Search for interaction partners of GGDEF/EAL proteins involved in antibiotic adaptation

Abstract

Following the genomic era, a large number of genes coding for enzymes predicted to synthesize and degrade 3'-5'-cyclic diguanylic acid (c-di-GMP) was found in most bacterial genomes and this dinucleotide emerged as an important intracellular signal molecule. This molecule has been related to pathogenicity and adaptation of various bacteria mainly by regulates the bacterial behavior, coordinating the expression of genes involved in virulence, motility and biofilm formation. Diverse molecular mechanisms have been described as targets for c-di-GMP, however several questions remain to be addressed. Pseudomonas aeruginosa is a versatile gamma-proteobacterium that behaves as an opportunistic pathogen to a broad range of hosts, being a major cause of chronic infections in patients with cystic fibrosis. The genome of P. aeruginosa strain PA14 presents forty genes coding for proteins associated with c-di-GMP metabolism, highlighting a broad regulatory role of this di-nucleotide in this bacterial species. An association between high levels of c-di-GMP and antibiotic resistance is largely assumed, since high c-di-GMP upregulates biofilm formation and the biofilm mode of growth leads to enhanced antibiotic resistance; however, a clear understanding of this correlation is missing. Recently, we have found a link between c-di-GMP levels and antibiotic adaptation uncoupled of biofilm formation in Pseudomonas aeruginosa PA14, but the exactly mechanism of this phenomenon is still to be revealed. Preliminary results have shown that strains with mutations in c-di-GMP-related proteins present growth distinct from the wild type in sub inhibitory antibiotics concentrations. These proteins will be used as baits in a bacterial two-hybrd screening, searching for interaction partners that participate in those c-di-GMP dependent pathways. The interactions will be further validated by coimmunoprecipitation assays. Thus, this project will contribute for a deeper understanding of how c-di-GMP intracellular concentrations modulates antibiotic fitness in bacteria. (AU)