Cellular localization of Pseudomonas aeruginosa proteins related to the second messenger c-di-GMP and the type IV pilus

Abstract

One of the problems of healthcare globally is the extreme difficulty of containing the spread of multidrug-resistant pathogens, resulting in the hard-to-treat infections, especially in hospital settings. In this scenario, one of the main pathogens that infect immunocompromised and burn patients, individuals in assisted ventilation or with cystic fibrosis refers to the proteobacterium Pseudomonas aeruginosa, which is a highly resistant opportunistic pathogen adaptable to a variety of environmental conditions. Thus, a better understanding of the mechanisms and proteins linked to bacterial growth in the presence of drugs can indicate new targets for the development of more effective therapeutic alternatives. Biofilms are multicellular bacterial structures associated with the antibiotic resistance, and its regulation occurs by the second messenger c-di-GMP, so that high intracellular concentrations of this molecule determine the formation of the biofilm and, thus, proteins related to its metabolism can be potential targets for the development of new treatments. In addition, the intracellular levels of c-di-GMP are also related to the lifestyle (sessile or planktonic) adopted by the bacterium. The diguanilate cyclase DgcP is one of the main proteins responsible for the synthesis of this second messenger and previous studies have determined its cell polar location and its interaction with FimV, an essential protein for the assembly of pilus type IV (T4P), responsible for twitching motility and the initial stages of biofilm formation. Thus, this project aims to analyse the interactions and cellular localization of DgcP with two proteins that are also part of the T4P regulation and bind c-di-GMP. FimX with bound c-di-GMP binds to ATPase PilB, the ATPase that promotes pilus extension, and FimW is necessary for the initial adhesion of the cell to a surface, since it activates the function of T4P after binding c-di-GMP. Also, to more accurately characterize the role of DgcP and its relationship with T4P, the sensitivity of P. aeruginosa mutants to ZC01 bacteriophage infection will be evaluated. Another protein responsible for the metabolism of the second messenger c-di-GMP and which has potential as a possible target for alternative treatments isphosphodiesterase DipA, which is related to the decrease of c-di-GMP levels and the dispersion of the biofilm, and its role in the asymmetric division of the bacterium will be verified by analyzing the relative localization relative to DgcP. To achieve the goals of this project, genetic, biochemical, and molecular biology approaches will be combined, both with methodologies already established by our group, as well as with collaborations with important groups nationally and internationally.(AU)