Functional activation reveals a repressor-gated fourth type VI secretion system in clinical Pseudomonas aeruginosa

Wu, L.; Liu, Y.; Huang, R.; Zhao, Y.; Liu, J.; An, Y.; Su, J.; Zhang, Y.; Wang, X.; Zheng, H.; Liu, X.; Pei, T.; Liang, X.; Xia, Y.; Zheng, M.; Liu, R.; Li, Y.; Wang, H.; Qu, J.; Yang, L.; Zhang, M.; Dong, T.

doi:10.64898/2026.02.12.705469

bioRxiv · 10.64898/2026.02.12.705469

Functional activation reveals a repressor-gated fourth type VI secretion system in clinical Pseudomonas aeruginosa

Wu, L.·Liu, Y.·Huang, R.·Zhao, Y.·Liu, J.·An, Y.·Su, J.·Zhang, Y.·Wang, X.·Zheng, H.·Liu, X.·Pei, T.·Liang, X.·Xia, Y.·Zheng, M.·Liu, R.·Li, Y.·Wang, H.·Qu, J.·Yang, L.·Zhang, M.·Dong, T.

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Abstract

Pseudomonas aeruginosa is a major opportunistic pathogen in which the type VI secretion system (T6SS) contributes to interbacterial competition and virulence. While most strains encode three T6SSs, we here functionally characterize a fourth system (H4-T6SS) in a clinical P. aeruginosa isolate. The H4-T6SS gene cluster is transcriptionally silent under laboratory conditions. By promoter rewiring, we demonstrate that the H4-T6SS assembles a contractile machine and mediates effector secretion and robust inter- and intraspecific antagonism. We identify a dominant pore-forming effector and its secretion pathway and a silent yet conserved nuclease effector. Genetic and biochemical analyses show that H4-T6SS expression is activated by an H4-encoded transcriptional regulator and repressed by H-NS family proteins. Analysis of over 1,200 clinical isolates reveals that the H4-T6SS is distributed across lineages and geographic regions. Finally, we observe functional crosstalk between H4- and H2-T6SSs. Together, these findings establish H4-T6SS as a functional and repressor-gated secretion system that expands the competitive arsenal of clinical P. aeruginosa.

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Wu, L., Liu, Y., Huang, R., Zhao, Y., Liu, J., An, Y., Su, J., Zhang, Y., Wang, X., Zheng, H., Liu, X., Pei, T., Liang, X., Xia, Y., Zheng, M., Liu, R., Li, Y., Wang, H., Qu, J., Yang, L., Zhang, M., Dong, T.. 2026-02-12. Functional activation reveals a repressor-gated fourth type VI secretion system in clinical Pseudomonas aeruginosa. https://doi.org/10.64898/2026.02.12.705469

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