25×105 cells/well in 24-well tissue culture plates and incubated for 24 hr.

For measuring the activation of NFκB by B. pseudomallei wildtype (KHW) and mutants, the cells were transfected with 100 ng of pNFκB -SEAP plasmid using jetPRIME DNA & siRNA transfection reagent (Polyplus Transfection). After another 24 hr., the media were replaced with A-1210477 chemical structure antibiotics-free VX-689 mw media. The cells were then infected with mid log-phase cultures of B. pseudomallei at required MOI. Following infection, plates were centrifuged at 200 x g for 5 min to allow maximum bacteria to cell contact. Two hr. post infection, 250 μg/ml CA-4948 kanamycin was added to kill off extracellular bacteria. Cells without infection were included as control. Supernatant was collected at various time points and SEAP activity was measured. For measuring the activation of NFκB by B. pseudomallei T3SS3 effectors, the cells were co-transfected with 100 ng of pNFκB -SEAP plasmid and up to 400 ng of plasmid harbouring B. pseudomallei T3SS3 effector gene or 400 ng of empty plasmid using jetPRIME DNA & siRNA transfection

reagent. Total amount of DNA transfected were kept constant at 500 ng. After another 24 hr., supernatant was collected and SEAP activity was measured. SEAP activity was measured using Phospha-Light Sitaxentan kit (Life Technologies) according to the instructions of the manufacturer. Relative NFκB activation was calculated by averaging the raw luminescence values obtained using the Phospha-Light kit and converting them to fold activation with respect to uninfected cells or cells transfected with empty vector.

Intracellular bacterial count HEK293T cells were seeded and infected as described above. Two hr. post infection, cells were washed twice with 1x PBS before addition of fresh culture medium with 250 μg/ml kanamycin. At respective time points, infected cells were washed with 1x PBS and lysed with 0.1% (v/v) Triton X-100. Serial dilutions were performed on the lysates and subsequently plated on TSA agar and incubated at 37°C for 48 hr. Colony counts were used to calculate bacterial loads. Cytotoxicity of B. pseudomallei against HEK293T cells HEK293T cells (1.25 x 105 cells/well) were seeded and grown overnight in a 24 well plate.