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Identification and elucidation of proline-rich antimicrobial peptides with enhanced potency and delivery
Pin-Kuang Lai1, Daniel T Tresnak1, Benjamin J Hackel1
1Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota.
Abstract:
Proline-rich antimicrobial peptides (PrAMPs) kill bacteria via a nonlytic mechanism in which they permeate through the outer membrane, utilize protein-mediated transport across the inner membrane, and target the ribosome to inhibit protein synthesis. We previously reported that substitutions of oncocin ( ) with a pair of cationic residues improved the antimicrobial activity. In this study, we applied the design protocol to three other PrAMPs: apidaecin-1b, pyrrhocoricin, and bactenecin 7(1-16) and found that the substitutions (R4K and I8K/R) for apidaecin-1b improve the activity by twofold (p < .05) against nonpathogenic Escherichia coli. Moreover, the substitutions (L7K/R and R14K) for pyrrhocoricin improve the activity by 2-10-fold (p < .05) against some strains of E. coli and Salmonella Typhimurium. We also performed activity tests against inner membrane protein (SbmA or YgdD) knockout strains. The result is consistent with previous studies that SbmA is the major transporter for apidaecin-1b and pyrrhocoricin derivatives. However, bactenecin 7(1-16) functions independently of these transporters. In addition, several apidaecin-1b derivatives exhibit enhanced activity relative to wild-type only in the absence of SbmA, which is consistent with mutations that enhance transport across the inner membrane. A high performance liquid chromatography-based kinetic assay for cellular association and internalization demonstrates that the selected cationic mutations can improve cellular association in minimal media, but this enhanced association is not required for increased activity, which suggests the importance of inner membrane transport. These functional studies on cationic mutants of PrAMPs advance understanding of potency and mechanism and advance the ability to engineer improved antimicrobials as evidenced by the identification of the pyrrhocoricin mutant (L7R and R14K) with 10-fold elevated potency against pathogenic E. coli.
Insights
Engineered proline-rich antimicrobial peptides (PrAMPs) show enhanced antibacterial activity. Cationic substitutions improved potency by targeting bacterial inner membrane transport and protein synthesis inhibition.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Proline-rich antimicrobial peptides (PrAMPs) possess a nonlytic mechanism of action, involving bacterial membrane permeation and ribosome targeting to inhibit protein synthesis.
- Previous research demonstrated that cationic residue substitutions in oncocin enhance its antimicrobial activity.
- Understanding PrAMP transport and mechanism is crucial for developing novel antimicrobial agents.
Purpose of the Study:
- To investigate the impact of cationic residue substitutions on the antimicrobial activity and mechanism of apidaecin-1b, pyrrhocoricin, and bactenecin 7(1-16).
- To determine the role of specific inner membrane transporters (SbmA/YgdD) in the activity of modified PrAMPs.
- To advance the engineering of potent antimicrobial peptides through structure-activity relationship studies.
Main Methods:
- Site-directed mutagenesis to introduce cationic residues into selected PrAMPs.
- Antimicrobial activity assays against various bacterial strains, including wild-type and inner membrane protein knockout mutants.
- High-performance liquid chromatography (HPLC)-based kinetic assays to assess cellular association and internalization.
Main Results:
- Apidaecin-1b and pyrrhocoricin derivatives with cationic substitutions exhibited improved antimicrobial activity against *Escherichia coli* and *Salmonella Typhimurium*.
- SbmA was confirmed as a major transporter for apidaecin-1b and pyrrhocoricin derivatives, while bactenecin 7(1-16) functioned independently.
- Enhanced cellular association due to mutations did not directly correlate with increased activity, highlighting the importance of inner membrane transport.
Conclusions:
- Cationic substitutions can significantly enhance the potency of PrAMPs like apidaecin-1b and pyrrhocoricin.
- The SbmA transporter plays a critical role in the efficacy of certain PrAMP derivatives.
- These findings provide valuable insights for engineering improved antimicrobial peptides with enhanced efficacy against pathogenic bacteria.
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