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Heterologous production of transmembrane complexes of the pel operon from Pseudomonas aeruginosa for functional and structural studies

Grant number: 14/21864-6
Support type:Scholarships abroad - Research Internship - Master's degree
Effective date (Start): November 30, 2014
Effective date (End): May 29, 2015
Field of knowledge:Biological Sciences - Biophysics - Molecular Biophysics
Principal Investigator:Marcos Vicente de Albuquerque Salles Navarro
Grantee:Naiara Utimura Torres
Supervisor abroad: Rémi Fronzes
Home Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
Local de pesquisa : Institut Pasteur, France  
Associated to the scholarship:12/25217-0 - Structural and functional studies of PelF, a glycosyl transferase responsible for extracellular matrix production in biofilms from Pseudomonas aeruginosa and Xanthomonas axonopodis, BP.MS

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen capable to adapt and survive on a wide range of different environments. This adaptation ability is frequently associated with the formation of a sessile macrocolony embedded in a self-produced matrix, called biofilm. In Pseudomonas aeruginosa PA14, the major component of extracellular biofilm matrix is the PEL polysaccharide, a polymer produced by seven proteins of the pel operon (PelA-G). The proteins encoded by this gene cluster are predicted to form a transmembrane machinery responsible for the synthesis and translocation of PEL. This process is controlled by c-di-GMP, which interacts with the degenerate GGDEF domain of PelD, causing a large inter-domain reorganization. PelD cytoplasmic conformational change is transmitted to its transmembrane domain and probably triggers further structural changes in other members of the Pel complex. Concomitantly, the cytoplasmic glycosyltransferase encoded in the operon (PelF) must associate with the c-di-GMP-activated Pel complex in order to initiate the secretion of the polysaccharide. The aforementioned hypothesis is based on available experimental evidence performed only on individual soluble proteins or constructs (PelA, PelC, PelD and PelF), precise molecular mechanisms of PEL secretion and the structural organization of the Pel complex remain completely unknown. Thereby, this work aims the heterologous production of transmembrane Pel complexes for structural and functional studies, focusing on the entire Pel complex and the sub-complexes predicted to locate on the inner (PelDEG) or outer membrane (PelBC). The investigation presented in this work will help to elucidate molecular mechanisms that guide biofilm formation in this important human pathogen. (AU)