Shigella is a significant global pathogen that causes devastating infection rates every year. Shigella infects the gastrointestinal tract to cause diarrhea or dysentery. Our research aims to improve our understanding of infection to help develop more effective therapeutics like vaccines and other antimicrobial products.
First, animal models and emerging tissue culture techniques allow us to better understand the physiology of Shigella infection and faithfully reproduce the complexity of the human GI tract. Second, characterization of clinical Shigella isolates has helped to capture the diversity of this genus, identify important virulence genes and improve our understanding of infection. We have used gastrointestinal signals like bile salts and glucose to understand how Shigella survives transit in the small intestine and regulates virulence gene expression for infection in the large intestine.
We have demonstrated how Shigella resists bile salts and have shown that Shigella produces adherence proteins to help initiate contact with epithelial cells to start infection. These protocols are designed to look at key aspects of epithelial infection like adherence, invasion, and intracellular survival of Shigella. We're performing these protocols using a standard epithelial cell line, but have also adapted the methods for sophisticated human GI models that are now available.
These protocols consider each phase of infection independently, which is critical to understand the key steps in Shigella infection. They also highlight that combined analyses can facilitate a greater understanding of Shigella infection at a holistic level. Re-streak Shigella strains on agar plates containing Congo red dye to ensure colonies are virulent and to prevent the use of non-virulent colonies in infection assays.
To begin, take the overnight grown Shigella cultures and vortex them. Add 100 microliters of the culture to five milliliters of fresh TSB in a culture tube. Incubate the cultures at 37 degrees Celsius while shaking at 250 RPM until an optical density of 0.7 is reached at 600 nanometers.
Transfer two times 10 to the eight colony-forming units of subcultured Shigella to two milliliter microcentrifuge tubes. Pellet cells by centrifugation at 17, 000 G for two minutes at room temperature. Aspirate the supernatant.
Add one milliliter of warm PBS and resuspend the pellet thoroughly. After centrifuging the cell once again, resuspend the pellet in two milliliters of warm DMEM and vortex the cell suspension. Then add one milliliter of resuspended Shigella and one milliliter of DMEM to each well of HT-29 colonic epithelial monolayers in six-well plates.
To ensure bacterial contact with HT-29 cells, centrifuge the six-well plates at 2, 000 G for 10 minutes at room temperature or 37 degrees Celsius. Incubate six-well plates at 37 degrees Celsius and 5%carbon dioxide for 45 minutes. To determine the bacterial infection titer, prepare tenfold serial dilutions of resuspended Shigella cells in PBS.
Plate 100 microliters of the one times 10 to the negative five and one times 10 to the negative six dilutions onto Congo red plates with TSB and incubate overnight at 37 degrees Celsius. After incubation, aspirate media from each well. Add one milliliter of warm PBS to each well and wash gently.
Then add two milliliters of warm DMEM with 50 micrograms per milliliter of gentamicin and incubate for 30 minutes at 37 degrees Celsius with 5%carbon dioxide. Wash infected HT-29 cells three times with one milliliter of PBS. Add two milliliters of warm DMEM with gentamycin to each well and incubate for up to 24 hours for intracellular replication at 37 degrees Celsius with 5%carbon dioxide.
At the end of the incubation, wash cells two times with one milliliter of PBS. Add one milliliter of PBS with 1%triton X-100 to each well to lyse HT-29 cells. Then using a cell scraper or bent pipette tip, scrape the lysed cells from the well bottoms and transfer the mixture into a 1.7 milliliter microcentrifuge tube.
Vortex each tube for at least 30 seconds to further displace Shigella from the lysed eukaryotic cells. Prepare tenfold serial dilutions of the lysates in PBS. Plate 100 microliters of the serial dilutions onto Congo red plates with TSB and incubate overnight at 37 degrees Celsius.
