The multifaceted role of SsrB in S. Typhi H58 during THP-1 macrophage infection

S . Typhi, the causative agent of typhoid fever, remains less well understood due to its human-restricted nature compared to S . Typhimurium, which enjoys a broad host range. Herein, we investigated the acid response of S . Typhi and the role of the SPI-2 master regulator SsrB during infection of human THP-1 macrophages. Using fluorescence lifetime imaging of the pH-sensitive probe mCherryTYG, we demonstrate that the S . Typhi cytosol acidifies during infection of THP-1 macrophages, similar to S . Typhimurium. This acidification triggers a marked up-regulation of SsrB, with S . Typhi expressing significantly more SsrB molecules than S . Typhimurium, as quantified by photoactivatable localization microscopy. Despite increased SsrB expression, activation of the SsrB-dependent reporter P sifA was reduced, indicating serovar-specific differences in SsrB activity. Unexpectedly, deletion of ssrB in S . Typhi increased net intracellular replication and host cell cytotoxicity, contrasting with its essential role in S . Typhimurium. Using phosphorylation incompetent (D56A) and acid-sensing deficient (H12Q) mutants, we demonstrate that the reduction in cytotoxicity is phosphorylation-independent, whereas restriction of net intracellular replication requires both phosphorylation and acid-dependent DNA binding. The SPI-2 effector SifB partially mediates this restriction. Our findings show that SsrB contributes to two infection phenotypes with distinct genetic requirements: it limits host cytotoxicity in a manner that requires neither phosphorylation nor acid-enhanced DNA binding, and it restricts intracellular replication in a manner that requires both activities. Whether these two phenotypes are executed by separate or by shared downstream targets was not established here. These results challenge the assumption that SsrB is dispensable for S . Typhi pathogenesis and highlight serovar-specific virulence strategies.