Figure 2
Identification of cGAMP PDEs in V. cholerae (V-cGAPs) that inhibit DncV-induced IRF3 activation. (A) 293T cells were transiently transfected with 50 ng of DncV and 150 ng of the indicated Flag-tagged PDE candidates, along with STING and IFN-β-Luc reporter construct. Luciferase assay was performed after 24 h. Candidates with the most significant difference were marked with an asterisk (*) and selected for further analysis. Genes in red are HD-GYP proteins fully characterized in this study. (B) Flag-tagged PDE candidates as indicated were transfected into 293T cells, along with STING and DncV. p-IRF3 levels were then analyzed. (C) Chemically synthesized 3′3′-cGAMP was incubated with the indicated purified proteins (bottom) for 2 h. cGAMP activity assay (top) was performed in permeabilized THP-1 cells. (D, E) All indicated HD-GYP domain-containing proteins in V. cholerae were expressed and purified from E.coli (D, bottom), and incubation with 3′3′-cGAMP for 2 h. cGAMP activity assay (D, top) or HPLC assay (E) was then performed. P1: the first product; P2: the second product (also see Figure 4A).
