Structure and dynamics of the Pel exopolysaccharide secretion machinery of Pseudomonas aeruginosa

• Applicant: Professor Dr. Alexej A. Kedrov
• Subject Area: Structural Biology ; Biochemistry ; Biophysics ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 468785194

Biofilms of a critical human pathogen, Pseudomonas aeruginosa, are essential for colonization of host tissues and abiotic surfaces in medical institutions, as well as for resistance to antibiotics. Understanding the mechanisms of biofilm formation is of the highest biomedical importance. The assembly of the durable biofilm matrix relies on crosslinking of secreted exopolysaccharides, DNA and proteins. In P. aeruginosa, secretion of exopolysaccharides, such as alginate, Pel and Psl is an essential step for the matrix assembly, but for neither system the molecular mechanisms are known. Here, we set out to elucidate the architecture and dynamics of the multi-subunit machinery for Pel synthesis and secretion, PelDEG. A multi-disciplinary approach based on biochemical, biophysical and bioinformatics tools will be used (a) to determine the stoichiometry of the subunits within the complex and to identify the transmembrane conduit for Pel translocation; (b) to characterize the interactions of the secretion complex PelDEG and the cytoplasmic Pel synthase, PelF; (c) to decipher the regulatory effect of cyclic-di-GMP on Pel synthesis and translocation.Site-specific mutagenesis combined with the in vivo analysis of the biofilm architecture will serve to verify the results from in vitro analysis. The secretion complex PelDEG, as well as its individual subunits, will be used for structural analysis, where the both mid-resolution (small-angle scattering) and high-resolution approaches (cryo-electron microscopy, X-ray crystallography) will be combined. The integrated view on the secretory machinery gained along the project will be of fundamental importance for understanding Pel secretion in pathogens and will also facilitate understanding of homologous and more sophisticated systems.

• DFG Programme: Research Grants