Open Access Dissertation
Date of Award
Christine S. Chow
The development of short peptides that specifically bind to higher-order structures of ribosomal RNA is one promising way to address the problem of antibiotic resistance. However, the poor correlation between in vitro and in vivo activities of these peptides is one of the major questions in antibiotic peptide research. Therefore, one of the main objectives of this dissertation work was to utilize a plasmid-based system to in vivo express ribosome-targeting peptides and study their direct inhibitory effects on bacteria. A specific plasmid system was optimized to in vivo express oncocin, a prolin-rich antimicrobial peptide and its variants in bacteria. Our data showed that the in vivo-expressed peptide completely inhibited bacterial growth and displayed bactericidal activity. The in vivo dimethyl sulfate (DMS) footprinting data revealed interactions of oncocin with the PTC region of the bacterial ribosome. The optimized-plasmid system was utilized to in vivo express short peptides that are known to target the helix 69 (H69) region of the ribosome. In the first approach, peptides were expressed as GFP-fusion proteins, and in the second approach they were expressed as free peptides. In both systems, we found that the NQAANHQ peptide had slightly better inhibition compared to other H69-targeting peptides. Based on in vivo and in vitro data, we consider NQAANHQ as a potential drug lead, but it will need considerable modifications or alterations to improve its activity.
The bactericidal nature of 2-deoxystreptamine aminoglycoside antibiotics is still poorly understood despite decades of clinical use and biochemical studies. Previous work showed that modifications are important for efficient binding of aminoglycosides to H69. However, the effects of modifications on the bactericidal activity of aminoglycosides have not been examined. Antibacterial activities of 2-deoxystreptamine aminoglycosides were assessed by performing minimum inhibitory concentration (MIC) studies using wild-type and -deficient bacterial strains. Our data revealed that loss of modifications confered resistance to 4,6-linked-2-DOS aminoglycosides, gentamicin and kanamycin, whereas the effect was not significant with 4,5-linked-2-DOS aminoglycosides, neomycin and paromomycin. However, bacterial strains carrying mutant release factor 2 (RF2, Ala246Thr) showed resistance to neomycin and paromomycin in the -deficient background. The observed results could be a combined effect of loss of s and defective RF2 that perturb the ribosome-drug interactions. Collectively, the information gained from these studies provides deeper insight into the underlying mechanism of action of ribosome-targeting drugs, which is important for the development of unique antibiotics that target the bacterial ribosome at novel sites such as H69.
Muthunayake, Nisansala Sarangi Thilakarathne, "Investigation Of The In Vivo Activity Of Ribosome-Targeting Peptides And Aminoglycosides In Escherichia Coli" (2018). Wayne State University Dissertations. 2052.