Mechanism Of H-NS Regulation On T6SS Effectors EvpB And EvpC In Edwardsiella Tarda

Edwardsiella tarda, a rod-shaped Gram-negative pathogen, is one of the most harmful pathogenic bacteria in aquaculture industry. It has been reported that E. tarda utilizes various virulence factors to attack or be internalized into host cells. H-NS, an important global transcriptional regulator distributing in gram-negative bacteria, regulates expression of genes involved in virulence, metabolism and stress response of pathogens. Our previous study showed that H-NS negatively regulated the transcription of T6SS effector EvpP in E. tarda. 2D-electrophoresis proteomics analyses showed that expression of T6SS effectors EvpB and EvpC were up-regulated in hns deletion strains. This research aims to identify the molecular regulation mechanism of H-NS on EvpB and EvpC. The qRT-PCR assays showed that the transcription levels of evpB and evpC were significantly up-regulated in hns deletion strains while down-regulated in hns overexpression strains. The results of EMS A revealed that H-NS could bind directly to regions upstream of evpC ORF. DNase I footprinting experiments mapped the interaction sites of H-NS and region upstream of evpC ORF, and eight major interaction sites were identified distributing in evpB ORF. Our results supported that H-NS was able to bind simultaneously to several sites within the evpB ORF, and form a rigid nucleoprotein filament along DNA that down-regulates the transcription of evpB and evpC. Moreover, the binding sites were confirmed to be rich in AT content, acknowledged to be the H-NS preferred consensus structure. The results of site-directed mutagenesis showed that substitutions of Q84 and R86 with A led to declining or even abolished binding capacity of H-NS 1505. However, R86 was identified to play a more important role than Q84 in DNA binding. Moreover, invasion assays showed that H-NS negatively regulated internalization into BF-2 cells instead of adhesion.These results suggested that H-NS negatively regulated the expression of T6SS effectors EvpB and EvpC in E. tarda. This study will support to further understanding of E. tarda pathogenesis, and help to develop the virulence regulatory network.