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

ABSTRACT: 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.

ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S

PROVIDER: GSE239303 | GEO | 2023/08/05

REPOSITORIES: GEO

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Project description:The cholera disease bacterium V. cholerae, can adopt planktonic or biofilm lifestyles depending on the intracellular concentration of the second messenger cyclic diguanylic acid (c-di-GMP). Biofilm formation protects Vibrios from stressful conditions and facilitates disease transmission by enhancing infectivity. The histone-like nucleoid structuring protein (H-NS) is a global regulator of genes associated with pathogenicity and responses to environmental stresses. H-NS represses the transcription of genes vpsT, vpsA and vpsL, which are required for the biosynthesis of the biofilm exopolysacchide matrix. Here we demonstrate that the c-di-GMP-binding protein VpsT disrupts H-NS nucleoprotein complexes at the vpsA and vpsL promoters and that this effect is enhanced by c-di-GMP. We used ChIP coupled with Next Generation Sequencing (ChIP-Seq) and transcriptome analysis (RNA-Seq) to identify additional loci repressed by H-NS affecting biofilm formation. This study showed that H-NS directly represses the transcription of genes encoding proteins present in the biofilm matrix such as the rbmA-F cluster, hemolysin and chitinase. Similar to vpsA and vpsL, the promoter region of vpsU, rbmA and rbmF exhibited overlapping H-NS and VpsT binding motifs. Deletion of vpsT increased H-NS occupancy at the vpsU, vpsA, vpsL, rbmA and rbmF promoters. Conversely, artificially increasing the c-di-GMP pool diminished H-NS occupancy at the above promoters. Deletion of vpsT did not affect H-NS occupancy at its own promoter. However, deletion of genes encoding the regulators AphA and VpsR significantly increased H-NS occupancy at the vpsT promoter. In sum, our study shows that c-di-GMP enhances biofilm formation by acting through VpsT to activate an H-NS anti-repression cascade. The Binding profile of V. cholerae H-NS to the genome was determined by ChIP followed by Next Generation Sequencing (ChIP-Seq) using the Illumina HiSeq2000 platform. V. cholerae C7258 cells expressing H-NS-FLAG fusion protein from the hns transcription and translation signals were collected from LB cultures grown to mid-exponential phase (OD600 0.5). An anti-FLAG Immunoprecipitation (IP) and an Input samples were used for the analysis.

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

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