Deletion of the <i>hslV</i> gene alleviates the virulence and lethality of <i>Salmonella</i> Typhimurium in hosts by modulating <i>lon</i> and T3SS-1

<i>Salmonella</i> Typhimurium (<i>S</i>. Typhimurium) is a major foodborne pathogen that utilizes flagella-mediated biofilm formation and type III secretion systems (T3SSs) to initiate infections, contributing significantly to its virulence and antibiotic resistance. Despite its serious threat to public health, effective therapeutic strategies that simultaneously target T3SS effectors and biofilm remain underdeveloped. In this study, we deleted the <i>hslV</i> gene in <i>S</i>. Typhimurium (Δ<i>hslV</i>) and demonstrated that the Δ<i>hslV</i> strain exhibited significantly impaired bacterial adhesion, invasion, and intracellular proliferation in host cells. These defects were rescued upon genetic complementation. <i>In vivo</i> studies further revealed that the Δ<i>hslV</i> strain exhibited attenuated systemic spread and delayed lethality. These results demonstrate HslV plays an essential role in promoting <i>S</i>. Typhimurium virulence. Proteomics analysis, combined with SDS-PAGE and Western blot, showed the expression of T3SS-1 effectors (e.g. SipA, SipC) and flagellar protein FliC was down-regulated in the Δ<i>hslV</i> strain compared to the WT, while Lon expression was up-regulated. Notably, reduced flagellar protein expression in the Δ<i>hslV</i> strain likely contributes to motility and biofilm formation. Mechanically, HslV enhances the transcription of T3SS-1 virulence factors by suppressing <i>lon</i> expression. Functionally, it promotes bacterial virulence by facilitating flagellar assembly and synthesis to enhance chemotaxis, while also strengthening adhesion, invasion and biofilm formation against host cells. Consequently, HslV potentiates infection, inflammatory responses, and host lethality. In summary, this study provides the first evidence that HslV functions as a critical regulatory factor of virulence in <i>S</i>. Typhimurium, identifying it as a promising therapeutic target for combating infections and antibiotic resistance.