Fig. 3.

TssS is a Mn²⁺-binding micropeptide, and its Mn²⁺-binding activity is required for Yptb virulence. (A) The binding of divalent ions by TssS was detected by atomic adsorption spectrometry. (B) The binding of Mn²⁺ with Mn²⁺-free TssS protein or TssS* (TssS^{E30A/E36A/H43A/Q44A} mutated protein) was determined via ITC. (C) Stationary-phase Yptb WT, ΔtssS, mutant and ΔtssS(tssS) were exposed to 2.5 mM H₂O₂ for 40 min in M9 medium with 1 μΜ MnSO₄. Mn²⁺ associated with bacterial cells was measured by ICP-MS. (D) The viability of Yptb strains under oxidative stress was determined. Yptb WT, ΔtssS, ΔtssS(tssS), and ΔtssS(tssS*) were exposed to H₂O₂ (1 mM) for 40 min in M9 medium. (E) C57BL/6 mice were intragastrically inoculated with the Yptb WT, ΔtssS, ΔtssS-tssS, or ΔtssS-tssS*, respectively. Homogenates of different tissues were plated to determine the bacterial CFU counts per gram of organs at 72 h postinfection. (F) qRT-PCR analysis of the gene expression in PMs infected with Yptb WT, ΔtssS, ΔtssS-tssS, or ΔtssS-tssS*, respectively. Data in F were normalized to mock-infected control (Mock, set as 1), and Gapdh was used as the housekeeping gene. Error bars represent ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.