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Molecular Dynamic in type II secretion : the pseudopilus/piston paradigm – 3Dpilus

Submission summary

The gram negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen, able to secrete in the extracellular medium a wide range of toxins and hydrolytic enzymes. The Xcp Secreton, also called type 2 secretion system (T2SS) is a major pathway for the release of these virulence factors. The Xcp secreton is a macromolecular complex involving twelve different proteins (XcpA, XcpP-Z) assembled in the bacterial cell envelope. However, if individual Xcp proteins are well characterized, very little is known about their specific function during the secretion process. The Type 2 secretion apparatus shares similarities with another macromolecular complex, the type 4 pili biogenesis system. Type 4 pili are long fibrillar and adhesive structures present on the cell surface of various gram-negative bacteria. Five Xcp proteins possess characteristics similar to the major type 4 pilin subunit, therefore they have been called pseudopilins. Interestingly, we have shown that, when overproduced, the major pseudopilin XcpT is assembled by the Xcp secreton into a fibrillar structure that we called the type 2 pseudopilus suggesting common features between Type 2 secretion and Type 4 piliation. However, we do think that, during the secretion process, pseudopilus biogenesis must be tightly regulated to allow efficient secretion and that a mechanism involving the four minor pseudopilins (XcpU-X) might exist to control the assembly of this structure. In the present project, we propose to develop a multidisciplinary approach to investigate in detail the structure-function relationship between the five pseudopilines in order to understand their specific involvement in the dynamic of the Type II secretion process. The major investment will be dedicated to solve the high resolution structure of the five pseudopilins soluble domains, to acquire crucial information at the atomic level. This will be done in tight collaboration with the 2 structural biologists involved in this project. We will also study, using biochemical, biophysical and genetic tools, protein-protein interactions between the five pseudopilins in a systematic manner. Moreover, we have preliminary evidences that XcpV and XcpX could respectively act as initiator and terminator of pseudopilus assembly. By using targeted and random mutagenesis, we will try to localize in XcpV and XcpX the domains responsible for their specific function in the pseudopilus biogenesis. Finally, we will use the electron microscopy facilities of our institute to analyze at high resolution the architecture of the type 2 XcpT pseudopilus as well as the involvement and possible localization of the 4 minor pseudopilins in the XcpT pseudopilus. By using NMR and X-ray crystallography we will provide a massive effort to solve the structure of the 5 pseudopilins of the T2SS. We hope that the collected structural data combined with the experience on the T2SS field brought by members of the group will help to answer unresolved questions and will open new fields of investigation for a better understanding of the type 2 secretion process. The T2SS might be seen as one of the model system to understand membrane translocation in general and translocation across the gram-negative bacterial cell envelope in particular.

Project coordination

Romé VOULHOUX (Organisme de recherche)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


Help of the ANR 195,500 euros
Beginning and duration of the scientific project: - 48 Months

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