Characterization and inhibition of Pseudomonas aeruginosa Type III Secretion System

Orateur : Soutenance de Thèse de Tuan Dung NGO (BCI)

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes nosocomial diseases and infects cystic fibrosis patients. The Type III Secretion System (T3SS) is one of its most important virulence factors, allowing the direct injection of four exotoxins into the target eukaryotic cells. An important protein of T3SS is the conserved ATPase, named PscN that is involved in the assembly and functioning of this system. In this work, we demonstrated the interaction of PscN with T3SS secreted cargo proteins and with their chaperons in complex or alone using ELISA, HTRF and MST assays. Of important, MST (Microscale Thermophoresis) allowed us to determine the dissociation constants (Kd) of these proteins and PscN, showing the interaction preference of this enzyme for the cargo or complex proteins rather than for the corresponding chaperons alone. This confirm the hypothesis that the chaperons are released in the bacterial cytoplasm after the complex dissociation. Otherwise, we assess the Kd between the effector, translocator or needle complexes and PscN bound or not to the gate-keeper complex which is described as a regulator of substrate sorting for the secretion. The results showed that the binding of the gate-keeper to PscN dramatically increases its relative affinity for the needle complex, thus revealing a new role of the gate-keeper in the loading of the needle complex to the ATPase for the control of substrate hierarchical secretion in P. aeruginosa.

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