Functional studies of Rickettsia genes are crucial for understanding their role in pathogenesis, so we need dependable methods of altering the genes in a controlled fashion. Our protocol uses electroporation to introduce exogenous DNA into obligate intracellular bacteria of the genus Rickettsia, thus allowing us to study the effects of the introduced DNA. Demonstrating the procedure along with me today will be Lisa Price, a researcher from Dr.Munderloh and Dr.Kurtti's laboratory.
To begin, prepare a 90 to 100%Rickettsia parkeri-infected ISE6 cell culture as described in the text. Then, to determine the infection rate by Giemsa staining, dilute the cell suspension to a ratio of 1:5 with complete medium and deposit 100 microliters of the cell suspension onto a glass microscope slide by centrifugation at 113 G for five minutes. After letting the slide air-dry, fix it an absolute methanol for five minutes at room temperature.
Then, incubate the slide and Giemsa stain for 30 minutes at 37 degrees Celsius. Next, rinse the slide in water and once dry, view it under a light microscope with oil objectives. Determine the percentage of infected cells by counting the infected and non-infected cells.
To prepare the cell-free Rickettsia parkeri, pour about 0.2 milliliters of sterile 60 to 90 silicon carbide grit into two two-milliliter sterile microcentrifuge tubes and set the tubes aside. From a previously inoculated Rickettsia parkeri-infected flask containing five milliliters of medium, remove two milliliters of the medium, taking care not to disturb the cell layer. Then, resuspend the cells with the remaining three milliliters of the medium.
Divide this suspension equally between the two prepared silicone carbide grit tubes and vortex the tube at high speed for 30 seconds. Then, place them on ice. Next, prepare a sterile five-milliliter luer lock syringe by extending the plunger and insert the plunger end into a polystyrene base used for holding 15-milliliter conical tubes.
Using a barrier two-milliliter Pasteur pipette operated with a rubber bulb, carefully remove the supernatant of the vortex cells without aspirating any grit. Then, insert the pipette tip into the syringe hub opening and eject the contents into the syringe with gentle pressure. Next, filter the Rickettsia parkeri culture into sterile 1.5-milliliter tubes through a sterile two-micrometer pore size filter fitted on the syringe hub and centrifuge the tubes at 13, 600 G for five minutes at four degrees Celsius.
After removing the supernatant, wash the cell pellet two times with 1.2 milliliters of 300-millimolar ice-cold sucrose solution. Centrifuge the sample and remove the supernatant between washes. At the end of centrifugation, resuspend the pooled cell pellet with 100 to 150 microliters of ice-cold sucrose solution for two to three transformation samples, respectively.
Then, divide the sample into 50-microliter aliquots in pre-chilled, sterile, 1.5-microliter microcentrifuge tubes. For transformation at three micrograms of endotoxin-free pRAM18sSFA plasmid DNA to each tube containing 50 microliters of Rickettsia parkeri on ice and stir gently with a pipette tip. Transfer the mixture into a pre-chilled sterile electroporation cuvette with a 0.1-centimeter gap size.
Gently tap the cuvette to distribute the mixture evenly and incubate on ice for 10 to 30 minutes. Meanwhile, remove the medium from a fully-confluent previously cultured ISE6 cell culture flask and resuspend the cell layer by gently rinsing the cells with 1.5 milliliters of fresh medium containing sodium bicarbonate and HEPES buffer. After generating a homogenous cell suspension, transfer the whole volume into a single sterile two-milliliter microcentrifuge tube.
Next, electroporate the Rickettsia parkeri and plasmid DNA mixture using an electroporator. After electroporation, use a sterile extended fine-tip pipette to transfer a small amount of the ISE6 cell suspension into the cuvette and gently pipette up and down to recover the electroporated Rickettsia parkeri. Then, transfer the contents of the cuvette to the two-milliliter tube containing the remainder of the ISE6 cell suspension.
Next, centrifuge the sample at room temperature, first at 700 G for two minutes to pull down the ISE6 cells, and then at 13, 600 G for one minute to pull down the Rickettsia. Incubate the tube at 34 degrees Celsius for 15 minutes to one hour. After incubation, transfer the resuspended contents of one transformation into one 25 square-centimeter cell culture flask containing 3.5 milliliters of fresh medium with sodium bicarbonate and HEPES buffer.
Rock the flask to spread the mixture evenly and incubate at 34 degrees Celsius. After 16 to 24 hours, add 10 microliters each of spectinomycin and streptomycin into the incubated flask. Rock the flask to mix the antibiotics evenly into the medium before returning the flask to the incubator.
Successful propagation of wild-type Rickettsia parkeri and ISE6 cells was evident by Giemsa staining. The nuclei of the ISE6 cells stained dark purple with Giemsa in the intracellular as well as extracellular Rickettsi are visible. Transformation of Rickettsia parkeri expressing the red fluorescence protein in ISE6 cells was confirmed by confocal microscopy after seven days of incubation.
A substantial increase in the infection rate was observed after 10 days. Keep everything sterile during the whole procedure. Pay attention to detail and be patient, as some species of Rickettsia take a long time to transform, especially slow growing endosymbionts.
This protocol has enabled the study of multiple aspects of Rickettsia biology and their interactions with their arthropod vectors and mammalian hosts. For example, fluorescent protein-expressing Rickettsi have been used to track motility and symbiont tick cell interactions.
