Deciphering the route for Pel exopolysaccharide secretion in Pseudomonas aeruginosa: From synthesis to export

• 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

Pseudomonas aeruginosa is an opportunistic pathogen and a life-threatening risk factor for immunocompromised patients. Its infamous virulence and antibiotic resistance are enhanced upon formation of durable biofilms, which allow the bacterium withstanding mechanical stress and desiccation, and promote colonization of new areas. The biofilm stability is mediated by its composite matrix built of secreted exopolysaccharides (EPS) alginate, Psl and Pel complexed with extracellular DNA and structural proteins. Despite their biomedical relevance, the molecular insights on the EPS secretion machineries are scarce, and neither their architecture nor the functional dynamics are known. Understanding the secretion mechanisms of the biofilm-forming EPS would facilitate new approaches to combat the pathogen spreading, thus being of the highest biomedical importance. We set out to elucidate the biogenesis pathway of the Pel EPS used by P. aeruginosa at early stages of the biofilm formation. During the first funding period we have determined the high-resolution structure of the outer membrane export complex PelBC in lipid nanodiscs and made major advances in studying the multi-subunit machinery PelDEFG required for the EPS synthesis and translocation across the inner membrane. As an essential step forward, we now aim to elucidate the molecular mechanisms of the secretion system and reconstruct the route of the Pel EPS across the cell envelope. Towards those overarching goals, we propose to (i) visualize the Pel complexes in different states by cryo-electron microscopy to reconstruct their functional dynamics and trace the EPS; (ii) establish in vitro assays of the Pel EPS synthesis and translocation to study the pathway under tailored and well-controlled conditions; (iii) use biophysical tools to probe the interactions of the Pel EPS with the components of the secretion machinery and examine the role of electrostatic interactions in translocation; and (iv) investigate coupling of the Pel complexes in the inner and outer membranes, and the role of the nascent EPS in the assembly. The proposed biochemical, biophysical and structural analysis of the Pel machinery, complemented by molecular dynamics simulations and in vivo studies of the P. aeruginosa biofilm formation envisioned within collaborations should result in the comprehensive understanding of the pathogenicity-relevant and wide-spread EPS secretion system, thus being of the high value for microbiology and molecular medicine.

• DFG Programme: Research Grants