Project Title: New cofactor-independent dioxygenases to interfere with quorum sensing and thus virulence of Pseudomonas aeruginosa

Supervisor: Prof. Susanne Fetzner, University of Münster, Germany

Objectives: In order to set up a toolbox of dioxygenases for inactivation of the virulence-associated Pseudomonas aeruginosa signal molecule ‘PQS’, the natural genetic diversity of PQS dioxygenase genes will be exploited. The enzymes will be functionally characterized, especially with respect to affinity for PQS, stability, and catalytic efficiency. If applicable, enzyme engineering approaches will be applied to improve their kinetic parameters, thermostability, and stability towards proteases. For the most promising PQS dioxygenases, their potential to interfere with virulence factor production of P. aeruginosa will be assessed.

Methodology: The work will involve various techniques in microbiology, molecular biology, biochemistry and biophysics. For enzyme production and engineering, Escherichia coli will be used as host. Enzyme characterization will include: binding studies (fluorimetry), kinetic analyses (spectrophotometric assays for steady-state kinetics) and stability analyses (thermostability). Part of the enzyme characterization will be done in collaboration with Prof. Gerrit Poelarends. Homology models and, if applicable, crystal structures will be determined in collaboration with the group of Prof. Andrea Mattevi. For evaluating the potential of the enzymes to interfere with virulence, their effect on virulence factor production by P. aeruginosa cultures will be analyzed using physiological and analytical methods.

Expected Results: (i) Definition of a subfamily of cofactor-independent dioxygenases active toward the quorum sensing signal molecule PQS; (ii) identification of PQS dioxygenases efficient in quorum sensing interference and virulence attenuation.