Poster abstracts

Poster number 25 submitted by Rabiul Islam

Expression and in vivo characterization of a proline-rich antimicrobial peptide that targets 23S ribosomal RNA

Rabiul Islam (Department of Chemistry Wayne State University), Christine Chow (Department of Chemistry Wayne State University)

Abstract:
The development of bacterial resistance to antibiotics is a major challenge for clinicians. Naturally occurring proline-rich antimicrobial peptides (PrAMPs) have shown significant activity against pathogenic bacteria. One example is the 19-mer peptide oncocin, which has promising antibiotic action against multidrug-resistant species. Synthesis of oncocin using standard solid-phase synthesis protocols and in vitro testing are both time and resource intensive. In this study, we used an inducible expression system to generate oncocin and measure its antibiotic activity. We employed non-template PCR, cloning, and transformation, then the transformed cells were isolated and screened for antimicrobial activity upon induction of peptide expression with arabinose. In a similar manner, truncated versions of oncocin were produced to determine the contributions of the N- and C-termini to antibiotic activity upon in vivo expression. Dimethyl sulfate (DMS) was used to map the oncocin binding sites on the ribosome under cellular conditions. Total RNA was isolated from DMS-treated induced cells and reverse transcription was performed with radiolabeled primers. The DMS stop sites on rRNA were visualized by gel electrophoresis and quantified by densitometry. Our results reveal that oncocin binds at the peptidyl transferase center of 23S ribosomal RNA, consistent with previous high resolution structure studies. Overall, these studies show the promise of using in vivo expression to fine-tune antimicrobial peptides for improved activity along with mapping of the target rRNA binding sites in cellular milieu.

References:
Nisansala S. Muthunayake, Rabiul Islam, Ellen D. Inutan, Wesley Colangelo, Sarah Trimpin, Philip R. Cunningham, and Christine S. Chow, Biochemistry 2020, 59(36): 3380-3391

Keywords: PrAMPs, DMS probing, bacterial resistance