Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

The overall goal of the following experiment is to measure effector protein translocation into host cells via the vernia type three secretion system. This is achieved by incubating heela cells with yersinia mutants expressing effector TEM one beta lactamase fusions to allow for translocation of effector fusions into the HELOC cells. As a second step, the infected HELOC cells are loaded with a cell permeant fret reporter compound, which is processed by cellular estro to the charged in fluorescent CCF four and thereby trapped within the cell.

Next cleavage of CCF four by the trans located beta-lactamase results in disruption of fre, which is recorded by laser scanning microscopy in order to analyze donor and acceptor fluorescence intensities. The results show different amounts of trans located effector TEM one beta lactamase fusion based on different kinetics of rise of donor fluorescence. One of the main advantages of this technique over existing methods is that the intercellular processing of the CCF four compound provides a highly specific and sensitive tool for measuring translocation.

The method can help answer key questions in the field of bacterial pathogenesis, such as how translocation is fine tuned during the interaction of bacteria and host cells One day before infection. Inoculate three separate vials containing three milliliters of LB medium and the required antibiotics with one colony from each of the transformed strains of Y enterocolitica prepared as described in the accompanying text protocol. Also prepare plates containing heela cells the day before infection by seeding 15, 000 cells in 200 microliters of culture medium into nine wells of a 96 well plate incubate the cells in a 5%CO2 incubator at 37 degrees Celsius on the day of infection.

Inoculate two milliliters of the overnight culture into 40 milliliters of fresh lb medium without antibiotics. Incubate the bacteria for 90 minutes at 37 degrees Celsius in a shaker at 180 RPMs. This will induce expression of the type three secretion system after 90 minutes, transfer the cultures into a fresh tube and keep the cultures on ice.

From this point forward. Centrifuge the cultures for 10 minutes at 5, 000 G and four degrees Celsius to pellet the bacteria. Next, re suspend the pellet in two to three milliliters of ice cold PBS dilute the bacteria suspension to a concentration of 800 million colony forming units per milliliter, and then place the cultures on ice.

Then determine the corresponding optical density of each culture. Add 3.5 microliters of the bacterial suspension from each strain to different wells of the prepared 96. Well plate and mix by gently pipetting the medium twice, allow the bacteria to sediment onto the cells at a moty of infection of about 100 bacteria per cell.

Perform a time course by starting infections at 30 minute intervals and keeping the cells in a 5%CO2 chamber at 37 degrees Celsius. After each infection, 90 minutes after the first infection, carefully aspirate the medium without removing any of the hela cells. Then add 50 microliters of PBS containing 20 micrograms per milliliter chloramphenicol, and 2.5 millimolar pronic.

This will stop the expression of effectors and reduce the export of CCF four. To prepare microscopy, ensure continuous immersion by spreading immersion medium to the bottom of the wells, using a one milliliter syringe. Avoid trapping any air bubbles in the immersion medium.

Then transfer the 96 well plate onto the microscope stage and locate suitable spots for later analysis in each. Well finish this step within 10 minutes. Then add 70 microliters of CF 4:00 AM loading solution into each.

Well incubate the plate for five minutes at room temperature. Next, aspirate the loading solution and add 100 microliters of PBS containing 20 micrograms per milliliter chloramphenicol, and 2.5 millimolars pronic. Then rapidly start microscopy.

Place a 20 fold magnification high numerical aperture immersion objective into place and open the detector pinhole to 2.5 airy units and transfer the 96 well plate into the confocal laser scanning stage. Next, choose high sensitivity gallium arsenide phosphate detectors with simultaneously active donor and accepter channels. Eight bit depth threefold frame averaging and excite with 10%of a 405 nanometer diode laser.

To ensure correct quantification, set the detector gain of both channels to the same value and avoid any saturated pixels. Next, activate the transmitted light and recheck the representative fields of view in every, well adjust and resave the positions as needed using a calibrated automatic stage. Finally, set the time lapse to two minutes, the duration to two hours, and begin acquiring images.

Following image acquisition, import the dataset into software that allows arithmetic calculations of pixel matrices. Subtract the background in both channels using the same offset by first clicking on menu, and then go to image processing and select baseline subtraction. Then select each channel and enter the baseline value.

Correct the bleed through of the donor channel, channel one into the acceptor channel channel two with the predetermined correction factor, creating a new channel channel two prime to this end. Click on the menu and then select image processing followed by channel Arithmetics and enter the absolute value of channel two subtracted by point 33 times. Channel one afterwards, delete channel two and ensure that the bleed through corrected Accepter channel remains as the new channel two.

Then create a new channel with the operation shown here to determine the relative fret coefficient. Next, go back into the channel arithmetics menu and enter channel two divided by channel one plus channel two for proper visualization of the fret channel. In an eight bit image, create a fourth channel channel four, which is equal to 255 times channel three.

Change the channel color by going to the image properties menu, select channel four, then mapped color, followed by color table file, and select jet. Next, apply a three by three median filter to channels three and four. That will reduce the noise in the images by entering the image processing menu and selecting smoothing.

Followed by median filter, select both channels and apply a filter size three by three by one for quantification of cellular signals. Detect the cells in channel four by selecting surpass view and clicking on the icon. Add new surfaces.

Define the detection algorithm parameters in the surface window and activate the two check boxes. Segment only a region of interest and process entire image. Finally, next, define the region of interest by editing its dimensions.

Select channel four as the source channel and set the threshold by going to the thresholding menu and selecting on background subtraction. Set diameter to 10 micrometers the minimum intensity threshold manually to zero and the maximum threshold to maximum intensity. Filter the structures by area and set the minimum value to 187 square micrometers.

Then finish the surface detection. Finally, extract the mean values for donor fluorescence intensity in channel one and relative fret coefficient in channel three, along with their standard deviations in the cellular entity. To show the capability of this method to quantitatively analyze effector translocation into target cells, two yersinia strains with different translocation kinetics were studied using both donor fluorescence and relative fret coefficients.

The wild-type infected cells show an increase of fluorescence intensity over time. As expected, the maximum slope of fluorescence intensity is positively correlated with the length of infection, indicating that different concentrations of translated beta lactamase can be distinguished in accordance with previous studies. Cells infected with the yersinia YOPE deletion mutant exhibit more rapid increase of fluorescence intensity compared to the wild type strain.

Interestingly, expanding infection to 90 minutes did not further accelerate the rise of donor fluorescence compared to 60 minutes of infection While attempting this procedure, it's important to keep in mind that the processing of the CCR four compound starts immediately after its addition. Therefore, it is important to start image acquisition in time.