Fig. 6.

TssS acts as a Mn²⁺ chelator to suppress STING-mediated innate immune response. (A) qRT-PCR analysis of gene expression in PMs untreated (Con) or pretreated with 10 μΜ TssS and 50 μM EDDHA along with or without 100 μΜ Mn²⁺ for 24 h and then transfected with 20 μM c-di-GMP for 4 h. (B) 2′3′-cGAMP production was measured by high-performance liquid chromatography. 2′3′-cGAMP was produced in the presence of 0.2 mM ATP and 0.2 mM GTP by cGAS under 10⁻² mg ⋅ mL⁻¹ double-stranded DNA. (Middle) Added without (Con) or with 1 mM Mn²⁺, 10 μM TssS protein, 10 μM TssS* protein, or 500 μM EDDHA for 2 h. (Bottom) Added without (Con) or with 1 mM Mn²⁺ or the indicated concentrations of TssS protein for 2 h (as described in Materials and Methods). (Insets) Expanded views of the indicated products. (C) Viral titers in HeLa cells transduced with the pSIN-IRES-Puro-4×tssS overexpression vector. At 24 h post-transduction, the cells were infected with virus vaccinia at a multiplicity of infection of 0.01. Viral titers were determined at 24 h postinfection. (D) Mn concentrations in Raw264.7 cytoplasm. Raw264.7 was coincubated with TssS or TssS* protein (10 μΜ) for 30 min. The cytoplasmic Mn²⁺ concentration was measured by ICP-MS after removing His₆-tagged TssS or TssS* proteins with His•Bind Ni-NTA resin. Data in A were normalized to untreated, nontransfected control (Con, set as 1), and Gapdh was used as the housekeeping gene. Error bars represent ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001.