Functional characterization of the T6SS effector TseN in Yersinia pseudotuberculosis

Abstract:

[Background] The type VI secretion system (T6SS) with wide distribution in Gram-negative bacteria plays important roles in bacterial colonization, environmental adaptation, and bacterium-host interactions. T6SS is crucial for the pathogenicity of Yersinia pseudotuberculosis. [Objective] To investigate the physiological and biochemical characteristics and the biological function of the potential effector TseN encoded by the T6SS gene cluster of Y. pseudotuberculosis. [Methods] The bioinformatics analysis was performed to identify the potential T6SS effector TseN and its immune protein TsiN. Recombinant plasmids were constructed and transformed into Escherichia coli, and growth curves were established to evaluate the toxicity of TseN to bacteria and the immune activity of TsiN. The bacterial two-hybrid system was used to verify the interaction between TseN and TsiN. The in vitro DNA nuclease activity assay was employed to confirm the DNA nuclease activity of the purified TseN. Y. pseudotuberculosis mutant with tseN deletion (ΔtseN) was constructed by homologous recombination, and a complemented strain (ΔtseN(tseN)) was generated to examine the roles of TseN in bacterial competition and stress resistance. [Results] The expression of TseN significantly inhibited the growth of E. coli, which was restored in the strain co-expressing TseN and TsiN. The result indicated that TsiN neutralized the toxicity of TseN by direct interaction. TseN could bind to DNA and exhibited Mn²⁺-dependent DNA nuclease activity. Compared with the wild-type strain, ΔtseN showed significantly reduced ability in mediating intra- or inter-species bacterial competition, and its biofilm formation was significantly affected. In addition, ΔtseN showcased decreased survival rate under high salt, low pH, and other stress conditions, with partial recovery observed in the complemented strain. [Conclusion] TseN is a Mn²⁺-dependent DNA nuclease secreted by the T6SS of Y. pseudotuberculosis, with strong toxicity to bacteria. TsiN is the immune protein of TseN, protecting Y. pseudotuberculosis from the toxic effects of TseN. TseN play a role in the bacterial competition of Y. pseudotuberculosis by helping the bacterium occupy ecological niches, promoting biofilm formation, and enhancing tolerance to stress conditions such as high salt and low pH.

Key words:Yersinia pseudotuberculosis;type VI secretion system (T6SS);effector;immune protein;functional characterization