Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic Pathogen Klebsiella oxytoca

Evan M. Alexander; Dale F. Kreitler; Valeria Guidolin; Alexander K. Hurben; Eric Drake; Peter W. Villalta; Silvia Balbo; Andrew M. Gulick; Courtney C. Aldrich

doi:10.1021/acsinfecdis.0c00326

Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic Pathogen Klebsiella oxytoca

Evan M. Alexander
, Dale F. Kreitler
, Valeria Guidolin
, Alexander K. Hurben
, Eric Drake
, Peter W. Villalta
, Silvia Balbo
, Andrew M. Gulick
, Courtney C. Aldrich

Department of Structural Biology

University of Minnesota Twin Cities
SUNY Buffalo

Research output: Contribution to journal › Article › peer-review

35 Scopus citations

Abstract

Tilimycin is an enterotoxin produced by the opportunistic pathogen Klebsiella oxytoca that causes antibiotic-associated hemorrhagic colitis (AAHC). This pyrrolobenzodiazepine (PBD) natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway composed of three proteins: NpsA, ThdA, and NpsB. We describe the functional and structural characterization of the fully reconstituted NRPS system and report the steady-state kinetic analysis of all natural substrates and cofactors as well as the structural characterization of both NpsA and ThdA. The mechanism of action of tilimycin was confirmed using DNA adductomics techniques through the detection of putative N-2 guanine alkylation after tilimycin exposure to eukaryotic cells, providing the first structural characterization of a PBD-DNA adduct formed in cells. Finally, we report the rational design of small-molecule inhibitors that block tilimycin biosynthesis in whole cell K. oxytoca (IC50 = 29 ± 4 μM) through the inhibition of NpsA (KD = 29 ± 4 nM).

Original language	English
Pages (from-to)	1976-1997
Number of pages	22
Journal	ACS Infectious Diseases
Volume	6
Issue number	7
DOIs	https://doi.org/10.1021/acsinfecdis.0c00326
State	Published - Jul 10 2020

Keywords

Klebsiella oxytoca
adenylation
microbiome
nonribosomal peptide synthetase
pyrrolobenzodiazepine
tilimycin

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10.1021/acsinfecdis.0c00326

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title = "Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic Pathogen Klebsiella oxytoca",

abstract = "Tilimycin is an enterotoxin produced by the opportunistic pathogen Klebsiella oxytoca that causes antibiotic-associated hemorrhagic colitis (AAHC). This pyrrolobenzodiazepine (PBD) natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway composed of three proteins: NpsA, ThdA, and NpsB. We describe the functional and structural characterization of the fully reconstituted NRPS system and report the steady-state kinetic analysis of all natural substrates and cofactors as well as the structural characterization of both NpsA and ThdA. The mechanism of action of tilimycin was confirmed using DNA adductomics techniques through the detection of putative N-2 guanine alkylation after tilimycin exposure to eukaryotic cells, providing the first structural characterization of a PBD-DNA adduct formed in cells. Finally, we report the rational design of small-molecule inhibitors that block tilimycin biosynthesis in whole cell K. oxytoca (IC50 = 29 ± 4 μM) through the inhibition of NpsA (KD = 29 ± 4 nM).",

keywords = "Klebsiella oxytoca, adenylation, microbiome, nonribosomal peptide synthetase, pyrrolobenzodiazepine, tilimycin",

author = "Alexander, \{Evan M.\} and Kreitler, \{Dale F.\} and Valeria Guidolin and Hurben, \{Alexander K.\} and Eric Drake and Villalta, \{Peter W.\} and Silvia Balbo and Gulick, \{Andrew M.\} and Aldrich, \{Courtney C.\}",

note = "Publisher Copyright: Copyright {\textcopyright} 2020 American Chemical Society.",

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doi = "10.1021/acsinfecdis.0c00326",

language = "English",

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T1 - Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic Pathogen Klebsiella oxytoca

AU - Alexander, Evan M.

AU - Kreitler, Dale F.

AU - Guidolin, Valeria

AU - Hurben, Alexander K.

AU - Drake, Eric

AU - Villalta, Peter W.

AU - Balbo, Silvia

AU - Gulick, Andrew M.

AU - Aldrich, Courtney C.

PY - 2020/7/10

Y1 - 2020/7/10

N2 - Tilimycin is an enterotoxin produced by the opportunistic pathogen Klebsiella oxytoca that causes antibiotic-associated hemorrhagic colitis (AAHC). This pyrrolobenzodiazepine (PBD) natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway composed of three proteins: NpsA, ThdA, and NpsB. We describe the functional and structural characterization of the fully reconstituted NRPS system and report the steady-state kinetic analysis of all natural substrates and cofactors as well as the structural characterization of both NpsA and ThdA. The mechanism of action of tilimycin was confirmed using DNA adductomics techniques through the detection of putative N-2 guanine alkylation after tilimycin exposure to eukaryotic cells, providing the first structural characterization of a PBD-DNA adduct formed in cells. Finally, we report the rational design of small-molecule inhibitors that block tilimycin biosynthesis in whole cell K. oxytoca (IC50 = 29 ± 4 μM) through the inhibition of NpsA (KD = 29 ± 4 nM).

AB - Tilimycin is an enterotoxin produced by the opportunistic pathogen Klebsiella oxytoca that causes antibiotic-associated hemorrhagic colitis (AAHC). This pyrrolobenzodiazepine (PBD) natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway composed of three proteins: NpsA, ThdA, and NpsB. We describe the functional and structural characterization of the fully reconstituted NRPS system and report the steady-state kinetic analysis of all natural substrates and cofactors as well as the structural characterization of both NpsA and ThdA. The mechanism of action of tilimycin was confirmed using DNA adductomics techniques through the detection of putative N-2 guanine alkylation after tilimycin exposure to eukaryotic cells, providing the first structural characterization of a PBD-DNA adduct formed in cells. Finally, we report the rational design of small-molecule inhibitors that block tilimycin biosynthesis in whole cell K. oxytoca (IC50 = 29 ± 4 μM) through the inhibition of NpsA (KD = 29 ± 4 nM).

KW - Klebsiella oxytoca

KW - adenylation

KW - microbiome

KW - nonribosomal peptide synthetase

KW - pyrrolobenzodiazepine

KW - tilimycin

UR - https://www.scopus.com/pages/publications/85088488052

U2 - 10.1021/acsinfecdis.0c00326

DO - 10.1021/acsinfecdis.0c00326

M3 - Article

C2 - 32485104

AN - SCOPUS:85088488052

SN - 2373-8227

VL - 6

SP - 1976

EP - 1997

JO - ACS Infectious Diseases

JF - ACS Infectious Diseases

IS - 7

ER -

Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic Pathogen Klebsiella oxytoca

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