Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Mutant library screening and transcriptomic studies of intracellular pathogens can help answer key questions about genomic and regulatory adaptations of pathogens of interest that are used to survive inside the host. The main advantages of these techniques are that they are large scale investigation techniques, and that they provide an objective insight into adaptation to intracellular life. The implications of these techniques extend toward the therapy of Mycobacterium abscessus that associate to this disease by giving insight into potential targets for vaccine therapy.

Begin by culturing amoeba Acanthamoeba castellani, or Ac, in 30 milliliters of Peptone Yeast Glucose medium in 75 square centimeter tissue culture flasks at room temperature. After one hour use a 25 milliliter pipette to remove the supernatant, and wash the cells three times with 10 milliliters of Ac buffer without carbon or nitrogen per wash. After the last wash, detach the amoeba from the bottom of the flask with a single vigorous shake, and adjust the cells in a Falcon tube, to a five time 10 to the five amoebae per mil concentration in fresh Ac buffer.

Seed five times 10 to the four amoebae per well in a 96-well plate. Place the plate at 32 degrees celsius for one hour without shaking, to allow the floating amoeba tropho-zoites to adhere to the wells. Meanwhile, warm the bacteria on ice, and add five microliters of the thawed bacteria to each well at the end of the incubation.

After 1.5 hours at 32 degrees celsius, discard the supernatant by inverting the plate onto a stack of sterile gauze in a sterilized metal tray under a fume hood. Wash each well three times with 200 microliters of fresh Ac medium per well. After the last wash, incubate the co-culture with 20 microliters of fresh medium supplemented with amikacin per well, to eliminate any remaining extracellular mycobacteria for two hours.

Followed by three washes in fresh Ac medium, as just demonstrated. Then, add 200 microliters of medium, supplemented with fresh amikacin to each well, followed by the addition of five times 10 to the seven heat inactivated Escherichia coli bacteria. After 48 hours, lyse the cells with 10 microliters of 10%sodium dodecyl sulfate per well, for 30 minutes at 32 degrees celsius, and spread five microliters of the lysate on Columbia Agar plates containing 5%sheep blood.

Further dilute the lysates by adding 25 microliters of sterile water on each drop. When all of the lysates have been plated, seal the plates with plastic paraffin film, and incubate the cultures for three to four days at 37 degrees celsius. When colonies appear on the plates, photograph the cultures and identify the mutants impaired for intracellular growth by the deposits with the lowest number of bacterial colonies.

For intracellular mycobacteria RNA extraction, first grow M.abscessus in 7H9 medium supplemented with glycerol and glucose to the mid-exponential phase in 50 milliliter tubes at 120 RPM. At the end of the culture, wash the bacteria two times with 10 milliliters of Ac buffer per wash, and measure the O.D.600 to estimate the bacteria concentration. Next, add 8.5 times 10 to the eight mycobacteria to 8.5 times 10 to the six amoebae in 10 milliliters of fresh Ac medium for a one hour incubation at 30 degrees celsius and 80 RPM.

At the end of the incubation, harvests the cells by centrifugation, and wash the pellet three times in 10 milliliters of fresh Ac buffer per wash under the same centrifuge conditions. Then re-suspend the cells in 10 milliliters of fresh Ac buffer supplemented with amikacin to eliminate any extracellular bacteria, and incubate the cells for four hours at 30 degrees celsius and 80 RPM, followed by two washes in fresh Ac buffer. After the last wash, re-suspend the infected amoebae in four milliliters of cold solution three, supplemented with 280 microliters of beta mercaptoethanol to disrupt the amoebae on ice for 10 minutes.

At the end of the incubation, centrifuge the cell lysate to pellet the released intracellular bacteria, and re-suspend the bacterial pellet in one milliliter of cold solution three. It's important to remove all the supernatant to avoid contamination with amoeba RNA or proteases that might have made the samples. Harvest the released bacteria with a second centrifugation, and re-suspend the pellet in one milliliter of extraction buffer.

Then incubate for 24 hours at negative 80 degrees celsius. Transfer the mycobacterial solution into two milliliter screw tubes containing zirconium beads up to the 500 microliter gradation mark, and store the tubes for another 24 hours at negative 80 degrees celsius to facilitate RNA syn activation and cell dissolution. On the day of the analysis, thaw the samples on ice, and lyse the cells with two rounds of homogenization at 6000 RPM for 25 seconds, followed by one round homogenization at 6000 RPM for 20 seconds.

In between each round of homogenization, cool the samples on ice for one minute to avoid RNA degradation. After the third round of homogenization, cool the samples on ice for 20 minutes, and add 200 microliters of chloroform diluted in isoamyl alcohol to each tube. Vortex for one minute, then centrifuge the samples, and add 0.8 milliliters of cold isopropanol to the aqueous phase for a two to three hour incubation at negative 20 degrees celsius.

At the end of the incubation, centrifuge the samples again, and wash the pellets in 500 microliters of cold 70%ethanol without mixing. Then immediately remove the ethanol by centrifugation. Aspirate the supernatant for drying the pellets in a centrifugal concentrator, and re-suspend the pellets in 100 microliters of DNA/RNA-free water.

Mycobacterial MRNA extraction as demonstrated, allows the evaluation of induced and repressed gene families by grouping the genes according to their roles in responding to an environment limited in it's source of nutrients and minerals, or to a hypoxic or a acidic environment. In the resistance to oxidative and nitrosative stress, or in the expression of virulence factors in response to environmental amoeba. While attempting those procedures, it's important to remember to perform the culture and RNA isolation of the controlled and infected samples at the same time to avoid batch effects.

Following this procedure, other methods like dual RNA sequencing can be preformed to answer additional questions about host pathogen interactions. After it's development, this technique paved the way for researchers in the field of microbiology towards mycobacteria virulence in the amoeba host model. Don't forget that working with amoebae might be extremely hazardous, as the amoebae might transform into cysts if you do not follow the steps described in the procedure.