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Comprehensive Mutational Analysis of the Lasso Peptide Klebsidin
Ethan Hills1, Tyler J Woodward2, Stanley Fields1,3
1Department of Genome Sciences, University of Washington, Seattle, Washington 98195, United States.
Abstract:
Antibiotic resistance is a growing threat to public health, making the development of antibiotics of critical importance. One promising class of potential new antibiotics are ribosomally synthesized and post-translationally modified peptides (RiPPs), which include klebsidin, a lasso peptide from Klebsiella pneumoniae that inhibits certain bacterial RNA polymerases. We develop a high-throughput assay based on growth inhibition of Escherichia coli to analyze the mutational tolerance of klebsidin. We transform a library of klebsidin variants into E. coli and use next-generation DNA sequencing to count the frequency of each variant before and after its expression, thereby generating functional scores for 320 of 361 single amino acid changes. We identify multiple positions in the macrocyclic ring and the C-terminal tail region of klebsidin that are intolerant to mutation, as well as positions in the loop region that are highly tolerant to mutation. Characterization of selected peptide variants scored as active reveals that each adopts a threaded lasso conformation; active loop variants applied extracellularly as peptides slow the growth of E. coli and K. pneumoniae. We generate an E. coli strain with a mutation in RNA polymerase that confers resistance to klebsidin and similarly carry out a selection with the klebsidin library. We identify a single variant, klebsidin F9Y, that maintains activity against the resistant E. coli when expressed intracellularly. This finding supports the utility of this method and suggests that comprehensive mutational analysis of lasso peptides can identify unique and potentially improved variants.
Insights
Developing new antibiotics is crucial due to rising antibiotic resistance. This study analyzed klebsidin variants, identifying key mutations for potential new antibiotic development against resistant bacteria.
Area of Science:
- Microbiology
- Biochemistry
- Drug Discovery
Background:
- Antibiotic resistance poses a significant global health threat, necessitating the discovery of novel antimicrobial agents.
- Ribosomally synthesized and post-translationally modified peptides (RiPPs), such as the lasso peptide klebsidin, represent a promising class of antibiotics.
- Klebsidin inhibits bacterial RNA polymerases, making it a target for antibiotic development.
Purpose of the Study:
- To develop a high-throughput assay for analyzing the mutational tolerance of klebsidin.
- To identify specific amino acid positions within klebsidin that are critical for its activity.
- To discover novel klebsidin variants with enhanced or altered activity profiles, including resistance to bacterial resistance mechanisms.
Main Methods:
- A high-throughput growth inhibition assay using *Escherichia coli* was established.
- A comprehensive library of klebsidin variants was generated and analyzed using next-generation DNA sequencing to determine functional scores for single amino acid changes.
- A resistant *E. coli* strain was engineered to select for klebsidin variants active against resistant bacterial RNA polymerase.
Main Results:
- Mutational analysis revealed specific regions in klebsidin's macrocyclic ring and C-terminal tail that are intolerant to mutation, while the loop region shows high tolerance.
- Characterization confirmed that active klebsidin variants maintain a threaded lasso conformation.
- Extracellular application of active loop variants inhibited the growth of *E. coli* and *Klebsiella pneumoniae*.
- A specific variant, klebsidin F9Y, demonstrated sustained intracellular activity against a klebsidin-resistant *E. coli* strain.
Conclusions:
- Comprehensive mutational analysis is an effective strategy for understanding structure-activity relationships in lasso peptides.
- The developed high-throughput assay facilitates the identification of functionally important residues and potentially improved antibiotic variants.
- The discovery of klebsidin F9Y highlights the potential for developing novel RiPP-based antibiotics effective against resistant bacterial strains.
