INTRODUCTION

This study investigates the negative regulatory role of the global transcriptional regulator H-NS (Histone-like Nucleoid Structuring Protein) on the Type VI secretion system (T6SS) in (). We explored potential targets of H-NS mediated silencing or activation within the regulation of T6SS, along with the specific regulatory mechanisms involved, thereby providing a theoretical foundation for further research on invasive infections stemming from mixed infections and the development of therapeutic target.

METHODS

Using the plasmids pAT04 and pYMAb2-hyg, we constructed ATCC19606 strains with the gene knocked out (ABΔ) and overexpressed (AB+). We measured the expression of the T6SS-related gene in wild-type (AB WT), ABΔ, and AB+ strains using RT-qPCR, combined with a mouse sepsis model featuring mixed infections. We assessed their serum resistance, competitive ability against (), and blood invasion capability. Proteomic analysis identified differentially expressed proteins, and we further investigated the regulatory role of H-NS on T6SS using electrophoretic mobility shift assays (EMSA).

RESULTS

We successfully constructed both ABΔ and AB+ strains of ATCC19606. RT-qPCR results indicated that H-NS functions as a negative regulator of the T6SS-related gene in . Phenotypic assays for extracellular virulence revealed that the loss of enhanced both the competitive ability and serum resistance of ATCC19606. Results from the mouse sepsis infection model demonstrated that knockout of significantly increased the bacterium's blood invasion capability. Bioinformatics analysis of differentially expressed proteins identified elevated levels of T6SS-related proteins in the knockout strain. Furthermore, EMSAs confirmed that H-NS directly binds to multiple sites in the upstream region of .

CONCLUSION

H-NS inhibits the expression of T6SS-related proteins in by regulating relevant targets associated with the T6SS. This regulation influences the bacterium's pathogenicity, interspecies competitive ability, and serum resistance.