TY - JOUR
T1 - Comprehensive Mutational Analysis of the Lasso Peptide Klebsidin
AU - Hills, Ethan
AU - Woodward, Tyler J.
AU - Fields, Stanley
AU - Brandsen, Benjamin M.
N1 - Funding Information:
This work was supported by NIH grants P41GM103533 (to S.F.), F32GM122202 (to B.M.B.), and 5P20GM103427 (sub-award to B.M.B.). E.H. acknowledges support from the University of Washington Levinson fellowship, and T.J.W. acknowledges support from Creighton University Center for Undergraduate Research and Scholarship. We thank K. Severinov for plasmid pBAD- kleABCD, K. Prather for the CRISPR-Cas9 plasmids used for genome engineering (Addgene plasmids 62654, 62655, 62656), and N. Hanson for K. pneumoniae strain KP23.
Publisher Copyright:
© 2022 American Chemical Society. All rights reserved.
PY - 2022/4/15
Y1 - 2022/4/15
N2 - 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.
AB - 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.
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U2 - 10.1021/acschembio.2c00148
DO - 10.1021/acschembio.2c00148
M3 - Article
C2 - 35315272
AN - SCOPUS:85127600251
VL - 17
SP - 998
EP - 1010
JO - ACS Chemical Biology
JF - ACS Chemical Biology
SN - 1554-8929
IS - 4
ER -