Structural and functional studies of c-di-GMP transmembrane receptors involved in virulence and bacterial biofilm formation

Abstract

The formation of bacterial biofilms is a well-known phenomenon, characterized by the formation of a static bacterial community, embedded in an exopolymeric matrix. However, this process has only recently been elucidated at the molecular level, revealing a new signaling molecule, c-di-GMP, as a key regulator of mobility, cell adhesion and exopolysaccharide synthesis. Protein domains catalyzing the synthesis (GGDEF) and degradation (EAL and HD-GYP) of c-di-GMP have been identified in a large number of proteins in almost every bacterial genome sequenced to date. Together with c-di-GMP turnover enzymes, a broad spectrum of c-di-GMP effector proteins has also been identified. These include transcription factors, FleQ and VpsT, Pilz domains, "riboswitches", degenerate EAL and GGEDF domains. The latter class includes the protein LapD from Pseudomonas fluorescens, a transmembrane c-di-GMP receptor essential for biofilm formation under conditions of nutritional stress. LapD is composed of a periplasmic interaction domain (PD), followed by a single transmembrane helix (TM), a HAMP domain and a degenerate dual GGDEF-EAL module in the cytoplasmatic portion. Binding of c-di-GMP to the degenerate EAL domain increases the affinity of PD for the periplasmic protease LapG, preventing the degradation of the surface adhesin LapA, thus stabilizing the biofilm. Studies have partially demonstrated LapD molecular mechanism, however a series of functional characteristics of this sub-family of proteins with the architecture PD-TM-HAMP-GGEDF-EAL still need further elucidation. Accordingly, this project aims the structural determination and biophysical studies of conformational changes of the proteins PA14_45930 from P. aeruginosa (homologous to LapD) and STM3615 from Salmonella enterica, a protein with the same architecture of LapD that probably is involved in cell invasion and virulence. Using mainly X-ray crystallography and pulsed electron paramagnetic resonance, we expect to elucidate the molecular mechanisms of these proteins in signal transduction involved in biofilm formation and virulence. Given the absence of c-di-GMP in eukaryotes, the signaling pathways mediated by this molecule represents a great opportunity for the development of new therapies against chronic infections, and the study proposed in this project will increase our understanding of the molecular basis of virulence and biofilm formation in bacteria.