Ricerc@Sapienza

The opportunistic bacterium Pseudomonas aeruginosa causes serious infections in immunocompromised patients. One of the most worrisome characteristics of P. aeruginosa is its low antibiotic susceptibility and its ability to develop acquired resistance, thus the discovery of new antibacterial drugs is of paramount importance. In this context, a novel approach is to target bacterial ribosome maturation. Ribosome biogenesis is a complex and not fully understood process requiring the separate maturation the 30S and 50S subunits. Correct assembly of subunits proceeds through different sequential steps that need to be strictly controlled by the ribosomal assembly factors. Ribosome small subunit-dependent GTPase (RsgA) is a late-stage ribosome biogenesis factor involved in the 30S subunit maturation and has been recently identified in B. subtilis as one of the target of (p)ppGpp, a second messenger responsible for the bacterial switching to stringent response during stress conditions. Altogether, this information poses RsgA from P. aeruginosa (Pa-RsgA) a promising target for P. aeruginosa infection treatment.
This project aims at characterizing Pa-RsgA from both a structural and functional point of view. The recombinant protein has been already expressed and purified in our laboratory in the GDP-bound form. A protocol to obtain the nucleotide-free form of the protein was also set up. Specific goals of the project are: i) characterization of the thermodynamic properties of the protein; ii) determination of the GDP/GTP binding parameters by equilibrium and kinetic experiments; iii) analysis of the catalytic mechanism; iv) structural characterization by X-ray crystallography. An additional interesting goal will be the characterization of the RsgA-(p)ppGpp interaction recently hypothesized in gram-positive bacteria. Obtained results will pave the way to the development of a potential drug target for P. aeruginosa infection treatment.