Quantifying Yersinia pseudotuberculosis Type III Secretion System Activity Following Iron Starvation and Anaerobic Growth

Bacterial pathogens use virulence factors to colonize different niches within the host organism. We are working to understand how bacterial pathogens sense specific environmental cues in host tissues to regulate virulence factor expression. The human pathogen Yersinia pseudotuberculosis uses the type three secretion system virulence factor to inject effector proteins into host cells.

The type three secretion system allows the bacteria to subvert host defense mechanisms and colonize host tissues. However, expression of the Yersinia Type III secretion system is metabolically burdensome, so the expression of type three secretion system genes is strictly regulated in response to environmental cues such as temperature. Our previous publications showed that expression of the Yersinia Type III secretion system is controlled by iron availability and oxygen tension through a transcription factor called ISCR.

These findings are significant because Yersinia colonizes host tissues that vary in their iron content and oxygen tension. For example, the intestinal lumen contains sufficient iron for Yersinia growth but has low oxygen tension. In contrast, tissues like the blood have very low iron availability but higher oxygen tension.

Our method allows us to grow Yersinia in culture conditions that vary in the amount of available iron and oxygen, to study how these environmental cues impact expression of the type three secretion system. This method will help us determine the molecular mechanism by which iron and oxygen availability control expression of the type three secretion system, and help us understand how this controls the outcome of Yersinia infection. To begin, streak Yersinia Pseudo tuberculosis on Lysogeny broth agar plates.

Incubate the plates at room temperature for 48 hours. Once visible colonies form, inoculate a single isolated colony into four milliliters of M9 media. Culture it overnight at 26 degrees Celsius with aeration at 250 RPM.

Place a cuvette containing the culture in a spectrometer, then dilute the culture in 14 milliliters of sterile chelated M9 media without iron supplementation to achieve an OD600 value of 0.1. Place the flasks in a 26-degree Celsius shaker and incubate for eight hours. After the incubation is complete, use the culture to start three fresh cultures;two for anaerobic growth and one for aerobic growth.

For aerobic culturing, dilute the culture to an OD600 value of 0.1 in 14 milliliters of sterile chelated M9 media without iron supplementation. Then, culture with aeration at 26 degrees Celsius for 12 hours. For anaerobic culturing, dilute the culture in 14 milliliters of sterile chelated M9 media into two acid-washed glass tubes.

In the first tube, add filter-sterilized iron sulfate to a final concentration of one milligram per liter. In the second tube, add iron sulfate for a final concentration of 0.01 milligram per liter. Incubate both tubes in an anaerobic chamber at room temperature for 12 hours.

Then, induce type three secretion system activity by shifting the anaerobic cultures to 37 degrees Celsius, and continuing incubation for four hours. Next, dilute the aerobically growing cultures to an OD600 value of 0.2 by adding 14 milliliters of chelated M9 media into two acid-washed flasks. Add iron sulfate at the required final concentrations to the flasks.

Incubate both flasks with aeration at 26 degrees Celsius at 250 RPM for two hours. After two hours, shift the cultures to 37 degrees Celsius with aeration at 250 RPM for four hours to induce type three secretion system activity. After inducing the type three secretion system in Yersinia pseudotuberculosis, transfer normalized volumes of this culture incubated into 15 milliliter tubes.

Add four microliters of bovine serum albumin to each tube as a protein precipitation control. Centrifuge the cultures at 3, 200 G for 15 minutes at four degrees Celsius. Filter the supernatant into a fresh 15 milliliter tube using a 0.22 micron PVDF filter attached to a syringe.

Then, add trichloroacetic acid equivalent to 10%of the supernatant volume. Vortex the tubes vigorously for one minute. Incubate the tubes on ice in a cold room at four degrees Celsius overnight.

Next, add two milliliters of each sample to a two milliliter tube, then centrifuge at 21, 000 G for 15 minutes at four degrees Celsius. Aspirate the supernatant using a vacuum attachment. Repeat pipetting two milliliters of sample to the same tube, centrifuge, and aspirate the supernatant.

Consolidate the precipitation reactions into a single two milliliter tube. Wash the pellets with one milliliter of ice-cold 100%acetone followed by centrifugation and aspiration of supernatant. After removing the supernatant for the final time, dry the pellet on the bench for one hour.

The precipitated protein should look like a haze along the side of the tube. Then, resuspend each pellet in 50 microliters of F-S-B-DTT solution. Vortex thoroughly for one minute.

Next, boil the samples on a heat block at 95 degrees Celsius for 15 minutes. Then, centrifuge briefly at maximum speed at room temperature. Load 15 microliters of each anaerobic sample and 10 microliters of each aerobic sample to a 12.5%SDS-PAGE gel for analysis.

After the gel run is complete, use silver staining to visualize proteins on the gel. To begin, place an SDS-PAGE gel containing separated aerobic samples of Yersinia pseudotuberculosis in an imaging system. In the software, select the bands representing the type three secretion system effector protein, YopE, across all wells.

Set the reference YopE band to the appropriate sample. Export the software-calculated relative quantification values for all YopE bands. Then, select all BSA bands across all wells.

Ensure the reference BSA band matches the sample of the reference YopE band. Export the relative quantification values for all BSA bands. Divide the YopE value by the BSA value for each condition to produce normalized results.

Silver-stained SDS-PAGE gel revealed that in the anaerobic samples, 38-fold more YopE was present in the low iron samples compared to the high iron samples. In the aerobic samples, these proteins were secreted in nearly similar amounts.