EVALUATION OF STAPHYLOCOCCUS AURUES RNPA PROTEIN AS AN ANTIBACTERIAL TARGET

2019-08-13T19:39:49Z (GMT) by Lisha Ha

Staphylococcus aureus (S. aureus) is a Gram-positive pathogen that causes a wide range of infections in both hospitals and communities, of which the total mortality rate is higher than AIDS, tuberculosis, and viral hepatitis combined. The drug resistant S. aureus is a member of the “ESKAPE” pathogens that require immediate and sustained actions of novel method to combat. However, the current antimicrobial development against S. aureus is in stagnation, which underscores the urgent need for novel antimicrobial scaffolds and targets. S. aureus Ribonuclease P protein (RnpA) is an essential protein that plays important roles in both tRNA maturation and mRNA degradation pathways. The goal of this research was to evaluate RnpA as an antimicrobial target using biophysical methods. The crystal structures of wild-type RnpA in three different constructs were determined, among which the tag-free RnpA construct has a structural model of 2.0 Å resolution and Rcrys/Rfree= 0.214/0.234, and its crystals are reproducible. This crystal structure of tag-free S. aureus RnpA shows a globular representation with key structural motifs, including the “RNR” Ribonuclease P RNA binding region and a substrate binding central cleft, which shares high similarity to previously solved RnpA structures from other species despite of their low sequence identity. Meanwhile, in a screen of S. aureus RnpA mutants performed by our collaborator, RnpAP89A was found lacking the mRNA degradation activity while retaining the tRNA maturation function, and causing defects in cell viability. We therefore studied this mutant using differential scanning fluorimetry, crystallography, and circular dichroism. It was shown that RnpAP89A is thermally less stable than wild-type RnpA by ~2.0 ˚C, but no secondary structural or 3D conformational differences were found between the two proteins. Although the mutant RnpAP89A requires further characterization, the results of the studies in this thesis have begun to shed light on the relatively new role of S. aureus RnpA in mRNA degradation, and will serve as useful tools in future structure-based drug discovery for multi-drug resistant S. aureus treatment.