Indole produced during dysbiosis mediates host–microorganism chemical communication
- State key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, China
- Yunnan Provincial Key Laboratory of Molecular Biology for Sinomedicine, Yunnan University of Traditional Chinese Medicine, China
- Yunnan Key Laboratory of Vaccine Research Development on Severe Infectious Disease, Chinese Academy of Medical Sciences and Peking Union Medical College, China
Figures
Figure 1 with 1 supplement
Activation of DAF-16 by bacterial accumulation is involved in longevity and organismal fitness.
(A) The nuclear translocation of DAF-16::GFP in the intestine was increased in worms fed live E. coli OP50, but not in those fed heat-killed (HK) E. coli OP50. White arrows indicate nuclear localization of DAF-16::GFP. Scale bars: 50 μm. (B) Quantification of DAF-16 nuclear localization. These results are means ± standard deviation (SD) of three independent experiments (n > 35 worms per experiment). ***p < 0.001. (C) DAF-16 was also retained in the cytoplasm of the intestine of worms fed ampicillin-killed E. coli OP50 on days 4 and 7. These results are means ± SD of three independent experiments (n > 35 worms per experiment). ***p < 0.001. p-values (B, C) were calculated using the Chi-square test. (D) daf-16(mu86) mutants grown on live E. coli OP50 had a shorter lifespan than those grown on HK E. coli OP50. ***p < 0.001. ns, not significant. (E) daf-16(mu86) mutants grown on live E. coli OP50 had a shorter lifespan than those grown on ampicillin-killed E. coli OP50. ***p < 0.001. ns, not significant. A+, Ampicillin treatment; AmpR-OP50, E. coli OP50 containing an ampicillin resistance plasmid (PMF440). p-values (D, E) were calculated using log-rank test. (F, G) DAF-16 was involved in delaying the appearance of the aging markers, including pharyngeal pumping (F) and body bending (G), in worms fed live E. coli OP50, but not in those fed HK E. coli OP50. These results are means ± SD of five independent experiments (n > 20 worms per experiment). *p < 0.05. ns, not significant. (H) Representative images of intestinal permeability stained by food dye FD&C Blue No. 1 in worms. White arrows indicate the body-cavity leakages of worms. Scale bars: 100 μm. (I) Quantification of body-cavity leakages in animals fed on live E. coli OP50 or HK E. coli OP50 over time. These results are means ± SD of five independent experiments (n > 20 worms per experiment). ***p < 0.001. ns, not significant. p-values (F, G, I) were calculated using a two-tailed t-test.
-
Figure 1—source data 1
Lifespan assays summary and quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
The expression of DAF-16 target genes dod-3 and hsp-16.2 is significantly up-regulated in worms fed live E. coli OP50 on day 4.
(A) Diagram of the nematode–microbe interaction study. (B) Representative images of worms expressing dod-3p::gfp and hsp-16.2p::nCherry fed live or heat-killed (HK) E. coli OP50 on days 1 and 4. Scale bars: 100 μm. Quantification of fluorescent intensity of dod-3p::gfp (C) and hsp-16.2p::nCherry (D). These results are means ± standard deviation (SD) of three independent experiments (n > 30 worms per experiment). ***p < 0.001; *p < 0.05. ns, not significant. p-values (C, D) were calculated using a two-tailed t-test. (E) Starvation induced the nuclear translocation of DAF-16 in the intestine of worms on day 1. These results are means ± SD of three independent experiments (n > 35 worms per experiment). ***p < 0.001. p-values were calculated using the Chi-square test. STR, starvation.
-
Figure 1—figure supplement 1—source data 1
Quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig1-figsupp1-data1-v2.xlsx
Figure 2 with 4 supplements
Indole is involved in the nuclear translocation of DAF-16 in worms with age.
(A) High-resolution mass spectrum of indole. (B) Supplementation with indole promoted the nuclear translocation of DAF-16::GFP in the intestine of worms. These results are means ± standard deviation (SD) of three independent experiments (n > 35 worms per experiment). ***p < 0.001. (C) Colony-forming units (CFU) of E. coli OP50 were increased in worms over time, which was accompanied by an increase in the levels of indole in worms. These results are means ± SD of three independent experiments (n > 30 worms per experiment). *p < 0.05; **p < 0.01; ***p < 0.001. p-values (C) were calculated using a two-tailed t-test. (D) Deletion of tnaA significantly suppressed the nuclear translocation of DAF-16::GFP in the intestine of worms fed E. coli BW25113. These results are means ± SD of three independent experiments (n > 35 worms per experiment). ***p < 0.001. p-values (B, D) were calculated using the Chi-square test.
-
Figure 2—source data 1
Quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Indole is the active compound for activation of DAF-16.
The candidate compound for activation of DAF-16 was identified as indole with 1H NMR (A) and 13C NMR (B).
Figure 2—figure supplement 2
Indole treatment induces the expressions of DAF-16 target genes in worms.
Quantification of fluorescent intensity of dod-3p::gfp (A) or hsp-16.2p::nCherry (B) in young adult worms treated with indole (100 μM). These results are means ± standard deviation (SD) of three independent experiments (n > 35 worms per experiment). *p < 0.05；***p < 0.001. p-values throughout were calculated using a two-tailed t-test.
-
Figure 2—figure supplement 2—source data 1
Quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig2-figsupp2-data1-v2.xlsx
Figure 2—figure supplement 3
Quantitative analysis of indole in C. elegans by LC–MS.
(A) The levels of indole were not altered in worms fed heat-killed (HK) E. coli OP50 on days 1, 4, and 7. These results are means ± standard deviation (SD) of three independent experiments. ns, not significant. (B) Supplementation with indole (50–200 μM) significantly increased the indole levels in young adult worms after 24 hr treatment. These results are means ± SD of three independent experiments. ***p < 0.001. p-values throughout were calculated using a two-tailed t-test.
-
Figure 2—figure supplement 3—source data 1
Quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig2-figsupp3-data1-v2.xlsx
Figure 2—figure supplement 4
Functional validation of tnaA-deficient BW25113 strain.
The levels of tnaA mRNA (A) and indole (B) were undetectable in E. coli K-12ΔtnaA strains. These results are means ± standard deviation (SD) of three independent experiments. (C) DAF-16::GFP was mainly located in the cytoplasm of the intestine in worms fed live E. coli K-12ΔtnaA strains on day 4. However, supplementation with indole (100 μM) induced the nuclear translocation of DAF-16::GFP in the intestine of these worms. These results are means ± SD of three independent experiments. ***p < 0.001. p-value was calculated using the Friedman test (with Dunn’s test for multiple comparisons). (D) tnaA-rescued K12 ΔtnaA::tnaA strain could produce indole. (E) DAF-16::GFP was translocated to the nuclear of the intestine in worms fed live E. coli K12ΔtnaA:: tnaA strain on days 4 and 7. These results are means ± SD of three independent experiments. ***p < 0.001. p-values were calculated using the Chi-square test.
-
Figure 2—figure supplement 4—source data 1
Quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig2-figsupp4-data1-v2.xlsx
Figure 3
Indole is required for maintenance of normal lifespan via DAF-16 in worms.
(A) Wild-type (WT) worms fed E. coli K-12 tnaA strains had a shorter lifespan than those fed E. coli K-12 strain at 20°C. Supplementation with indole (100 μM) extended the lifespan of WT worms fed E. coli K-12, and rescued the short lifespan of WT worms fed E. coli K-12 ΔtnaA strain. **p < 0.01; ***p < 0.001. (B) Indole-mediated lifespan extension depended on DAF-16 in worms. ns, not significant. p-values (A, B) were calculated using log-rank test. (C, D) Colony-forming units (CFU) of E. coli K-12 or K-12tnaA were measured in WT worms (C) or daf-16(mu86) mutants (D). These results are means ± standard deviation (SD) of five independent experiments (n > 30 worms per experiment). *p < 0.05; **p < 0.01. ns, not significant. (E) Fluorescence images of worms exposed to E. coli K-12 or K-12tnaA expressing mCherry. Scale bars: 50 μm. (F, G) Quantification of fluorescent intensity of E. coli K-12 or K-12tnaA expressing mCherry in WT worms (F) or daf-16(mu86) mutants (G). These results are means ± SD of three independent experiments (n > 35 worms per experiment). *p < 0.05; **p < 0.01. ns, not significant. CFU of E. coli K-12 were measured in WT worms (H) or daf-16(mu86) mutants (I) in the presence of exogenous indole (100 μM). These results are means ± SD of five independent experiments (n > 30 worms per experiment). **p < 0.01. ns, not significant. p-values (C, D, F–I) were calculated using a two-tailed t-test.
-
Figure 3—source data 1
Lifespan assays summary and quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig3-data1-v2.xlsx
Figure 4 with 3 supplements
TRPA-1 is involved in indole-mediated DAF-16 nuclear translocation.
Knockdown of trpa-1 by RNAi suppressed the nuclear translocation of DAF-16::GFP in worms on days 4 and 7 (A), or in young adult worms treated with indole (100 μM) for 24 hr (B). EV, empty vector. These results are means ± standard deviation (SD) of three independent experiments (n > 35 worms per experiment). ***p < 0.001. ns, not significant. p-values (A, B) were calculated using the Chi-square test. (C) Knockdown of trpa-1 by RNAi significantly shortened the lifespan of worms treated with indole (100 μM). *p < 0.05; **p < 0.01; ***p <0 .001. (D, E) Colony-forming units (CFU) of E. coli K12 were significantly increased in worms subjected to trpa-1 RNAi on days 4 (E) and 7 (F). Meanwhile, supplementation with indole (100 μM) failed to suppress the increase in CFU in trpa-1 (RNAi) worms. These results are means ± SD of five independent experiments (n > 30 worms per experiment). **p < 0.01; ***p < 0.001. ns, not significant. p-values (D, E) were calculated using a two-tailed t-test.
-
Figure 4—source data 1
Lifespan assays summary and quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Indole promotes nuclear localization of DAF-16 independent of AHR-1 and SGK-1.
Knockdown of ahr-1 by RNAi did not affect the nuclear translocation of DAF-16 in worms fed E. coli K12 strain on day 7 (A) or young adult worms treated with indole for 24 hr (B). These results are means ± standard deviation (SD) of three independent experiments (n > 35 worms per experiment). (C) Knockdown of sgk-1 by RNAi did not affect the nuclear translocation of DAF-16 in young adult worms treated with indole (100 μM) for 24 hr. These results are means ± SD of three independent experiments (n > 35 worms per experiment). ns, not significant. p-values were calculated using the Chi-square test.
-
Figure 4—figure supplement 1—source data 1
Quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
Indole extends lifespan and inhibits bacterial accumulation via TRPA-1–DAF-16 axis.
(A) Knockdown of daf-16 by RNAi did not further shorten the lifespan of trpa-1(ok999) worms treated with indole (100 μM). ns, not significant. p-values were calculated using log-rank test. (B) Quantification of fluorescent intensity of E. coli K-12 expressing mCherry in trpa-1(ok999) mutant worms. ns, not significant. p-values were calculated using a two-tailed t-test.
-
Figure 4—figure supplement 2—source data 1
Lifespan assays summary and quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig4-figsupp2-data1-v2.xlsx
Figure 4—figure supplement 3
Supplementation with indole fails to induce nuclear localization of SKN-1 in worms.
(A) Representative images of SKN-1::GFP. Scale bars: 100 μm. (B) Quantification of SKN-1 nuclear localization. (C) Podocarpic acid failed to induce DAF-16 translocating to nuclear. These results are means ± standard deviation (SD) of three independent experiments (n > 35 worms per experiment). ***p < 0.001. ns, not significant. p-values were calculated using the Chi-square test.
-
Figure 4—figure supplement 3—source data 1
Quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig4-figsupp3-data1-v2.xlsx
Figure 5
TRPA-1 in neurons is involved in indole-mediated longevity.
Either neuronal- (A) or intestinal- (B) specific trpa-1 RNAi shortened the lifespan of worms. However, supplementation with indole (100 μM) significantly extended the lifespan of worms after knockdown of trpa-1 by RNAi in the intestine, but not in neurons. EV, empty vector. **p < 0.01; ***p < 0.001. ns, not significant. p-values (A, B) were calculated using log-rank test. Supplementation with indole (100 μM) no longer inhibited colony-forming units (CFU) of E. coli K12 in worms on days 4 (C) and 7 (D) after knockdown of trpa-1 by RNAi in neurons. These results are means ± standard deviation (SD) of five independent experiments (n > 30 worms per experiment). *p < 0.05; **p < 0.01. ns, not significant. Supplementation with indole (100 μM) significantly suppressed the CFU of E. coli K-12 in worms subjected to intestinal-specific trpa-1 RNAi on days 4 (E) and 7 (F). These results are means ± SD of five independent experiments (n > 30 worms per experiment). **p < 0.01; ***p < 0.001. ns, not significant. p-values (C–F) were calculated using a two-tailed t-test.
-
Figure 5—source data 1
Lifespan assays summary and quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig5-data1-v2.xlsx
Figure 6 with 2 supplements
LYS-7 and LYS-8 are required for maintenance of normal lifespan in worms.
The lifespans of worms fed either live E. coli OP50 (A) or heat-killed (HK) E. coli OP50 (B). The lys-7(ok1384); lys-8(ok3504) double mutants exhibited a shorter lifespan, than wild-type (WT) worms fed live E. coli OP50 (A). In contrast, the lifespan of the lys-7(ok1384); lys-8(ok3504) double mutants was comparable of that of WT worms fed HK E. coli OP50 (B). ***p < 0.001. ns, not significant. p-values (A, B) were calculated using log-rank test. (C–E) lys-7 and lys-8 were involved in delaying the appearance of the aging markers, including pharyngeal pumping (C), body bending (D), and body-cavity leakage (E) in worms fed live E. coli OP50. These results are means ± standard deviation (SD) of five independent experiments (n > 20 worms per experiment). *p < 0.05; **p < 0.01; ***p < 0.001. (F) Colony-forming units (CFU) of E. coli OP50 were significantly increased in lys-7(ok1384); lys-8(ok3504) double mutants on day 7. These results are means ± SD of five independent experiments (n > 20 worms per experiment). **p < 0.01. ns, not significant. (G) Quantification of fluorescent intensity of E. coli OP50 expressing mCherry in lys-7(ok1384);lys-8(ok3504) double mutants. These results are means ± SD of three independent experiments (n > 35 worms per experiment). *p < 0.05. ns, not significant. p-values (C–G) were calculated using a two-tailed t-test.
-
Figure 6—source data 1
Lifespan assays summary and quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
The roles of lysozyme genes in lifespan in worms.
The lifespans of lys-7(ok1384), or lys-8(ok3504) mutants, or lys-2 RNAi worms fed either live E. coli OP50 (A) or heat-killed (HK) E. coli OP50 (B). *p < 0.05. ns, not significant. (C) Knockdown of lys-2 by RNAi did not affect the lifespan of lys-7(ok1384); lys-8(ok3504) double mutants fed live (C) or HK E. coli OP50 (D). ns, not significant. p-values throughout were calculated using log-rank test.
-
Figure 6—figure supplement 1—source data 1
Lifespan assays summary.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig6-figsupp1-data1-v2.xlsx
Figure 6—figure supplement 2
Indole-mediated lifespan extension in worms depends on LYS-7 and LYS-8.
(A) Supplementation with indole (100 μM) no longer extended the lifespan of lys-7(ok1384); lys-8(ok3504) double mutants. p-values throughout were calculated using log-rank test. (B) Supplementation with indole failed to suppress the increase in colony-forming units (CFU) of K-12 in lys-7(ok1384); lys-8(ok3504) double mutants. These results are means ± standard deviation (SD) of five independent experiments (n > 30 worms per experiment). ns, not significant. p-values were calculated using a two-tailed t-test.
-
Figure 6—figure supplement 2—source data 1
Lifespan assays summary and quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig6-figsupp2-data1-v2.xlsx
Figure 7 with 2 supplements
The expressions of lys-7 and lys-8 were up-regulated by the indole/TRPA-1/DAF-16 signaling.
The expression of either lys-7p::gfp (A) or lys-8p::gfp (B) was significantly suppressed after knockdown of daf-16 or trpa-1 by RNAi in worms fed live E. coli OP50 on days 4 and 7. EV, empty vector. The expression of either lys-7p::gfp (C) or lys-8p::gfp (D) was reduced in worms fed E. coli K-12 ΔtnaA strain on days 4 and 7, compared with that in age-matched worms fed E. coli K-12. Indole (100 μM) remarkably increased the expression of either lys-7p::gfp (E) or lys-8::gfp (F) in young adult worms after 24 hr of treatment. These results are means ± standard deviation (SD) of three independent experiments (n > 35 worms per experiment). *p < 0.05; **p < 0.01; ***p < 0.001. ns, not significant. p-values (A–F) were calculated using a two-tailed t-test.
-
Figure 7—source data 1
Quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
The expressions of lys-7p::gfp and lys-8p::gfp are up-regulated in worms with age.
Representative images of lys-7p::gfp (A) and lys-8p::gfp (C) in worms fed live E. coli OP50. Scale bars: 100 μm. Quantification of fluorescent intensity of lys-7p::gfp (B) and lys-8p::gfp (D). The expressions of lys-7p::gfp and lys-8p::gfp were stable in worms treated with indole. These results are means ± standard deviation (SD) of three independent experiments (n > 35 worms per experiment). *p < 0.05; **p < 0.01. The mRNA levels of lys-7 (E) and lys-8 (F) were up-regulated in wild-type (WT) worms fed live E. coli OP50, but not heat-killed (HK) E. coli OP50, on day 4. These increases in the mRNA levels of lys-7 (E) and lys-8 (F) were abolished by a mutation in daf-16(mu86). **p < 0.01. These results are means ± SD of three independent experiments. p-values (B) and (D–F) were calculated using a two-tailed t-test.
-
Figure 7—figure supplement 1—source data 1
Quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig7-figsupp1-data1-v2.xlsx
Figure 7—figure supplement 2
TRPA-1 in neurons is required for the expression lys-7 and lys-8.
The mRNA levels of lys-7 and lys-8 were significantly down-regulated in worms subjected to neuronal specific (A), but not intestinal specific (B), knockdown of trpa-1 by RNAi. These results are means ± standard deviation (SD) of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. ns, not significant. Supplementation with indole (100 μM) up-regulated the mRNA levels of lys-7 and lys-8 in worms subjected to neuronal specific (C), but not intestinal specific (D), knockdown of trpa-1 by RNAi. These results are means ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. ns, not significant. p-values (A–D) were calculated using a two-tailed t-test.
-
Figure 7—figure supplement 2—source data 1
Quantification results.
- https://cdn.elifesciences.org/articles/85362/elife-85362-fig7-figsupp2-data1-v2.xlsx
Figure 8
Schematic model of indole as a microbial signal of gut dysbiosis in maintaining organismal fitness.
This study demonstrates that intestinal accumulation of E. coli in C. elegans with age leads to elevated levels of indole, which activates FOXO/DAF-16 transcription factor in the intestine via a cold-sensitive TRP channel TRPA-1 in neurons. The transcription factor in turn up-regulates the expression of lysozyme genes, thereby limiting the bacterial overproliferation in the gut to support organismal fitness in worms.
Author response image 1
DAF-16 nuclear translocation is independent of DAF-2.
(A) The mRNA levels of daf-2 were gradually increased in worms with age. **P < 0.01; ***P < 0.001; ns, not significant. (B) The mRNA levels of daf-2 were not altered after treatment with indole for 24 hours. ns, not significant.
Author response image 2
Sequence alignment between Arabidopsis ABCG37 and 15 C. elegans ABC transporters.
Author response image 3
Production of indole is associated with induction of DAF-16 nuclear translocation in E. coli, not in other three bacteria.
(A) The nuclear translocation of DAF-16::GFP was increased in the intestine of worms fed E. coli and S. Typhimurium, but not P. aeruginosa PA14 or S. aureus on day 4. These results are means ± SD of three independent experiments (n > 35 worms per experiment). ***P < 0.001. (B) The levels of indole in worms fed S. Typhimurium, P. aeruginosa, and S. aureus were much lower than those in worms fed E. coli OP50 on day 4. These results are means ± SD of three independent experiments. *P < 0.05. (C) The levels of indole in the culture supernatants of S. Typhimurium, P. aeruginosa, and S. aureus were much lower than those of E. coli OP50. ***P < 0.001.
Author response image 4
The effect of indole on the expression of p38 target genes.
Supplementation with indole only upregulated the expression of Y37a1a.2, but not T24B8.5, F35e12.5, nlp-29, and F08g5.6. *P < 0.05.
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/85362/elife-85362-mdarchecklist1-v2.pdf
-
Supplementary file 1
Table S1. The 1H and 13C NMR spectroscopic data of indole at 600 MHz for 1H NMR and 150 MHz for 13C NMR with reference to the solvent signals.
- https://cdn.elifesciences.org/articles/85362/elife-85362-supp1-v2.docx
-
Supplementary file 2
Table S2. The data of activity-guided isolation.
- https://cdn.elifesciences.org/articles/85362/elife-85362-supp2-v2.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Indole produced during dysbiosis mediates host–microorganism chemical communication
eLife 12:e85362.
https://doi.org/10.7554/eLife.85362
