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 (K D = 29 ± 4 nM).

Design, Synthesis, and Biophysical Evaluation of Mechanism-Based Probes for Condensation Domains of Nonribosomal Peptide Synthetases

“…We elected to use salicylic acid in the probe since it is also a competent substrate for EntF forming a salicyl-Ser-S∼EntF intermediate that cannot undergo elongation to the trilactone . Additionally, we discovered salicyl-containing probes are more stable than the corresponding DHB probes, which we hypothesize undergo facile oxidation to reactive ortho -quinones leading to complex mixtures presumably through polymerization and/or adduct formation.…”

Characterization and quantitation of busulfan DNA adducts in the blood of patients receiving busulfan therapy

“…15 N-labeled Escherichia coli DNA is a useful tool to further confirm the putative BU-DNA adducts listed in Table 1 . 29 A 1:1 mixture of unlabeled and 15 N-labeled E. coli DNA exposed to BU was hydrolyzed and purified following the same approach described for the analysis of CT-DNA. The hydrolysate was analyzed by the same adductomic DDA-CNL/MS 3 method, with the exception that 15 N-labeled deoxyribonucleosides were included as monitored neutral losses.…”

“…Data-dependent MS 2 parameters were dynamic exclusion of 30 s, mass tolerance of ±5 ppm, repeat count of 1, minimum intensity of 2 × 10 4 , and a cycle time of 3 s. An MS 2 exclusion list of 18 masses, consisting of unmodified deoxyribonucleosides and their electrostatically bound dimer ions, was included in the method with a mass tolerance of ±5 ppm. 29 The MS 2 scan events were triggered on the basis of an intensity threshold of 5 × 10 3 . The MS 2 fragmentation was performed with a quadrupole isolation width of m/z 1.5, higher-energy C-trap dissociation (HCD) collision energy 30%, automatic gain control (AGC) value of 400%, maximum injection time of 54 ms, and Orbitrap detection at a resolution setting of 30,000.…”

cAMP Receptor Protein Positively Regulates the Expression of Genes Involved in the Biosynthesis of Klebsiella oxytoca Tilivalline Cytotoxin

“…K. oxytoca is a pathobiont of the intestinal microbiota that can produce TV cytotoxin ( Beaugerie et al, 2003 ; Högenauer et al, 2006 ; Zollner-Schwetz et al, 2015 ; Glabonjat et al, 2021 ). After penicillin treatment, alteration of the enteric microbiota occurs, and overgrowth of K. oxytoca is originated in the colon causing a severe dysbiosis ( Schneditz et al, 2014 ; Hajishengallis and Lamont, 2016 ; Dornisch et al, 2017 ; von Tesmar et al, 2018 ; Alexander et al, 2020 ). The imbalance in the gut microbiota and the production of TV cytotoxin result in AAHC ( Högenauer et al, 2006 ; Unterhauser et al, 2019 ).…”