Cellular Stress Upregulates Indole Signaling Metabolites in Escherichia coli

Abstract

Escherichia coli broadly colonize the intestinal tract of humans and produce a variety of small molecule signals. However, many of these small molecules remain unknown. Here, we describe a family of widely distributed bacterial metabolites termed the "indolokines." In E. coli, the indolokines are upregulated in response to a redox stressor via aspC and tyrB transaminases. Although indolokine 1 represents a previously unreported metabolite, four of the indolokines (2-5) were previously shown to be derived from indole-3-carbonyl nitrile (ICN) in the plant pathogen defense response. We show that the indolokines are produced in a convergent evolutionary manner relative to plants, enhance E. coli persister cell formation, outperform ICN protection in an Arabidopsis thaliana-Pseudomonas syringae infection model, trigger a hallmark plant innate immune response, and activate distinct immunological responses in primary human tissues. Our molecular studies link a family of cellular stress-induced metabolites to defensive responses across bacteria, plants, and humans.

Keywords: E. coli; IL-6; antibiotic tolerance; host-bacteria interactions; indole; innate immunity; persister cell; signaling; stress response.

Figure 1.. Structure of indolokine metabolites and characterization of their genetic origins in E. coli.

A, Structures of characterized bacterial metabolites (indolokines 1-5) and their plant-derived analogs (6-9). B, Comparison of metabolite production levels between wild-type E. coli BW25113 and its tyrB, aspC, and tyrB-aspC mutants. n = 3 biological replicates. Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n.s., not significant; n.d., not detected. Two-tailed t-test.

Figure 2.. Production of indolokines 1-5 in various microbial strains and mouse fecal samples.

A, Production of indolokines 1-5 from E. coli Nissle 1917, E. coli LF82, E. coli MG1655, E. coli BW25113, S. enterica serovar Typhimurium, K. pneumoniae ATCC 700603, X. bovienii str. feltiae Moldova (with and without 25 μM of erythromycin (ERM) as a ribosomal stressor), V. cholerae El Tor N16961 ΔctxAB, V. parahaemolyticus, P. aeruginosa PAO1, vancomycin-resistant E. faecalis (VRE), E. gallinarum, L. parafarraginis F0439, L. rhamnosus LMS2-1, L. reuteri CF48-3A, B. subtilis BR151, methicillin-resistant S. aureus ATCC BAA-1717 (MRSA), S. cerevisiae, and mouse fecal samples. n = 3 (bacteria) or 5 (mouse fecal samples) biological replicates. Data are presented by mean ± s.d. of MS intensities (counts) observed in bacterial culture extracts subtracted by the mean of the media background signals. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., not significant; n.d., not detected. Two-tailed t-test between bacterial metabolite production level signals and respective media control signals. B, Dose-dependent regulation of 1-5 in E. coli Nissle 1917 by paraquat. n = 3 biological replicates. Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n.s., not significant. Two-tailed t-test between no stress controls and paraquat stress conditions.

Figure 3.. Proposed biosynthesis of the indolokines.

A, Key NMR correlations for novel small molecule 1. COSY (blue bold); HMBC (red arrows); LR-HSQMBC (green arrows). B, Proposed pathway of 1-5 in E. coli. Ox., oxidation. C, Biomimetic synthesis of 2 and 3 in M9 medium (see Figure S3 for corroborating LB medium data). Metabolites 2 and 3 were produced only when both l-Cys and I3P were present. n = 3 biological replicates. Data are mean ± s.d.; n.d., not detected. D, Time-course analysis for production of metabolites 2-5 in E. coli Nissle 1917.

Figure 4.. Evaluation of indolokine 1-9 biological activities.

A, Persister cell formation of E. coli BW25113 pretreated with or without 1-5 in the presence of gentamicin. n = 4 biological replicates. Data are mean ± s.d. **P < 0.01, ***P < 0.001. Two-tailed t-test. B, Growth analysis of the virulent plant pathogen P. syringae pv. tomato (Pto) DC3000 in surface-inoculated adult leaves of A. thaliana. 5-week-old leaves were pre-infiltrated (pre-immunized) with 1 μM flg22 and 1 μM compounds (1-9) 24 h prior to infiltration with OD₆₀₀ = 0.0002 bacteria. n = 6 (2, 5-9, ICN, and 4-OH-ICN) or 12 (DMSO, 3, and 4) biological replicates. Data are mean ± s.d. *P< 0.05, ****P< 0.0001. n.s., not significant. Two-tailed t-test. C, BioMAP^® Phenotypic Profiling assay with metabolites 1-9 (21 μM). Cell types and stimuli used in each system are as follows: 3C system [HUVEC + (IL-1β, TNFα and IFNγ)], 4H system [HUVEC + (IL-4 and histamine)], LPS system [PBMC and HUVEC + LPS (TLR4 ligand)], SAg system [PBMC and HUVEC + TCR ligands (1×)], BT system [CD19+ B cells and PBMC + (α-IgM and TCR ligands (0.001×))], BF4T system [bronchial epithelial cells and HDFn + (TNFα and IL-4)], BE3C system [bronchial epithelial cells + (IL-1β, TNFα and IFNγ)], CASM3C system [coronary artery smooth muscle cells + (IL-1β, TNFα and IFNγ)], HDF3CGF system [HDFn + (IL-1β, TNFα, IFNγ, EGF, bFGF and PDGF-BB)], KF3CT system [keratinocytes and HDFn + (IL-1β TNFα and IFNγ)], MyoF system [differentiated lung myofibroblasts + (TNFα and TGFβ)] and /Mphg system [HUVEC and M1 macrophages + Zymosan (TLR2 ligand)]. D, AhR activating properties of 2-5 and ITE (positive control). n = 3 biological replicates. Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Non-marked data in panel D were not significant relative to controls. Two-tailed t-test.

Scheme 1.

Synthesis of indolokines 2-5.

Scheme 2.

Synthesis of indolokine 6-9.