Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

The overall goal of this experimental procedure is to examine the impact of specific host processes on the ability of bacterial pathogens to translocate effector proteins. This method can help answer key questions in the field of cellular microbiology. Specifically how the host is involved in either blocking or promoting the delivery of bacterial virulence effectors into the host cell.

The main advantage of this technique is that it combines two established techniques. Namely, siRNA gene silencing and reporter essay to measure bacterial protein translocation. To investigate the interplay between pathogens and the eukaryotic cell.

This method provides insight into coxiella pathogensis and can also be applied to investigate the host pathogen interactions of other bacteria that depend on efficient protein translocation. For example, chlamydia, salmonella, and attaching interfacing E.coli. After culturing C.burnetii expressing CBU0077, fuse the betalactamase according to the text protocol in a 25 centimeter squared culture flask with a vented cap.

Inoculate 10 milliliters of pre-warmed ACCM2 containing three micrograms per milliliter of chloramphenical with 20 microliters of the bacteria. Grow the culture at 37 degrees celsius with five percent carbon dioxide and 2.5 percent oxygen for seven days. To reverse transfect hela cells with siRNA and after preparing the siRNA according to the text protocol, place DMEM plus 10%FCS, 05 percent trypsin EDTA, and PBS in a 37 degrees celsius water bath for 30 minutes to warm prior to use.

For each experimental siRNA transfection, combine 0.4 microliters of transfection reagent and 63.6 microliters of reduced serum media in an individual microfuge tube. Vortex all microfuge tubes and allow the components to complex for five minutes at room temperature. Add 16 microliters of each experimental siRNA to the corresponding microfuge tubes containing the transfection complex.

Then vortex and incubate at room temperature for 20 minutes. During the 20 minutes incubation, add 20 microliters of the transfection reagent, reduced serum medium and siRNA mix into the appropriate wells of a sterile black, flat, clear bottomed 96 well tray. Also during the 20 minute incubation, use 10 milliliters of pre-warmed PBS to wash a monolayer of confluent or subconfluent hela cells in a 75 centimeter squared flask.

Add one milliliter of 05 percent trypsin EDTA solution to the flask and incubate at 37 degrees celsius and five percent carbon dioxide for three to five minutes to detach the cells. Collect the cells in nine milliliters of pre-warmed DMEM with 10%FCS. Using a hemocytometer to count the cells.

Then with DMEM 10%FCS, bring the cells to a final density of 4.9 times 10 to the four cells per milliliter. Immediately following the 20 minute incubation, pipette 80 microliters of the cells into each well of the 96 well plate containing the siRNA transfection mixture which will bring the cell density to 3.92 times 10 to the third cells per well. This is the most critical step in the procedure.

It is very important that the hela cells are added to the siRNA transfection mixture immediately following the 20 minute incubation. Otherwise transfection efficiency can be compromised. Ensure the blank wells contain only 80 microliters of DMEM plus 10%FCS.

Incubate the plate for 24 hours. Then, with a multi-channel adapter attached to a vacuum source, and a pipette, replace the medium with 100 microliters of fresh DMEM plus 10%FCS. Incubate the cells for an additional 48 hours.

After seven days of growth in ACCM2, centrifuge the 10 milliliters of C.burnetii P BlaM CBU0077 culture at 15, 000 times g and room temperature for 15 minutes. After removing the supernatant, use 10 milliliters of pre-warmed DMEM FCS to re-suspend the pellet. Then use water to prepare one in ten and one in one hundred dilutions in a final volume of 100 microliters of the culture and set up QPCR according to the text protocol.

After calculating the total genomes per milliliter in the C.burnetii P BlaM CBU0077 culture, use pre-warmed DMEM FCS to adjust the volume of the culture to two milliliters for a concentration of 1.88 times 10 to the eight bacteria per milliliter. Next, remove the medium from the blank and reverse transfected cells. Then add 50 microliters of the diluted bacteria to the appropriate wells and add 50 microliters of DMEM FCS to the blank and uninfected wells.

After preparing the solutions for the BlaM substrate according to the text protocol, prepare a master mix by combining 1.8 microliters of solution A and 16.2 microliters of solution B.Mix the tube well by vortexing. Then add 237 microliters of solution C and 45 microliters of 0.1 molar probenecid. Vortex the tube again.

Add 10 microliters of the Six X loading solution directly into all the blank and reverse transfected wells without removing the existing medium. Incubate the plate in the dark at room temperature for two hours. To determine the level of translocation, on a microplate reader, create a fluorescence intensity protocol using Endpoint as the reading mode.

Select 96 well microplate, then under optic settings, select two multichromatics and for the first number preset, input 410-10 nanometers excitation and 520-10 emission. For the second number preset, input 410-10 nanometers excitation and 450-10 emission. Then ensure Well multichromatics is selected.

Next, select Orbital Averaging with a diameter of three millimeters. Under Optic, select Bottom optic. Then under Speed and Precision, select Precise.

This corresponds to eight regions per well. Adjust the plate layout by selecting the appropriate wells as blanks and sample wells. Then, select Start measurement.

Remove the lid and place the tray into the plate reader. Ensure the gain value is adjusted to avoid reaching maximum fluorescence values by performing a gain adjustment on one of the infected wells that has been reverse transfected with OTP siRNA. Select Start measurement to measure all the appropriate wells using Bottom fluorescence at an excitation of 410 nanometers and emission of both 450 nanometers and 520 nanometers for blue and green fluorescence respectively.

Analyze the results according to the text protocol. This figure demonstrates an example of the cycle threshold value expected from both standards and samples following QPCR to determine the total number of genomes per milliliter in the seven day C.burnetii culture. Based on this data, there were 2.31 times 10 to the eight genomes per milliliter in the 10 milliliter re-suspended bacterial culture.

The result to silencing either Rab5A or Rab7A on effector translocation from three independent experiments after incubation of the reversed transfected cells with BlaM substrate is shown here. A significant decrease in the level of BlaM CBU0077 translocation occurred during silencing of both Rab5A and Rab7A compared to OTP control. As demonstrated in this graph, although treatment with either Rab5A or Rab7A results in a reduction in cell viability compared to OTP control, this reduction is not as severe as treatment with PLK1, a gene known to cause cell death when silenced.

After watching this video, you should have a good understanding of how to silence specific host targets using siRNA, and subsequently investigate the impact this has on bacterial effector protein translocation using FRET based reporter assay. This protocol has been optimized for both silencing of Rab GTPases in coxieller infection of hela cells. To utilize this method to target other host proteins, transfection conditions require optimization to ensure efficient gene silencing.

Similarly to use this method with other bacterial pathogens, infection condition require optimization.