Typhoid toxin retro-translocation assay

Abstract

Typhoid toxin encoded by typhoidal Salmonella plays a critical role in the pathogenesis of typhoid fever by intoxicating a variety of cell types. This toxin belongs to the AB toxin family and possesses two A subunits CdtB and PltA, and a homopentameric complex comprised of the B subunit PltB. The B subunit (PltB) mediates the cellular entry of typhoid toxin through the retrograde transport pathway. This protocol described a measurement to analyze the toxin release from the ER into the cytosol in intoxicating cells. 

Materials and Reagents

-DPBS

-Purified typhoid toxin

-Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Gibco®) containing 10% FBS

-HCN buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM CaCl₂, 0.04 % Digitonin, and 1X of a protease inhibitor cocktail (Roche)

-Elution buffer containing 200 mM imidazole and 0.15 M Tris-HCl (pH 6.8)

Equipment

10 cm cell culture dishes

Centrifuge

Microcentrifuge

Cell scraper

Laminar flow hood

Pipetteman

Procedure

Day 1: HEK293T cells (1x10⁷) are seeded on 10 cm dishes.

Day 2:  

1. HEK293T cells are treated with 1 μg of purified His-tagged typhoid toxin at 37 ˚C for 30 minutes. 
2. Wash the cells with DPBS once and then incubate in media containing 10% FBS. 
3. Collect cells at indicated times and resuspend in 500μL of HCN buffer for 10 min at 4 ˚C.
4. Centrifuge the eppendorf at 14,000 rpm for 10 min at 4 ˚C to separate cytosolic (soluble) and membrane (pellet) fractions.
5. Pellets are resuspended in 2 x Laemmli buffer and analyzed by western blot.
6. Soluble fractions are incubated with 20 μL of nickel resin at 4˚C overnight. 

Day 3: 

1. The toxin from the soluble fractions is recovered in 30 μL elution buffer for 20 min at room temperature.
2. Centrifuge the tube at 1,000 rpm for 1 min to pellet the nickel resin.
3. Collect the supernatant and analyze by western blot.

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