Fig. 5.

TssS inhibits STING activity to suppress host innate immune responses. (A and B) PMs were transduced with the pSIN-IRES-Puro-4×tssS overexpression vector. At 24 h post-transduction, the cells were pretreated with or without 100 μM MnCl₂ for 24 h. At 48 h post-transduction, cells were lipofectamine transfected with 4 μM 2′3′-cGAMP (A) or 20 μM c-di-GMP (B). Gene expression of ISGs were determined at 4 h post c-di-GMP or 2′3′-cGAMP transfection. (C) 293T cells were transfected with STING-Flag plasmid. Cells were further untreated (Con) or pretreated with 100 μM MnCl₂ and along with or without 10 μΜ TssS or TssS* protein for 24 h and then lipofectamine transfected with 20 μM c-di-GMP. Cell lysates were immunoblotted with an antibody against STING after sodium dodecyl sulfate–polyacrylamide gel electrophoresis (PAGE) and native PAGE. (D) qRT-PCR analysis of gene expression in C57BL/6 or Tmem173^−/− PMs mock infected or infected with the Yptb WT or ΔtssS strain. (E) C57BL/6 or Tmem173^−/− mice were intragastrically inoculated with Yptb WT or ΔtssS. Homogenates of different tissues were plated to determine the bacterial CFU numbers per gram of organs at 72 h postinfection. Data in A and B were normalized to untreated control (Con, set as 1). Data in D were normalized to mock-infected C57BL/6 PMs and mock-infected Tmem173^−/− PMs, respectively (Mock, both set as 1). Gapdh was used as the housekeeping gene. Error bars represent ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.