Interactions of PA14_04420 with proteins involved in cell growth and envelope synthesis in Pseudomonas aeruginosa

Abstract

The regulation of bacterial infectious processes, such as biofilm formation, is modulated by the intracellular levels of the second messenger cyclic bis-(3',5')-diguanosine monophosphate (c-di-GMP). The synthesis and degradation of c-di-GMP are coordinated by the antagonistic enzymatic activities of diguanylate cyclases (DGCs), which contain the GGDEF domain, and phosphodiesterases (PDEs), which harbor the EAL or HD-GYP domains. About 40 proteins with such domains are found in the opportunistic pathogen Pseudomonas aeruginosa, however the function of many of these proteins remains unknown. Among these is the protein PA14_04420, which has GGDEF and PAS sensory domains predicted by its sequence. Previous experiments in our laboratory showed a putative interaction of PA14_04420 with proteins involved in cell division, cell wall formation and lipopolysaccharide (LPS) synthesis. Therefore, this project will investigate the role of the PA14_04420 protein in bacterial physiology and its possible implication in cell division in P. aeruginosa. For this, potential interaction partners will be confirmed by Bacterial Adenylate Cyclase-based Two Hybrid (BACTH) assays. Comparative phenotypic analyzes between wild type, mutant and PA14_04420 overexpression strains, under different conditions, will be evaluated for differences in motility, biofilm formation, generation time, adaptation, tolerance and/or resistance to antibiotics. Assays to define the cellular localization of PA14_04420 and co-localization with interacting partners will be performed. Furthermore, the diguanylate cyclase catalytic activity will be examined by measuring intracellular levels of c-diGMP and enzymatic activity in vitro. Thus, elucidating the role of PA14_04420 may imply a better understanding of one of the fundamental bacterial processes and may indicate a new target for the development of therapeutic strategies to fight bacterial infections.