DgcP: a study of enzymatic activity, structure and integration with type IV pilus.

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen, afflicting vulnerable individuals, but not healthy ones. Studies about the organism are extremely important because this gamma-proteobacterium is responsible for 20% of nosocomial infections and is frequently associated with pneumonia in cystic fibrosis patients and in patients on assisted ventilation, besides its high intrinsic antibiotic resistance.P. aeruginosa is able to switch between planktonic and sessile life styles, each one with its respective gene regulation and signaling pathways. The second messenger c-di-GMP is the main mediator responsible for the transition between these states , so that high levels of this molecule promote the expression of genes and other mechanisms characteristic of a sessile lifestyle that also characterize chronic infections. The sessile lifestyle is marked by biofilm formation that starts when bacteria adhere to an abiotic surface or host cells, starting their cell differentiation and forming small clusters, the microcolonies. Following differentiation, bacteria lose their flagella and start secreting an extracellular matrix formed predominantly by polysaccharides, extracellular DNA and certain proteins, therefore forming the biofilm. Adhesion and twitching motility at the beginning of the process is mediated by the type IV pilus (T4P), a complex machinery composed of dozens of proteins, all fundamental for the adhesion and movement of the cells. Among these proteins is FimV, which has a transmembrane region and domains in both periplasmic and cytoplasmic regions. Previous studies of our group disclosed that the cytoplasmic portion of FimV interacts and increases the activity of the diguanylate cyclase DgcP, one of the enzymes responsible for the production of c-di-GMP. Despite being a potential regulator of cell differentiation in P. aeruginosa, little is known about the biochemistry of this protein. This project aims to investigate the enzymatic and structural properties of the DgcP protein, as well as to identify the nature of its interaction withFimV. Initially, both proteins will be produced in Escherichia coli and purified. Purified, DgcP will be submitted to enzymatic activity assays alone or in the presence of FimV. The structural analyses will be carried out initially in silico using appropriate software for the analysis of primary structure in search of homology with other proteins already characterized and the prediction of tertiary structures. If the characterization of the interaction between the proteins results in consistent data, the project can be extended with experimental structure determination and identification of domains related to the interaction DgcP/FimV.