Access Type
Open Access Dissertation
Date of Award
January 2021
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Chemistry
First Advisor
Christine Chow
Abstract
The threat of bacterial resistance throughout the world has created tremendous challenges to clinicians. Some bacterial species have acquired resistance to not only single antibiotics, but also to multiple compounds, which is called multidrug resistance. Since the early discovery of resistance to penicillin, scientists have been developing new drugs to combat antibiotic resistance. In some cases, it took less than a decade to develop resistance to a new antibiotic. The World Health Organization (WHO) reported several deadly multi-drug-resistant bacteria, some of which cause life-threating illnesses. Each year more than 35,000 people in the United States die from antibiotic-resistant bacterial infections. The number of deaths caused by antibiotic resistance is increasing, making this a significant health challenge. Antimicrobial peptides have promise as alternative therapeutics for treating resistant pathogenic bacteria. Naturally occurring proline-rich antimicrobial peptides (PrAMPs) have shown activity against several resistant bacterial strains. Replacement of amino acid residues at different positions in the PrAMPs increases their inhibitory activity by up to 100-fold against multidrug-resistant species. In addition, by incorporating unnatural amino acids into the PrAMPs, they have increased serum stability. These studies indicate that positional variances offer an opportunity to produce peptide derivatives with improved antimicrobial activities. However, the standard solid-phase peptide synthesis and in vitro testing methods are both time and resource intensive. In this study, we used an in vivo expression system in E. coli DH5α in which the corresponding DNA sequence for the PrAMP was placed under an inducible promoter pBAD. Upon addition of arabinose, pBAD is activated and peptide expression is turned on, the in vivo expressed peptides then bind to target sites within the cellular environment. To determine the antimicrobial properties of in vivo expressed peptides, we performed bacterial growth in the presence and absence of arabinose and compared the growth inhibition. A footprinting assay with dimethyl sulfate (DMS) was performed in vivo to confirm peptide binding to the intracellular rRNA target site. We first studied oncocin, a 19mer PrAMP, and its variants to determine the antimicrobial properties within the cellular environment. We performed deletion mutations from both the C and N termini, as well as conservative and null mutations in the 19mer oncocin. From the deletion studies, we showed that the first 15 residues on the N terminus are required for the antibacterial activity of oncocin. Both arginine and lysine scans revealed that charged residues are well tolerated at varying amino acid positions of the 15mer. Double and triple cationic amino acid substitutions in the 19mer oncocin led to peptides with slightly improved antimicrobial activities. DMS chemical probing was used to demonstrate that the 19mer, 16mer, and several cationic mutants of oncocin bind to the peptidyl transferase center (PTC) of the ribosome, as well as neighboring regions. We generated a library of 19mer oncocin variants using the same in vivo inducible expression plasmid system. Monosubstituted derivatives were generated by replacing each amino acid with all 20 natural amino acids in E. coli DH5α. The production of all peptide variants and the effects on growth were monitored concurrently. We also studied the antibacterial properties of other PrAMPs, such as apidaecin (api137), pyrrhocoricin, metalnikowin, bactenecin 7 (bac7), tur1A, and riptocin, Pyrrhocoricin, metalnikowin, and bac7 were shown to be potent inhibitors within the cellular sytem. DMS chemical probing results reveal that all of these PrAMPs have similar interactions in the PTC region of the ribosome. We also developed a laboratory teaching tool for undergraduate biochemistry students using our in vivo peptide expression approach. Students learned basic molecular biology techniques while carrying out an original research to understand the length and sequence dependence of PrAMP antibacterial activity. Overall, our results from site-directed mutation studies, small library generation and selection of oncocin variants, and studies with other PrAMPs suggest that the approach of in vivo expression can be used to identify peptides with improved antibacterial activities.
Recommended Citation
Islam, Rabiul, "An In Vivo Inducible Expression System To Determine Antibacterial Activity Of Peptides" (2021). Wayne State University Dissertations. 3541.
https://digitalcommons.wayne.edu/oa_dissertations/3541