High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

The overall goal of the following experiment is to screen and evaluate genes required for invasion and or survival inside macrophage cells in vitro, in a high throughput fashion, and identify mechanistic genes of salmonella pathogenesis. This is achieved by adding wild type or mutant salmonella strains to macrophage cell cultures. In 96, well plates for 30 minutes as an in vitro infection as a second step after the 30 minute incubation, salmonella that have not attached to the macrophage cells are washed away and fresh.

Medium containing a high concentration of Gentamycin antibiotic is added for one hour, which kills all the extracellular bacteria that are not internalized. Next, the macrophage cell culture is washed again and fresh medium containing a low concentration of gentamycin is added for an additional 22.5 hour incubation in order to maintain a bacteria free extracellular environment and allow only the intracellular salmonella to survive and proliferate. The results show that by comparing the mutant salmonella with the wild type strain, the in vitro phenotype of the mutant strains for association invasion and replication is determined based on the measurement of colony formation units per macrophage cells obtained by the serial dilution plating assay for each time point.

The main advantage of our technique is that it measures association, invasion and replication of salmonella and phagocytic cells and that it can measure multiple strains simultaneously for higher throughput. Demonstrating the procedure will be Dr.Jing Wu postdoctoral research fellow in my laboratory, and Mrs. Roberta Pugh, research associate in my laboratory To begin row low passage number mirroring macrophage cells raw 2 64 0.7 in a T 75 cell culture flask with a vented filter cap in delcos modified eagles medium supplemented with 10%fetal bovine serum 0.5%sodium bicarbonate and 1%100 x non-essential amino acids NEAA at 37 degrees Celsius in a 5%carbon dioxide incubator.

When the cells reaches 60 to 80%confluence, harvest the cells in DMEM with a cell scraper, then count the cells in a hemo cytometer and calculate the cell concentration, dilute the cell suspension to a concentration of 2.5 times 10 to the fifth cells per milliliter of fresh DMEM cell culture. Medium plate 200 microliters of the diluted cell suspension using a multi-channel pipette into each well of a 96 well cell culture plate plate cells into three separate 96 well plates for one set of phagocytic cell invasion assay and label each plate with association invasion and replication. Four wells per plate are used for each salmonella strain.

Place the plates in a 5%carbon dioxide incubator at 37 degrees Celsius overnight to allow the macrophage cells to attach to the bottom of the wells on the same day. The macrophage raw 2 6 4 0.7 cells are plated into 96 well plates. Pick single colonies of wild type salmonella entera serotype, tyria 1 0 4 2 8 delta INVA mutant delta four P mutant and the desired test mutant strains culture.

Each of the salmonella strains in five milliliters of LB broth and supplement the LB broth for the mutant strains With 50 micrograms per milliliter of can mycin grow the bacteria for 14 hours at 37 degrees Celsius with shaking at 220 RPM in loosely capped 14 milliliter polypropylene round bottom tubes. After the overnight incubation at 37 degrees Celsius subculture each of the salmonella strains at a ratio of one to 50 into five milliliters of the respective lb broth. Grow the cultures for an additional four hours at 37 degrees Celsius with shaking at 220 RPM.

Read the OD 600 values of each bacterial culture on a spectrophotometer. Each culture should have an OD 600 value between 0.6 and 1.2 to optimize salmonella pathogenicity island one to three secretion system SPI one type three secretion system gene expression for invasion. Calculate the bacterial concentration using the formula one OD 600 equals 7.5 times 10 to the eighth colony forming units per milliliter.

CFU then dilute the bacteria to a concentration of five times 10 to the six, the FU per milliliter and three milliliters of fresh DMEM cell culture medium Remove 3 96 well cell culture plates containing macrophages from the carbon dioxide incubator and wash each well once with 200 microliters of one XPBS. After the PBS wash is completely removed from each, well add 200 microliters of bacteria in DMEM medium into each well. This results in one times 10 to the six salmonella cells in each well and a multiplicity of infection MOI of 20 to one centrifuge the plates in a sealed container at 1000 times G for 10 minutes.

Then place the plates in a 5%carbon dioxide incubator at 37 degrees Celsius for 30 minutes. Once the 30 minute incubation is complete, remove the plates from the incubator and wash each well three times with 200 microliters of one XPBS to remove unassociated bacteria. Next at 200 microliters of DMEM medium containing 100 micrograms per milliliter of gentamycin to each well of the plates marked with invasion and replication in order to kill the extracellular salmonella.

Then return the plates to a 5%carbon dioxide incubator at 37 degrees Celsius for an additional hour. For the association plate, save one well from each infected strain for recording macrophage cell count and treat the remaining wells with 200 microliters of one XPBS containing 1%tritton X 100 for 10 minutes. To slice the cells, harvest the salmonella from each well and place them into 1.5 Milliliter eend orph tubes Make three tenfold cereal dilutions with 900 microliters of one XPBS on the harvested samples from each well and vortex between each dilution vortex the third dilution, 10 to negative three for each bacteria and place 10 microliters of the sample onto either LB plates for wild type or LB plates.

With can mycin for the mutant strains. Repeat this step four times for a total of five 10 microliter drops spaced evenly on the plates after the drops soak into the agar, turn the plates over and incubate overnight In a 37 degrees Celsius incubator, treat the remaining wells saved for counting the macrophages with 150 microliters of one XPBS containing 0.25%TRIPSIN EDTA for 10 minutes. After trypsin ization transfer macrophages into 1.5 milliliter einor tubes and treat with 50 microliters of FBS to neutralize the tryin EDTA solution.

Then remove 10 microliters of the cell suspension from one EOR tube into a new tube. Add 10 microliters of 0.4%trian blue to stain the cells and count them in a hemo cytometer when the invasion and replication plates have completed the one hour incubation in the carbon dioxide incubator. Remove the plates from the incubator and wash each well three times with 200 microliters of one XPBS.

Next, add 200 microliters of DMEM medium containing 10 micrograms per milliliter of Gentamycin to the replication plate to maintain clearance of extracellular salmonella in the medium. Then place the plate in a 5%carbon dioxide incubator at 37 degrees Celsius for an additional 22.5 hours. Then harvest and reflate the salmonella from the invasion plate onto agar plates and count the macrophages from one well of each infected strain following the steps described for the association plate on the next day.

Remove the replication plate from the incubator and wash each well three times with 200 microliters of one XPBS. Then harvest and reflate the salmonella from the replication plate onto agar plates and count the macrophages from one well of each infected strain following the steps described for the association plate. After the agar plates have incubated overnight in a 37 degrees Celsius incubator, remove the plates from the incubator and score the number of bacterial colonies per plate.

When counting each plate, colony distribution should fall between 10 and 100 colonies. If the count is too high or too low, then reflate the sample with tenfold higher or lower dilution as needed. The phagocytic cell invasion assay appropriately identifies the phenotype of salmonella deletion mutants as is shown for the delta INVA invasion control strain, a known invasion defective mutant, and the delta four P replication control strain.

A known replication defective mutant mutant A and mutant B are two representative strains assay Using this protocol that display altered phenotypes for replication in macrophages 20 of 38. Salmonella mutants tested so far in the phagocytic cell invasion assay display variable but significant defects in association invasion and or replication in raw 2 6 4 0.7 macrophages. This protocol was only tested for some nala and host effect static cells.

Thus modification and optimization will be necessary for other bacteria and host cells.