Date of Award

1-1-2020

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biological Sciences

Content Description

1 online resource (xi, 141 pages) : color illustrations.

Dissertation/Thesis Chair

Paul F Agris

Committee Members

Alexander Shekhtman, Douglas Kitchen, Cara Pager, Pan Li

Keywords

Antibiotic, Antibiotic resistance, Drug development, Gram-positive, RNA, T-box, Gram-positive bacteria, Antibiotics, Genetic transcription, Transcription factors, Drug resistance in microorganisms

Subject Categories

Biochemistry | Biology | Microbiology

Abstract

Bacterial infections and the rise of antibiotic resistance, especially multidrug resistant strains, have generated a clear need for discovery of novel therapeutics. Most antibiotics in use today are derivatives of previous antibiotics to which resistance mechanisms already exist, and traditionally they have a single target: either a protein or rRNA. Gram-positive bacteria regulate the expression of several essential genes or operons using a mechanism called the T-box. The T-box is a structurally conserved riboswitch-like gene regulator in the 5’-untranslated region (UTR) of numerous essential genes of Gram-positive bacteria. T-boxes are stabilized by cognate, unacylated tRNA ligands, allowing the formation of an anti-terminator hairpin in the mRNA that enables transcription of the gene. In the absence of an unacylated cognate tRNA, transcription is halted due to the formation of a thermodynamically more stable terminator hairpin. Since T-boxes regulate multiple genes or operons, targeting the T-box mechanism allows for simultaneous inhibition of several genes, making resistance formation unlikely. PKZ18 targets the site of the codon/anticodon interaction of the conserved Stem I Specifier Loop and reduces T-box controlled gene expression. Here we show that novel analogs of PKZ18 have improved minimum inhibitory concentrations (MICs), bactericidal effects against methicillin resistant Staphylococcus aureus (MRSA), and increased efficacy in nutrient limiting conditions. The analogs also have reduced cytotoxicity against eukaryotic cells compared to PKZ18. The PKZ18 analogs acted synergistically with aminoglycosides to significantly enhance the efficacy of the analogs and aminoglycosides, further increasing their therapeutic windows. RNA sequencing showed that the analog PKZ18-22 affects expression of most T-box controlled genes, but not other 5’UTR regulated genes in MRSA. Very low levels of resistance further support the existence of multiple T-box targets for PKZ18 analogs in the cell. Together the multiple targets, low resistance, and synergy make PKZ18 analogs promising drugs for development and future clinical applications.

Download

Share

COinS