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.
P. aeruginosa (PA) is a major gram-negative pathogen causing infection, especially in immunocompromised patients and it is often responsible for nosocomial infections such as pneumonia and urinary tract infections. In addition, PA infections are very common, and even lethal, in people with cystic fibrosis. PA infections are difficult to treat since this bacterium is particularly resistant to many available antibiotic therapies. PA resistance derives both from its highly versatile metabolism and its ability to form biofilms.
In the current situation of increasing antibiotic resistance, the discovery of new targets of antibiotic action is mandatory [1] and represents a critical strategy in combating multi-drug resistance in bacteria [2]. In this context, a new emerging approach is to target ribosomal assembly. Several proteins have been identified as crucial in the ribosomal biogenesis process. Among them, some specific GTPases are small, soluble, and amenable to structural analysis, such as X-ray crystallography, and to cryo-electron microscopy to determine their interaction with the ribosome. This offers hope that structure-based drug design might guide the development of specific GTPases inhibitors. Interstingly, Some of these GTPases are involved in multiple processes in the cell, thus, the pleiotropic effects of their inhibition may offer multiple ways to inhibit cell growth through a single target, an attractive feature that might limit the emergence of resistance [1].
We therefore decided to focus on the biochemical and structural characterization of RsgA from PA (Pa-RsgA).
To date, three crystal structures of RsgA from different pathogenes have been solved [3,4,5] showing a great similarity in terms of secondary and tertiary structures. Our aim is to obtain the structure of Pa-RsgA, not yet available, and to investigate the conformational changes that occur in the Pa-RsgA structure upon GTP hydrolysis.
Moreover, we want to investigate Pa-RsgA-alarmone interaction, a novel aspect that is attracting much interest since (p)ppGpp is critical for regulating bacterial adaptative response and virulence. (p)ppGpp is required for biofilm formation and might also decrease the sensitivity of biofilm to the treatments of antibiotics. Moreover, (p)ppGpp plays a role in regulating the quinolone signaling circuit in P. aeruginosa, as it modulates the expression of different genes involved in virulence factor production, biofilm formation and antimicrobial defenses [6,7]. An interesting hypothesis is that (p)ppGpp can interact with GTPases involved in ribosomal assembly to inhibit the association of the 50S and 30S subunits. In this way, these GTPases control ribosome assembly and protein synthesis.
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