TY - JOUR
T1 - A Platform for Deep Sequence-Activity Mapping and Engineering Antimicrobial Peptides
AU - Dejong, Matthew P.
AU - Ritter, Seth C.
AU - Fransen, Katharina A.
AU - Tresnak, Daniel T.
AU - Golinski, Alexander W.
AU - Hackel, Benjamin J.
N1 - Publisher Copyright:
© 2021 American Chemical Society.
PY - 2021/10/15
Y1 - 2021/10/15
N2 - Developing potent antimicrobials, and platforms for their study and engineering, is critical as antibiotic resistance grows. A high-throughput method to quantify antimicrobial peptide and protein (AMP) activity across a broad continuum would be powerful to elucidate sequence-activity landscapes and identify potent mutants. Yet the complexity of antimicrobial activity has largely constrained the scope and mechanistic bandwidth of AMP variant analysis. We developed a platform to efficiently perform sequence-activity mapping of AMPs via depletion (SAMP-Dep): a bacterial host culture is transformed with an AMP mutant library, induced to intracellularly express AMPs, grown under selective pressure, and deep sequenced to quantify mutant depletion. The slope of mutant growth rate versus induction level indicates potency. Using SAMP-Dep, we mapped the sequence-activity landscape of 170 »000 mutants of oncocin, a proline-rich AMP, for intracellular activity against Escherichia coli. Clonal validation supported the platform's sensitivity and accuracy. The mapped landscape revealed an extended oncocin pharmacophore contrary to earlier structural studies, clarified the C-terminus role in internalization, identified functional epistasis, and guided focused, successful synthetic peptide library design, yielding a mutant with 2-fold enhancement in both intracellular and extracellular activity. The efficiency of SAMP-Dep poises the platform to transform AMP engineering, characterization, and discovery.
AB - Developing potent antimicrobials, and platforms for their study and engineering, is critical as antibiotic resistance grows. A high-throughput method to quantify antimicrobial peptide and protein (AMP) activity across a broad continuum would be powerful to elucidate sequence-activity landscapes and identify potent mutants. Yet the complexity of antimicrobial activity has largely constrained the scope and mechanistic bandwidth of AMP variant analysis. We developed a platform to efficiently perform sequence-activity mapping of AMPs via depletion (SAMP-Dep): a bacterial host culture is transformed with an AMP mutant library, induced to intracellularly express AMPs, grown under selective pressure, and deep sequenced to quantify mutant depletion. The slope of mutant growth rate versus induction level indicates potency. Using SAMP-Dep, we mapped the sequence-activity landscape of 170 »000 mutants of oncocin, a proline-rich AMP, for intracellular activity against Escherichia coli. Clonal validation supported the platform's sensitivity and accuracy. The mapped landscape revealed an extended oncocin pharmacophore contrary to earlier structural studies, clarified the C-terminus role in internalization, identified functional epistasis, and guided focused, successful synthetic peptide library design, yielding a mutant with 2-fold enhancement in both intracellular and extracellular activity. The efficiency of SAMP-Dep poises the platform to transform AMP engineering, characterization, and discovery.
KW - antimicrobial peptide
KW - deep mutational scanning
KW - oncocin
KW - protein engineering
KW - ribosome
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UR - http://www.scopus.com/inward/citedby.url?scp=85115660469&partnerID=8YFLogxK
U2 - 10.1021/acssynbio.1c00314
DO - 10.1021/acssynbio.1c00314
M3 - Article
C2 - 34506711
AN - SCOPUS:85115660469
SN - 2161-5063
VL - 10
SP - 2689
EP - 2704
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
IS - 10
ER -