ChIP-seq and structural analyses delineating the regulatory mechanism of master regulator EsrB in Edwardsiella piscicida

As a response regulator of the EsrA-EsrB two-component system, EsrB is conserved in Hafniaceae and plays a crucial role in virulence and pathogenicity. EsrB possesses DNA binding abilities, enabling it to regulate the transcription of virulence genes to confront different stresses and achieve systematic infections. Here, ChIP-seq analysis of EsrB in Dulbecco's Modified Eagle's Medium (DMEM) (mimicking in vivo environments) revealed that EsrB preferred to bind to virulence-associated promoters with a distinct 7'-4-7'' pseudopalindromic DNA motif and interact with metabolic-related promoters with a high AT DNA motif. The crystal structure of the C-terminal of EsrB (EsrB_C) was solved at 2.20-Å resolution. Specifically, Lys¹⁸¹ enabled the DNA-binding affinity of EsrB and promoted the in vitro and in vivo pathogenicity of Edwardsiella piscicida. Moreover, EsrB directly regulated the expression of genes associated with basal metabolism, including iron and tricarboxylic acid (TCA) cycles. Furthermore, EsrB enhanced iron transport capability and the enzyme activity of succinate dehydrogenase and pyruvate dehydrogenase in DMEM. Collectively, our structural and ChIP-seq analysis provides valuable insights into the DNA binding mechanism of EsrB which will facilitate our understanding of EsrB coordinating virulence and basal metabolism gene expression.

Importance: As a crucial virulence regulator, EsrB possesses a LuxR-like superfamily domain at the C-terminal, which is conserved within the canonical NarL family regulators. Due to its critically important role in virulence and pathogenicity in fish hosts, the DNA binding ability has been believed to allow EsrB to regulate genes associated with the invasion process of host cells and basal metabolism in response to environmental stimuli. The lack of EsrB's crystal structure has been a major obstacle in understanding the molecular mechanisms of EsrB-DNA interaction which choreographs EsrB-mediated pathogenic behavior. Here, we conducted ChIP-seq and solved the crystal structure of the C-terminal of EsrB (EsrBC) at 2.20-Å resolution, which revealed that EsrB preferred to bind to virulence-associated promoters with a distinct 7'-4-7' pseudopalindromic DNA motif and interacted with metabolic-related promoters with a high AT DNA motif in Dulbecco's Modified Eagle's Medium (DMEM) (mimicking in vivo environments). Our results facilitate a detailed understanding of EsrB's regulatory role in Edwardsiella piscicida pathogenesis and expand our knowledge of virulence regulators in the family Hafniaceae.