«(Über die Bedeutung der bakteriellen Genomplastizität für die Adaptation und Evolution asymptomatischer Bakteriurie (ABU) Escherichia coli ...»
4.3. 2D protein gel electrophoresis 4.3.1. Isolation of intracellular proteins and rehydration Bacteria were grown at 37 °C without agitation in 400 ml of pooled human urine until OD600 = 0.2 and harvested by centrifugation (10,000 X g, 10 min, 4°C). Pellets were washed three times with decreasing volumes of TE buffer (1 x 50 ml TE, 1 x 25 ml TE, 1 x 10 ml TE) and were then resuspended in 700 µl TE buffer. Cell lysis was done using the FastPrep (MP™) device twice for 30 s at 5.5 x g with an incubation on ice for 1 min in between, followed by a two-step centrifugation for 10 min at 13,000 rpm at 4 °C. The supernatant was each time transferred into a fresh Eppendorf tube. The isolated intracellular proteins were frozen at -80 °C or used immediately for 2D gel electrophoresis.
300 µg of proteins were passively rehydrated on 17-cm, pH 4 to 7 immobilized pH gradient strips (Amersham Biosciences) in 330 µl of rehydration buffer in a total volume of 350 µl.
When needed, the vacuum centrifuge was used to increased the protein concentration of the samples.
4.3.2. Isolation of outer membrane proteins and rehydration Bacteria were grown in 800 ml of pooled human urine at 37 °C in 2 l flasks, without agitation until OD600 = 0.2 and harvested by centrifugation (10,000 x g, 10 min, 4 °C). Pellets were washed and resuspended in 10 ml of 10 mM HEPES, pH 7.0, containing 250 U of benzoesane ultrapure nuclease (Sigma) and 400 µl of 25x complete EDTA-free protease inhibitor stock solution (Roche, # 11873580001). Following four passages through the chilled French
68 Methods – Phenotypic Characterisation
pressure cell at 15,000 psm, lysates were centrifuged (6000 x g, 10 min, 4 °C) to remove unbroken cells and cell debris, the supernatants were diluted to 60 ml with 0.1 M carbonate buffer, pH 11, and incubated with stirring at 4 °C for 1 h. Carbonate-insoluble membranes were collected by ultracentrifugation (120,000 x g for 1 h at 4 °C). The resulting outer membrane pellet was rinsed with 10 mM HEPES, pH 7.0 and solubilized in 800 µl rehydration solution I at room temperature for 30-60 min. Soluble OMPs were quantified using the Roti® Nanoquant solution (Roth) and either immediately used for 2D gel electrophoresis or stored at -80°C.
4.3.3. Determination of protein concentrations
Protein concentrations were determined using the Roti-Nanoquant solution (Roth) following the manufacturer’s instructions. 200 μl protein samples were mixed with 800 μl 1 x assay solution, and absorbance was measured at 590 nm and 450 nm. Protein concentrations were then calculated using the following formula (based on a bovine serum albumin (BSA)
4.3.4. Protein rehydration 17-cm, pH 4 to 7 immobilized pH gradient strips (Amersham Biosciences) were passively rehydrated overnight with 300 µg of outer membrane proteins in 315 µl rehydration solution-I and 35µl rehydration solution-II.
During this step proteins were separated according to their isoelectric points (pI). The pI is the pH at which a protein will not migrate in an electric field and is determined by the charged groups in the protein. Proteins can carry positive, negative or no charge depending on their local pH, and for every protein there is a specific pH at which its net charge is zero; this is their isoelectric point. pI's generally fall in the range of 3 to 12, with most being between 4 to
7. When a protein is placed in a medium with a pH gradient and subjected to an electric field, it will initially move towards the electrode with the opposite charge. During migration through the pH gradient, the protein will pick up or lose protons. As it migrates, the net charge and the mobility will decrease and the protein will slow down. Eventually the protein will arrive at the point in the pH gradient which is equal to its pI. Here, it will be uncharged and hence stop migration. If the protein should happen to diffuse to a region outside its pI it will pick up a charge and hence move back to the position where its charge is neutral. In this way proteins are focused into sharp bands.
Rehydrated IPG strips were placed with the plus mark to the anode in a Protean IEF Multiphor cell. On both ends of the strips connecting papers soaked with water were placed.
The IPG strips were placed in cover oil. Isoelectric focussing was performed with a 500-V linear ramp for 1 h, a 1000-V linear ramp for 1 h, and a 3500-V rapid ramp for 22 h. During the run, the connecting papers were exchanged 3 to 4 times in order to improve protein separation.
An equilibration step is necessary to saturate the IPG strip with SDS buffer system before the second dimension separation. Equilibration is a multi-step process to ensure that the proteins are suitable for SDS-PAGE analysis. This ensures that the strip has the correct pH suitable for
70 Methods – Phenotypic Characterisation
subsequent analysis and preserves the fully denatured state of the protein. Glycerol ensures that the proteins are adequately transferred from the first to the second dimension and reduces electroendoosmosis in the buffer upon application of the electrical field. Electroendoosmosis is the movement of buffer within the IPG (immobilized pH gradient) strip and is due to the fixed charge associated with the ampholytes present in the strip. Electroendoosmosis can interfere with protein transfer from the IPG strip to the second dimension. A second equilibration alkylates any thiol groups in the protein preventing their reoxidation.
Reoxidation can result in protein smears within the gel. It also alkylates any remaining DTT to prevent smears of proteins and other artefacts which may occur during protein staining within the gel.
Prior to separation in a second-dimension, the immobilized pH gradient strips were subsequently equilibrated in 50 ml equilibration buffer-I and 50 ml equilibration buffer-II each time for 25 min under gentle agitation.
Preparation of equilibration buffers:
Basic buffer composition (100 ml):
4.3.7. Second dimension - separation based on size Proteins were subsequently separated on the basis of their molecular mass using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). SDS binds to most proteins in a constant fashion (about 1.4 grams of SDS per gram of protein) and also masks any charge of the protein by forming large anionic complexes. SDS also disrupts any hydrogen bonds, blocks many hydrophobic interactions and partially unfolds the protein molecules minimising differences based on secondary or tertiary structure. Proteins move through the gel towards the anode during electrophoresis. The rate at which they move is inversely proportional to their molecular mass.
Equilibrated IPG stripes with proteins resolved in the first dimension were applied to a second dimension gel and covered by 1 % agarose in 2 x electrophoresis buffer avoiding air bubbles.
The lower electrophoresis unit was filled in with 1 x electrophoresis buffer and the upper unit with 2 x electrophoresis buffer. Second dimension separation was performed overnight at 6 Watt (400 V, 300 mA).
4.3.8. Proteins staining After the second dimension, the gels were fixed by incubation for 1 to 2 h in fixing solution followed by washing in dH2O (2 x 10 min). Afterwards, the gels were incubated for 24 h in a Coomassie staining solution. On the next day, gels were washed 3 x 1 h in dH2O, wrapped in a plastic sleeve and scanned.
4.3.9. Analysis of 2-D Gels with the Delta-2D® Software (Decodon) The image analysis was performed with the Delta-2D® Software (http://www.decodon.com), which is based on dual-channel image system. Before, gels were scanned and saved as a gray scale TIFF image. An overlay of two wrapped 2-D gels was visualized by either green, red or yellow false colour code representing proteins expressed in the wild type, the mutant, or in both strains, respectively. In addition, a statistical analysis was performed based on spot intensities from at least 3 gel replicates.
4.3.10. Protein identification by MALDI-TOF-MS In order to identify proteins separated during the 2-D electrophoresis, gels were washed in dH2O for 1 h followed by spot excision. Gel pieces with a diameter not bigger than 2 mm
from different parts of the spot were subjected to MALDI-TOF-MS (Matrix-Assisted Laser Desorption-Ionization – Time Of Flight Mass Spectrometer) at the Institute for Microbiology and Molecular Biology of the University of Greifswald. Proteins were digested with trypsin, mixed with a matrix solution and allowed to co-crystallise on a target plate. Laser-pulsed voltage was applied to the target plate to accelerate the ionised sample towards a time-offlight mass analyser. This peptide mass fingerprint was used to search databases to identify the protein.
4.4. Analysis of lipopolysaccharides (LPS)
4.4.1. Isolation of LPS For analysis of the LPS composition, cells from an agar plate or pellets of liquid cultures were used. After weight measurement, the cells were resuspended in an adequate amount of water so that the concentration was 1 mg cells per 30 μl suspension. 30 μl samples (i.e. 1 mg cells) were mixed with 10 μl 4 x SDS-sample buffer and incubated at 100 °C for 10 min. After brief cooling, 20 μl 1 x SDS-sample buffer supplemented with 100 μg proteinase K were added to the samples, which then were incubated at 60 °C for 1 h for removal of proteins. 30 μl of the LPS preparations were used for electrophoresis.
4.4.2. Electrophoresis and staining with silver nitrate After electrophoresis, the polyacrylamide gels were stained with AgNO3. All used devices were carefully washed with double-destilled water, and gloves were worn throughout the experiment. The gels were fixed over night in 100 ml 1 × fixation solution. The next day, the solution was replaced by 100 ml 1 × periodate solution and the gels were incubated for 5 min in for oxidation. Subsequently, the gels were washed three times for 30 min with dH2O, and then incubated for 10 min in silver nitrate solution. After three more washes for 10 min with H2O, gels were developed in developing solution preheated to 60 °C. As soon as the intensity of the appearing bands was satisfying, the reaction was stopped by incubation in 50 M EDTA solution for 10 min.
4.5. Phenotypic assays 4.5.1. Detection of type 1 fimbrial expression Overnight cultures of the strains to be tested and of a positive (E. coli strain Nissle 1917) and of a negative (E. coli strain AAEC189) control were grown. The mannose-dependent yeast agglutination assay was carried out by mixing 10 μl of the different bacterial overnight cultures with 10 μl yeast cell suspension (1 mg/ml Saccharomyces cerevisiae cells diluted in 0.9 % (w/v) NaCl, with or without 2 % (w/v) mannose) on microscope slides (75:25:1 mm).
The slides were kept on ice until the aggregation of yeast and bacterial cells was observed in absence of mannose.
4.5.2. Detection of F1C and P fimbrial expression
Overnight cultures of the strains to be tested and of a positive (E. coli strain Nissle 1917) and of a negative (E. coli strain AAEC189) control were grown. For the immunoagglutination assay, a polyclonal α-F1C or P fimbriae rabbit antibody was used (provided by Dr. S. Kahn, Würzburg), that was diluted 1:1000 in 1 × PBS. The immunoagglutination assay was carried out by mixing 10 μl of the bacterial overnight culture with 10 μl of the α-F1C or P fimbriae antibody solution on microscope slides (75:25:1 mm) and incubation on ice until the aggregation of the bacterial cells was clearly observed.
4.5.3. Detection of secreted α-hemolysin To test hemolytic activity, cells from E. coli colonies were spread onto sheep blood agar plates (Oxoid) with a toothpick and incubated over night at 37 °C. Lysis of the blood cells by α-hemolysin was detected by formation of clear halos around the colonies after incubation.
4.5.4. Detection of biofilm forming abilities Biofilm formation was assessed in a microtiter plate assay modified after O’Toole and Kolter (1998). Bacteria were grown over night in pooled human urine or M63 medium at 37 °C with agitation.
In M63 defined media On the next morning, 158 μl fresh minimal medium were distributed in a microtiter plate (8 wells per strain) and inoculated with 1.6 μl of the overnight cultures. 96 well plates were incubated statically at 37 °C for 24 h. Subsequently, were carefully washed twice with 1 x PBS and dried for 1 h at 80 °C. Biofilm was stained with 0.1 % crystal violet for 10 min.
Staining solution was discarded and plates were rinsed twice with 1 x PBS. To quantitate the biofilm formation, wells were destained with 180 μl destaining solution (80 % ethanol / 20 % acetone) for 10 min. Remaining biofilm was dissolved by pipetting the solution up and down several times, before diluting the solution 10 times in dH2O and measuring absorbance at 570 nm.
In pooled, sterile human urine
To access the biofilm forming ability in human urine, 24-well flat bottom polystyrene plates (# 83.1836 SARSTEDT, Sarstedt Tissue) were used. In each well 1.5 ml sterile urine was inoculated with 15 µl bacterial overnight culture. The plates were incubated statically at 37 °C for 24 h. Washing and staining procedure as described above.
4.6. Continous culture of E. coli in microfermenters Long term in vitro bacterial culture was established in order to propagate bacteria over 2000
generations (Fig. 8; Fig. 9). Bacteria were grown under four different culture conditions: