ChIP-mini: a low-input ChIP-exo protocol for elucidating DNA-binding protein dynamics in intracellular pathogens

초록

Genome-wide identification of binding profiles for DNA-binding proteins from the limited number of intracellular pathogens in infection studies is crucial for understanding virulence and cellular processes but remains challenging, as the current ChIP-exo is designed for high-input bacterial cells (>1010). Here, we developed an optimized ChIP-mini method, a low-input ChIP-exo utilizing a 5,000-fold reduced number of initial bacterial cells and an analysis pipeline, to identify genome-wide binding dynamics of DNA-binding proteins in host-infected pathogens. Applying ChIP-mini to intracellular Salmonella Typhimurium, we identified 642 and 1,837 binding sites of H-NS and RpoD, respectively, elucidating changes in their binding position and binding intensity during infection. Post-infection, we observed 21 significant reductions in H-NS binding at intergenic regions, exposing the promoter region of virulence genes, such as those in Salmonella pathogenicity islands-2, 3 and effectors. Furthermore, we revealed the crucial phenomenon that novel and significantly increased RpoD bindings were found within regions exhibiting diminished H-NS binding, thereby facilitating substantial upregulation of virulence genes. These findings markedly enhance our understanding of how H-NS and RpoD simultaneously coordinate the transcription initiation of virulence genes within macrophages. Collectively, this work demonstrates a broadly adaptable tool that will enable the elucidation of DNA-binding protein dynamics in diverse intracellular pathogens during infection.

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