H-NS binding to evpB and evpC and repressing T6SS expression in fish pathogen Edwardsiella piscicida

Edwardsiella piscicida is an important causative agent of hemorrhagic septicemia in fish and infects both cultured and wild fish species. Type VI secretion system (T6SS) was proved to play important roles in pathogenesis of E. piscicida. In this study, it was demonstrated that the expression of T6SS genes evpB and evpC was under control of the global regulator H-NS in E. piscicida and the transcriptional level of evpB and evpC was significantly down-regulated by H-NS. Compared to the wild type, the transcriptional levels of evpB and evpC were up-regulated in hns null mutant, while down-regulated in hns overexpression strain. The results of EMSA and DNase I footprinting revealed that H-NS protein directly bound to upstream region of evpC at multiple sites. A high-affinity motif with a 9-nucleotide sequence 5'-ATATAAAAT-3' was defined for H-NS preferential recognition based on the feature of the binding sites. These results indicated that H-NS acted cooperatively to form extended nucleoprotein filaments on target DNA. Site-directed mutagenesis of H-NS further showed that R86 played an essential role in T6SS gene binding. These findings highlighted the mechanisms underlying the complex regulation network of T6SS by H-NS in E. piscicida.