A c-di-GMP binding effector STM0435 modulates flagellar motility and pathogenicity in Salmonella

Flagellum plays a crucial role in the invasion process of Salmonella, while also functioning as a significant antigen to trigger host pyroptosis. The regulation of flagella biogenesis is essential for both pathogenicity and immune escape of Salmonella. Here, we identified the conserved and unknown function protein STM0435 as a new flagellar regulator. ∆stm0435 strain exhibit higher pathogenicity in both cellular and animal infection experiments compared to wild-type Salmonella. Conjoint analysis of proteomics and transcriptomics demonstrated that almost all the flagellar genes were dramatically increased in a ∆stm0435 strain compared to wild-type Salmonella. Microscale thermophoresis assay showed that the purified STM0435 protein bound c-di-GMP with a ~194 µM KD affinity. The crystal structures of apo-STM0435 and STM0435&c-di-GMP complex were determined. Structural analysis revealed that R33, R137 and D138 of STM0435 are essential for c-di-GMP binding. Mutant that is unable to bind to c-di-GMP loses its ability to regulate motility, indicating that STM0435 affected the Salmonella flagellar biogenesis process by binding to c-di-GMP. Comparative gene expression profiling analysis of RNA-seq data for Salmonella typhimurium ATCC 14028s and Salmonella typhimurium ATCC 14028s (STM0435 knock-out),and each set has 3 repetitions .