Preparation of Mycobacterium tuberculosis Culture Filtrate to Understand TB Pathogenesis

The secretum of Mtb culture filtrate would give insight into the virulence factors or other proteins secreted by the pathogen for therapeutic interventions. Here, we're trying to find out the bacterial secreted proteins that would give an idea about the secretion system of Mtb, its unknown virulence factors, and their impact on host modulation for the survival of the pathogen inside the host. In the Mtb field of research, drugs like bedaquiline, delamanid can be used for treating MDR and exterior strains.

Research is still going on on developing drugs on MDR and XDR, and also vaccines for Mtb. Mtb is acquiring resistance more and more because of the incompliance of drug regimens. Treating the MDR, XDR strains is the most challenging task in the field of Mtb research.

Our group has established multiple significant findings in this field. Our lab showed the impact of phosphorylation of CFP-10 in Mtb virulence, and established the phospho-proteome, phospho-secretum, and secretum network of Mtb. Additionally, a paper from the lab has shown that the SecA2 secretor mediates the DNA damage in the host.

Treating cells with culture-filtered proteins instead of bacteria changes the cytokine levels and other immunomodulatory factors. With the help of secretum study, one can identify new virulent factors and their role in virulence. To begin, remove four independent Mtb glycerol stalks from minus 80 degrees Celsius and thaw them at room temperature.

Pour 10 milliliters of 7H9 medium containing 10%albumin dextrose catalase into a 100-milliliter autoclave conical flask with a screw cap. Inoculate the entire thawed glycerol stalk into the media to revive the culture which serves as the primary culture. Incubate the culture flask at 37 degrees Celsius in an incubator shaker at 100 revolutions per minute for three to four days.

On the fifth day, measure the absorbance at 600 nanometers. When the culture reaches an optical density between 0.8 to one, use it to inoculate the secondary culture in 100 milliliters of 7H9 medium containing 10%ADC in a 500-milliliter flask. Grow the secondary culture at 37 degrees Celsius in an incubator shaker at 100 revolutions per minute for two to three days.

When the optical density at 600 nanometers reaches approximately 0.8, withdraw 10 microliters of the culture using a pipette with a filter tip, and spot it on an LB auger plate without antibiotics inside the bacterial hood. Incubate the plate at 37 degrees Celsius for 12 to 16 hours to check for bacterial contamination. Now inoculate the bacteria from the contamination-free secondary culture into 100 milliliters of Sauton's medium in a 500-milliliter flask.

Grow the culture at 37 degrees Celsius for three to four days until the optical density reaches approximately 0.8. Use the culture grown to an optical density of approximately 0.8 in Sauton's medium to inoculate new 300-milliliter cultures for filtrate preparation. To begin, obtain 300 milliliters of log phase Mtb culture grown in Sauton's media.

Pellet down the cells in a 50-milliliter tube at 2, 100 G for 10 minutes at four degrees Celsius. Using a 25-milliliter serological pipette, transfer 45 milliliters of supernatant to a new tube, and set the untouched pellet aside for whole cell lysate preparation. Repeat the centrifugation step with the supernatant at 2, 100 G for 10 minutes at four degrees Celsius.

Transfer 40 milliliters of the supernatant to a new tube, leaving five milliliters untouched. Now filter the culture supernatant using a 0.2-micron syringe filter into new tubes. Inoculate one-milliliter aliquots of the culture filtrate onto an auger plate, and incubate it at 37 degrees Celsius for four weeks to check for contamination.

Resuspend the pellet in one milliliter of 1X PBS G containing phenylmethylsulfonyl fluoride, and 1X protease inhibitor cocktail. Transfer the lysate into a two-milliliter screw cap B beading tube filled to one third with 0.1 millimeters zirconia beads. Perform eight cycles of one minute bead beating, followed by two minutes of incubation on ice.

Centrifuge the tube at 18, 000 G for 15 minutes at four degrees Celsius. Collect the supernatant into a fresh 1.5-milliliter microcentrifuge tube placed on ice. After repeating the centrifugation step, filter the supernatant through a 0.2-micrometer syringe filter to eliminate pathogenic bacterial contamination.

Store the lysates at minus 80 degrees Celsius. To begin, add 15 milliliters of the Mtb culture filtrate to a three-kilodalton centricon filter unit. Centrifuge the tube at 2, 100 G for one hour at four degrees Celsius.

Repeat the centrifugation step 15 to 16 times until the culture filtrate reduces to approximately one milliliter. Aliquot 100 microliters of the concentrated culture filtrate proteins into 10 low-protein binding microcentrifuge tubes. Estimate protein concentration using the bicinchoninic acid protein assay kit.

Add 175 microliters of the mixed reagent as per the manufacturer's instructions. After mixing the samples, incubate them at 37 degrees Celsius for one hour. Measure absorbance readings at 562 nanometers using an ELISA reader.