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bioRxiv · 10.64898/2025.12.24.696360

Molecular Insights into the bactericidal Toxin Tle1 of Pseudomonas aeruginosa: Interaction with VgrG, its adaptor, and immunity protein

Lefebvre, D.·Soscia, C.·Schimtt, L.·Goulet, A.·Ize, B.·Bleves, S.
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Abstract

The Type VI secretion system (T6SS) delivers a wide range of antibacterial effectors, including phospholipases of the Tle family. Here, we characterize Tle1 from Pseudomonas aeruginosa and demonstrate that it functions as a bactericidal toxin with moderate activity when associated to the membranes in the periplasm. Bacterial two-hybrid assays revealed specific protein-protein interactions within the tle1 locus, involving the immunity protein Tli1a, the chaperone/adaptor Tla1, and the spike protein VgrG4a. These interactions were independently validated by co-purification assays. Structural modeling with AlphaFold 3 produced a high-confidence ternary complex in which a VgrG4a trimer accommodates one Tle1 monomer and one Tla1 monomer. The three predicted interfaces (Tle1-Tla1, Tla1-VgrG4a, and Tle1-VgrG4a) were confirmed experimentally in vivo and important charged residues mediating these interfaces were identified. Furthermore, modeling of the Tle1-Tli1a complex suggests an inhibition mechanism that does not occlude the catalytic pocket. Consistently, Tli1a was localized to the outer membrane of P. aeruginosa, supporting in silico predictions of an outer membrane lipoprotein and positioning it ideally to neutralize periplasmic Tle1 activity. We did not observe the second candidate immunity protein, Tli1b, either in P. aeruginosa or in E. coli. Together, these findings elucidate the molecular interactions underlying Tle1 delivery and inhibition and highlight the role of Tli1a as a dedicated immunity protein that protects P. aeruginosa from self-intoxication.

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Lefebvre, D., Soscia, C., Schimtt, L., Goulet, A., Ize, B., Bleves, S.. 2025-12-25. Molecular Insights into the bactericidal Toxin Tle1 of Pseudomonas aeruginosa: Interaction with VgrG, its adaptor, and immunity protein. https://doi.org/10.64898/2025.12.24.696360

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