Cloning and Characterization of the Tyrosyl‐S and Tyrosyl‐Z tRNA Synthetases from Pseudomonas aeruginosa

Abstract

IntroductionPseudomonas aeruginosa, a Gram‐negative opportunistic pathogen, is a leading cause of nosocomial infections. The ability of this bacteria to form resilient biofilms on implanted medical devices results in high mortality rates. Patients with cystic fibrosis (CF) are especially at risk as P. aeruginosa colonizes the lungs of these patients forming biofilms which leads to chronic infections and is a leading cause of death. Aminoacyl‐tRNA synthetases (aaRSs) catalyze the covalent attachment of amino acids to their cognate tRNAs. These enzymes are essential for protein synthesis in bacteria and serve as validated targets for the development of new anti‐infectives. Unlike in other bacteria, the P. aeruginosa genome contains two genes (tyrS and tyrZ) which encode two distinct TyrRS enzymes, which are very different at the amino acid level containing only 41.1/26.8 percent similar/conserved residues.Methods and ResultsThe gene encoding P. aeruginosa TyrRS‐Z was cloned and overexpressed in E. coli cells, and purified to homogeneity. Amplification of tyrS by PCR was unsuccessful, therefore the gene sequence was optimized for codon expression and synthetically synthesized. Cloning of the optimized gene resulted in expression of large amounts of TyrRS‐S. Both forms of TyrRS were active in aminoacylation. The kinetic parameters for the interaction of these enzymes with their three substrates (tRNA, ATP, tyrosine) were determined using timed aminoacylation and ATP:PPi exchange assays. Both forms of TyrRS were developed into screening platforms using scintillation proximity assay (SPA) technology. Using this assay, a synthetic compound library from ChemDiv, Inc. containing over 2000 distinct compounds was screened to detect compounds with the ability to inhibit the function of TyrRS. A number of inhibitory compounds were confirmed and are currently being characterized for the ability to inhibit both the enzymatic activity (IC50) of both forms of TyrRS and growth (MIC) of a panel of problematic pathogens in culture. They are also being analyzed to determine the potential mechanism of inhibition relative to substrate binding, global mode of action of inhibition of growing cultures of bacteria, as well as toxicity to human cell cultures.ConclusionTyrRS‐S and TyrRS‐Z from P. aeruginosa were cloned, expressed, characterized and used for development of screening platforms to identify compounds that have the potential for development as antibacterial agents against drug resistant pathogenic organisms.Support or Funding InformationNational Institutes of Health (grant number: 1SC3GM098173)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.