campostrini N, Antonioli P, Astner H: Critical survey of quantitative proteomicsin two dimensional electrophoretic approaches. Journal of.Chromatography.A 2004; 1051:3-17.
15. SarkaBeranova-Giorgianni: Proteome analysis by two-dimensional gel-electrophoresis and mass spectrometry: strengths and limitations. Trends in Analytical Chemistry 2003; 22 :(5), 273-281.
16. Leakey A.D.B, Ainsworth E.A, Bernard S.M, Cody Markelz R.J, Ort D.R, Placella S.A, Rogers A, Smith M.D, Sudderth E.A, Weston D. J, Wullschleger S.D, and  Yuan S: Gene Expression profiling: Opening the black box of plant ecosystem responses to global change. Global Change Biology 2009; 15: 1201-1213.
17. Polit, D. F., & Beck, C. T: Nursing research: Generating and assessing evidence for nursing practice (8th Ed.). Philadelphia: Wolters Kluwer Health 2008.

How to cite this article:

Chandrasekhar K., Sreevani S. and Kumari JP:Identification of E.coli nissle 1917 proteins by using 2-d gel electrophoresis under the influence of Cocos nucifera sap and wine. Int J Pharm Sci Res 2014; 5(7): 2763-71.doi: 10.13040/IJPSR.0975-8232.5 (7).2763-61.

All © 2014 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License