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

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

21 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 (US)
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

Bibliographical note

Funding Information:
This work was supported by grants (GM116957 and GM136235 to A.M.G.) from the National Institutes of Health. Mass spectrometry for tilivalline quantitation was carried out in the Center for Mass Spectrometry and Proteomics. Mass spectrometry for DNA adductomics was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, University of Minnesota, and supported in part by the U.S. National Institutes of Health and National Cancer Institute (Cancer Center Support Grant CA-77598). Isothermal titration calorimetry was carried out using an ITC-200 microcalorimeter, funded by the NIH Shared Instrumentation Grant S10-OD017982. GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (P41GM103393).

Publisher Copyright:
Copyright © 2020 American Chemical Society.

Keywords

Klebsiella oxytoca
adenylation
microbiome
nonribosomal peptide synthetase
pyrrolobenzodiazepine
tilimycin

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10.1021/acsinfecdis.0c00326

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@article{53e54c41afef4078986c1e5d7585f473,

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 = "Funding Information: This work was supported by grants (GM116957 and GM136235 to A.M.G.) from the National Institutes of Health. Mass spectrometry for tilivalline quantitation was carried out in the Center for Mass Spectrometry and Proteomics. Mass spectrometry for DNA adductomics was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, University of Minnesota, and supported in part by the U.S. National Institutes of Health and National Cancer Institute (Cancer Center Support Grant CA-77598). Isothermal titration calorimetry was carried out using an ITC-200 microcalorimeter, funded by the NIH Shared Instrumentation Grant S10-OD017982. GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (P41GM103393). Publisher Copyright: Copyright {\textcopyright} 2020 American Chemical Society.",

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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.

N1 - Funding Information: This work was supported by grants (GM116957 and GM136235 to A.M.G.) from the National Institutes of Health. Mass spectrometry for tilivalline quantitation was carried out in the Center for Mass Spectrometry and Proteomics. Mass spectrometry for DNA adductomics was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, University of Minnesota, and supported in part by the U.S. National Institutes of Health and National Cancer Institute (Cancer Center Support Grant CA-77598). Isothermal titration calorimetry was carried out using an ITC-200 microcalorimeter, funded by the NIH Shared Instrumentation Grant S10-OD017982. GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (P41GM103393). Publisher Copyright: Copyright © 2020 American Chemical Society.

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

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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

Abstract

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Keywords

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