1. Biochemistry and Chemical Biology
  2. Microbiology and Infectious Disease

Indole produced during dysbiosis mediates host–microorganism chemical communication

  1. Rui-Qiu Yang
  2. Yong-Hong Chen
  3. Qin-yi Wu
  4. Jie Tang
  5. Shan-Zhuang Niu
  6. Qiu Zhao
  7. Yi-Cheng Ma  Is a corresponding author
  8. Cheng-Gang Zou  Is a corresponding author
  1. State key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, China
  2. Yunnan Provincial Key Laboratory of Molecular Biology for Sinomedicine, Yunnan University of Traditional Chinese Medicine, China
  3. Yunnan Key Laboratory of Vaccine Research Development on Severe Infectious Disease, Chinese Academy of Medical Sciences and Peking Union Medical College, China
https://doi.org/10.7554/eLife.85362
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Cite this article
  1. Rui-Qiu Yang
  2. Yong-Hong Chen
  3. Qin-yi Wu
  4. Jie Tang
  5. Shan-Zhuang Niu
  6. Qiu Zhao
  7. Yi-Cheng Ma
  8. Cheng-Gang Zou
(2023)
Indole produced during dysbiosis mediates host–microorganism chemical communication
eLife 12:e85362.
https://doi.org/10.7554/eLife.85362
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Abstract

An imbalance of the gut microbiota, termed dysbiosis, has a substantial impact on host physiology. However, the mechanism by which host deals with gut dysbiosis to maintain fitness remains largely unknown. In Caenorhabditis elegans, Escherichia coli, which is its bacterial diet, proliferates in its intestinal lumen during aging. Here, we demonstrate that progressive intestinal proliferation of E. coli activates the transcription factor DAF-16, which is required for maintenance of longevity and organismal fitness in worms with age. DAF-16 up-regulates two lysozymes lys-7 and lys-8, thus limiting the bacterial accumulation in the gut of worms during aging. During dysbiosis, the levels of indole produced by E. coli are increased in worms. Indole is involved in the activation of DAF-16 by TRPA-1 in neurons of worms. Our finding demonstrates that indole functions as a microbial signal of gut dysbiosis to promote fitness of the host.

Editor's evaluation

This fundamental study provides compelling evidence for a new mechanism of host-microbe interaction, with indole, produced by proliferating bacteria in the C. elegans digestive system, signalling through the host via the transcription factor DAF-16 to induce the expression of genes controlling bacterial growth in the gut. The work is relevant to a wide audience as it invites deeper research into this mechanism, while also serving as a template for similar microbiome/host interactions in other systems.

https://doi.org/10.7554/eLife.85362.sa0

Introduction

The microbiota in the gut has a substantial impact on host nutrition, metabolism, immune function, development, behavior, and lifespan (Bana and Cabreiro, 2019; Johnson and Foster, 2018; Lee and Brey, 2013). Microbial community disequilibria, so-called dysbiosis, have been implicated in a broad range of human diseases, such as obesity, insulin resistance, autoimmune disorders, inflammatory bowel disease (IBD), aging, and increased pathogen susceptibility (Honda and Littman, 2012; Wu et al., 2015). Therefore, understanding of the mechanisms that modulate host–microbe interactions will provide important insights into treatment of these diseases by intervening the microbial communities.

The genetically tractable model organism Caenorhabditis elegans has contributed greatly to understand the role of host–microbiota interactions in host physiology (Cabreiro and Gems, 2013; Zhang et al., 2017). In the bacterivore nematode, the microbiota in the gut can be easily manipulated, making it an excellent model for studying how the microbiota affects host physiology in the context of disease and aging at a single species (Cabreiro and Gems, 2013). For instance, the neurotransmitter tyramine produced by intestinal Providencia bacteria can direct sensory behavioral decisions by modulate multiple monoaminergic pathways in C. elegans (O’Donnell et al., 2020). On the other hand, C. elegans-based studies have revealed a variety of signaling cascades involved in the innate immune responses to microbial infection (Irazoqui et al., 2010b), including the p38 mitogen-activated protein kinase (MAPK)/PMK-1, ERK MAPK/MPK-1, the heat shock transcription factor (HSF-1), the transforming growth factor (TGF)β/bone morphogenetic protein (BMP) signaling (Zugasti and Ewbank, 2009), and the forkhead transcription factor DAF-16/FOXO pathway (Garsin et al., 2003; Kim et al., 2002; Singh and Aballay, 2006; Zou et al., 2013). In Drosophila and mammalian cells, activation of FOXOs up-regulates a set of antimicrobial peptides, such as drosomycin and defensins (Becker et al., 2010), implicating that the role for FOXOs in innate immunity is conserved across species. Furthermore, disruption of these innate immune-related pathways, such as the p38 MAPK pathway and the TGF-β/BMP signaling cascade, turns beneficial bacteria commensal to pathogenic in worms (Berg et al., 2019; Montalvo-Katz et al., 2013). These results indicate that the integrity of immune system is also essential for host defense against non-pathogenic bacteria.

Escherichia coli (strain OP50) is conventionally used as a bacterial food for culturing C. elegans. In general, most of E. coli is efficiently disrupted by a muscular grinder in the pharynx of the worm. However, very few intact bacteria may escape from this defense system, and enter in the lumen of the worm intestine (Gupta and Singh, 2017). The intestinal lumen of worms is frequently distended during aging, which accompanied by bacterial proliferation (Garigan et al., 2002; McGee et al., 2011). Blockage of bacterial proliferation by treatment of UV, antibiotics, and heat extends lifespan of worms (De Arras et al., 2014; Garigan et al., 2002; Hwang et al., 2014), implicating that progressive intestinal proliferation of E. coli probably contributes to worm aging and death. Thus, age-related dysbiosis in C. elegans provides a model to study how host responses to altered gut microbiota to maintain fitness (Ezcurra, 2018).

Accumulating evidence has indicated that genetic inactivation of daf-16 accelerates tissue deterioration and shortens lifespan of wild-type (WT) worms grown on E. coli OP50 (Garigan et al., 2002; Li et al., 2019; Portal-Celhay and Blaser, 2012; Portal-Celhay et al., 2012). The observation that DAF-16 is activated during bacterial accumulation in older worms (Li et al., 2019) prompt us to investigate the role of DAF-16 in dysbiosis in the gut of worms. We found that activation of DAF-16 was required for maintenance of longevity and organismal fitness in worms, at least in part, by up-regulating two lysozyme genes (lys-7 and lys-8), thus limiting bacterial accumulation in the gut of worms during aging. Meanwhile, we identified that indole produced by E. coli was involved in the activation of DAF-16 by TRPA-1.

Results

Activation of DAF-16 is required for normal lifespan and organismal fitness in worms

To study the role of DAF-16 in age-related dysbiosis, all the experiments started from the young adult stage, which was considered day 0 (0 day) (Figure 1—figure supplement 1A). Consistent with a recent observation that DAF-16 is activated in worms with age (Li et al., 2019), we found that DAF-16::GFP was mainly located in the cytoplasm of the intestine in worms expressing daf-16p::daf-16::gfp fed live E. coli OP50 on day 1 (Figure 1A, B). The nuclear translocation of DAF-16 in the intestine was increased in worms fed live E. coli OP50 on days 4 and 7, but not in age-matched WT worms fed heat-killed (HK) E. coli OP50 (Figure 1A, B). To further confirm these results, we tested the expression of two DAF-16 target genes dod-3 and hsp-16.2 via the transcriptional reporter strains of dod-3p::gfp and hsp-16.2p::nCherry. As expected, the expression of either dod-3p::gfp or hsp-16.2p::nCherry was significantly up-regulated in worms fed live E. coli OP50 on day 4, but not in age-matched worms fed HK E. coli OP50 (Figure 1—figure supplement 1B–D). Likewise, DAF-16 was also retained in the cytoplasm of the intestine in worms fed ampicillin-killed E. coli OP50 on days 4 and 7 (Figure 1C). In contrast, starvation induced the nuclear translocation of DAF-16 in the intestine of worms on day 1 (Figure 1—figure supplement 1E). Thus, either HK or antibiotic-killed E. coli OP50 as a food source does not induce a starvation state in worms. Taken together, these results indicate that the activation of DAF-16 is mainly attributed to the presence of live E. coli, but not by age itself, in worms.

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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

Consistent with the previous observations (Li et al., 2019; Portal-Celhay et al., 2012), we found that a mutation in daf-16(mu86) shortened the lifespan of worms fed live E. coli OP50 at 20°C (Figure 1D). In contrast, the mutation in daf-16(mu86) had no impact on the lifespan of worms fed either HK (Figure 1D) or ampicillin-killed E. coli OP50 (Figure 1E). Next, we examined the effects of DAF-16 on phenotypic traits, such as the pharyngeal-pumping rate, body bending, and integrity of intestinal barrier, which are associated with aging in worms. Both the rates of pharyngeal pumping (Figure 1F) and body bending (Figure 1G) were reduced in daf-16(mu86) mutants on day 7 as compared to those in WT worms fed live E. coli OP50. In contrast, the rates of pharyngeal pumping and body bending were comparable in WT worms and daf-16(mu86) mutants grown on HK E. coli OP50 on day 7. Furthermore, we used food dye FD&C Blue No. 1 to evaluate the integrity of intestinal barrier (Ma et al., 2020). The body-cavity leakage in daf-16(mu86) mutants on days 7 and 10 were higher than those in age-matched WT worms fed live E. coli OP50, but not HK E. coli OP50 (Figure 1H, I). Taken together, these results demonstrate that the activation of DAF-16 by bacterial accumulation is required for normal lifespan and the maintenance of organismal fitness.

Indole produced from E. coli activates DAF-16

As DAF-16 is activated by bacterial accumulation, we hypothesized that this activation is probably due to bacterially produced compounds. To test this idea, culture supernatants from E. coli OP50 were collected, and isolated by high-performance liquid chromatograph (HPLC), silica gel G column and Sephadex LH-20 column chromatography. A candidate compound was detected by activity-guided isolation, and further identified as indole with mass spectrometry and NMR data (Figure 2A, Figure 2—figure supplement 1A, B; Supplementary file 1). The observation that indole secreted by E. coli OP50 could activate DAF-16 was further confirmed by analyzing commercial HPLC grade indole. Supplementation with indole (50–200 μM) not only significantly induced the nuclear translocation of DAF-16 (Figure 2B), but also up-regulated the expression of either dod-3p::gfp or hsp-16.2p::nCherry in young adult worms after 24 hr of treatment (Figure 2—figure supplement 2A, B). Next, we found that the levels of indole were 30.9, 71.9, and 105.9 nmol/g dry weight, respectively, in worms fed live E. coli OP50 on days 1, 4, and 7 (Figure 2C). The elevated indole levels in worms were accompanied by an increase in colony-forming units (CFU) of live E. coli OP50 in the intestine of worms with age (Figure 2C), suggesting that accumulation of live E. coli OP50 in the intestine is probably responsible for increased indole in worms. These data also raised a possibility that exogenous indole produced by E. coli OP50 on the nematode growth media (NGM) plates could increase the levels of indole in worms with age. However, we found that the levels of indole were 28.2, 31.6, and 36.1 nmol/g dry weight, respectively, in worms fed HK E. coli OP50 on days 1, 4, and 7 (Figure 2—figure supplement 3A), indicating that indole was not accumulated in worms fed HK E. coli OP50 even for 7 days. Thus, the increase in the levels of indole in worms results from intestinal accumulation of live E. coli OP50, rather than exogenous indole produced by E. coli OP50 on the NGM plates. The observation that DAF-16 was retained in the cytoplasm of the intestine in worms fed live E. coli OP50 on day 1 (Figure 1A, B) also indicated that exogenous indole produced by E. coli OP50 on the NGM plates is not enough to activate DAF-16. Supplementation with indole (50–200 μM) significantly increased the indole levels in young adult worms on day 1 (Figure 2—figure supplement 3B), which could induce nuclear translocation of DAF-16 in worms (Figure 2B).

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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

In bacteria, indole is biosynthesized from tryptophan by tryptophanase (tnaA) (Lee et al., 2015). To determine the effect of indole produced by bacteria in the gut, worms were fed E. coli K-12 BW25113 strain (called K-12), and tnaA-deficient strain BW25113 tnaA (called K-12 ΔtnaA), respectively. We found that both tnaA mRNA and indole levels were undetectable in the K-12 ΔtnaA strain (Figure 2—figure supplement 4A and B). Furthermore, disruption of tnaA significantly suppressed the nuclear translocation of DAF-16 (Figure 2D). The nuclear translocation of DAF-16::GFP was mainly located in the cytoplasm of the intestine in worms fed live K-12 ΔtnaA strains on day 4. However, supplementation with indole induced the nuclear translocation of DAF-16::GFP in the intestine of these worms (Figure 2—figure supplement 4C). Moreover, we rescued the expression of tnaA in the K-12ΔtnaA strains, and found that the production of indole in the K12ΔtnaA::tnaA strains bacterial solution was 34.1 μmol/l, similar to the K12 strains (Figure 2—figure supplement 4D). DAF-16::GFP was mainly located in the nuclear of the intestine in worms fed live K12ΔtnaA::tnaA strains on days 4 and 7 (Figure 2—figure supplement 4E). Taken together, our results suggest that indole is involved in the activation of DAF-16 in worms with age.

Indole produced by bacteria in the gut is required for normal lifespan

It has been shown that exogenous indole extends lifespan in C. elegans at 16°C (Sonowal et al., 2017). We found that adult worms fed E. coli K-12 tnaA strains exhibited a shortened lifespan at 20°C, compared with those fed E. coli K-12 strain (Figure 3A). Supplementation with indole in adults not only rescued the shortened lifespan of WT worms fed E. coli K-12 ΔtnaA strain, but also significantly extended the lifespan of WT worms fed E. coli K-12 strain (Figure 3A). In contrast, the lifespan of daf-16(mu86) mutants fed E. coli K-12 ΔtnaA strain was comparable to that of daf-16(mu86) mutants fed E. coli K-12 strain (Figure 3B). Supplementation with indole (100 μM) did not affect the lifespan of daf-16(mu86) mutants fed either E. coli K-12 or K-12 tnaA strain (Figure 3B). Moreover, the CFU of E. coli K-12 ΔnaA strain were significantly higher than those of E. coli K12 strain in WT worms on days 4 and 7 (Figure 3C). In contrast, the CFU of E. coli K-12 ΔtnaA strain were similar to those of E. coli K12 strain in daf-16(mu86) mutants on days 4 and 7 (Figure 3D). Likewise, the accumulation of E. coli K-12 ΔtnaA strain expressing mCherry was significantly higher than that of E. coli K12 strain in WT worms, but not daf-16(mu86) mutants, on days 4 and 7 (Figure 3E–G). Finally, supplementation with indole (100 μM) inhibited the CFU of E. coli K-12 in WT worms, but not daf-16(mu86) mutants, on days 4 and 7 (Figure 3H and I). These results suggest that indole produced by bacteria in the gut inhibits the proliferation of E. coli through DAF-16, thereby maintaining normal lifespan in worms.

Figure 3
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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

TRPA-1 in neurons is required for indole-mediated longevity

How did worms detect bacterially produced indole during aging? Previously, Sonowal et al. have identified that C. elegans xenobiotic receptor AHR-1, which encodes an ortholog of the mammalian AHR, mediates indole-promoted lifespan extension in worms at 16°C (Sonowal et al., 2017). However, we found that RNAi knockdown of ahr-1 did not affect the nuclear translocation of DAF-16 in worms fed E. coli K12 strain on day 7 (Figure 4—figure supplement 1A) or young adult worms treated with indole (100 μM) for 24 hr (Figure 4—figure supplement 1B). A recent study has demonstrated that bacteria-derived indole activates the transient receptor potential ankyrin 1 (TRPA-1), a cold-sensitive TRP channel, in enteroendocrine cells in zebrafish and mammals (Ye et al., 2021). As C. elegans TRPA-1 is an ortholog of mammalian TRPA-1 (Kindt et al., 2007; Venkatachalam and Montell, 2007), we tested the role of TRPA-1 in DAF-16 activation by indole. We found that RNAi knockdown of trpa-1 significantly inhibited the nuclear translocation of DAF-16 in worms fed E. coli K12 strain on days 4 and 7 (Figure 4A) or young adult worms treated with indole (100 μM) for 24 hr (Figure 4B). These results suggest that TRPA-1 is involved in indole-mediated DAF-16 activation. Previously, Xiao et al., 2013 have demonstrated that TRPA-1 activated by low temperatures mediates calcium influx, which in turn stimulates the PKC-2-SGK-1 signaling to promote the transcription activity of DAF-16, leading to lifespan extension in worms. However, we found that RNAi knockdown of sgk-1 did not influence the nuclear-cytoplasmic distribution of DAF-16 in the presence of indole (Figure 4—figure supplement 1C). Mutant worms lacking trpa-1 exhibited a shorter lifespan than did WT worms at 20°C (Xiao et al., 2013). Consistent with this observation, knockdown of trpa-1 by RNAi significantly shortened the lifespan of worms fed E. coli K12 strain (Figure 4C). Supplementation with indole no longer extended the lifespan of worms after knockdown of trpa-1 by RNAi or in trpa-1(ok999) mutants (Figure 4C; Figure 4—figure supplement 2A). In trpa-1(ok999) mutants, knockdown of daf-16 by RNAi did not further reduce the lifespan of worms (Figure 4—figure supplement 2A). Furthermore, the CFU of E. coli K-12 strain were significantly increased in worms subjected to trpa-1 RNAi on days 4 and 7 (Figure 4D, E). Supplementation with indole failed to suppress the increases in CFU in these worms. In addition, supplementation with indole also failed to suppress the increases in E. coli K-12 strain expressing mCherry in trpa-1(ok999) mutants subjected to empty vector or daf-16 RNAi (Figure 4—figure supplement 2B). These results suggest that indole exhibits its function in extending the lifespan and inhibiting bacterial accumulation primarily via TRPA-1. It has been shown that podocarpic acid, a TRPA-1 agonist, activates the SEK-1/PMK-1/SKN-1 pathway (Chaudhuri et al., 2016), a signaling cascade involved in C. elegans defense against pathogenic bacteria (Kim et al., 2002). However, we found that supplementation with 0.1 mM indole failed to induce nuclear localization of SKN-1::GFP in the intestine of the transgenic worms expressing skn-1p::skn-1::gfp (Figure 4—figure supplement 3A, B), suggesting that indole cannot activate SKN-1 in worms. Furthermore, 0.02 mM of podocarpic acid did not induce the nuclear localization of DAF-16::GFP (Figure 4—figure supplement 3C). These results suggest that TRPA-1 activation via podocarpic acid and indole happens largely through distinct mechanisms.

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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

As overexpression of trpa-1 in both the intestine and neurons is sufficient to extend the lifespan of worms (Xiao et al., 2013), we determined tissue-specific activities of TRPA-1 in the regulation of longevity mediated by indole. Consistent with this observation (Xiao et al., 2013), we found that both neuronal- and intestinal-specific knockdown of trpa-1 by RNAi significantly shortened the lifespan of worms (Figure 5A, B). However, supplementation with indole (100 μM) only extended the lifespan of worms subjected to intestinal specific (Figure 5B), but not neuronal specific, trpa-1 RNAi (Figure 5A). Likewise, knockdown of trpa-1 in either neurons (Figure 5C, D) or the intestine (Figure 5E, F) increased the CFU of E. coli K-12 in worms on days 4 and 7. However, supplementation with indole failed to inhibit the increase in the CFU of E. coli K-12 in worms subjected to neuronal-specific trpa-1 RNAi (Figure 5C, D). In contrast, supplementation with indole significantly suppressed the CFU of E. coli K-12 in these worms subjected to intestinal-specific trpa-1 RNAi (Figure 5E, F). These results suggest that TRPA-1 in neurons is involved in indole-mediated longevity.

Figure 5
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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

LYS-7 and LYS-8 functions as downstream molecules of DAF-16 to maintain normal lifespan in worms

C. elegans possesses a variety of putative antimicrobial effector proteins, such as lysozymes, defensin-like peptides, neuropeptide-like proteins, and caenacins (Dierking et al., 2016). Of these antimicrobial proteins, lysozymes are involved in host defense against various pathogens (Boehnisch et al., 2011; Irazoqui et al., 2010a; Mallo et al., 2002; O’Rourke et al., 2006; Visvikis et al., 2014). A previous study has demonstrated that expressions of lysozyme genes, such as lys-2, lys-7, and lys-8, are markedly up-regulated in 4-day-old worms, which is dependent on DAF-16 (Li et al., 2019). We thus determined the role of these lysozyme genes in the lifespan of worms. We found that a single mutation in lys-7(ok1384) slightly but significantly reduced the lifespan of worms fed live E. coli OP50 (Figure 6—figure supplement 1A), but not HK E. coli OP50 (Figure 6—figure supplement 1B), which was consistent with a previous observation (Portal-Celhay et al., 2012). In contrast, whereas either a single mutation in lys-8(ok3504) or RNAi knockdown of lys-2 did not affect the lifespan when worms were grown on live E. coli OP50, or HK E. coli OP50 (Figure 6—figure supplement 1A, B). However, lys-7(ok1384); lys-8(ok3504) double mutants exhibited a shortened lifespan when worms were fed live E. coli OP50 (Figure 6A). In contrast, the lifespan of lys-7(ok1384); lys-8(ok3504) double mutants was comparable to that of WT worms fed HK E. coli OP50 (Figure 6B). Furthermore, knockdown of lys-2 by RNAi did not affect the lifespan of lys-7(ok1384); lys-8(ok3504) double mutants grown on live E. coli OP50, or HK E. coli OP50 (Figure 6—figure supplement 1C, D). Both the rates of pharyngeal-pumping (Figure 6C) and body bending (Figure 6D) were reduced in 8-day-old lys-7(ok1384); lys-8(ok3504) double mutants fed live E. coli OP50. Moreover, the body-cavity leakage was increased in the double mutants fed live E. coli OP50 on day 10 (Figure 6E). However, these age-associated markers in lys-7(ok1384); lys-8(ok3504) double mutants were comparable to those in age-matched WT worms fed HK E. coli OP50. In addition, double mutations in lys-7(ok1384); lys-8(ok3504) increased the CFU of E. coli OP50 (Figure 6F) as well as the accumulation of E. coli OP50 expressing RFP in worms on day 7 (Figure 6G). Finally, we found that supplementation with indole no longer extended the lifespan of worms and failed to suppress the increase the CFU of E. coli K-12 in lys-7(ok1384); lys-8(ok3504) double mutants (Figure 6—figure supplement 2A, B).

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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

Using the transgenic worms expressing either lys-7p::gfp or lys-8p::gfp, we found that the expressions of lys-7p::gfp and lys-8p::gfp were significantly up-regulated in worms fed live E. coli OP50 on days 4 and 7 (Figure 7—figure supplement 1A–D), but not in age-matched worms fed HK E. coli OP50 (Figure 7—figure supplement 1A–D). RNAi knockdown of daf-16 also significantly suppressed the expressions of lys-7p::gfp and lys-8p::gfp in these worms fed live E. coli OP50 (Figure 7A, B). Similar results were obtained by measuring the mRNA levels of lys-7 and lys-8 in daf-16(mu86) mutants using quantitive real-time PCR (qPCR) (Figure 7—figure supplement 1E, F). Furthermore, we found that expression of either lys-7p::gfp (Figure 7C) or lys-8p::gfp (Figure 7D) was reduced in worms fed E. coli K-12 ΔtnaA strain on days 4 and 7, compared to those in age-matched worms fed E. coli K-12 strain. Likewise, indole (100 μM) remarkably increased the expression of either lys-7p::gfp or lys-8p::gfp in young adult worms after 24 hr treatment (Figure 7E, F). Meanwhile, RNAi knockdown of trpa-1 significantly inhibited the expression of either lys-7p::gfp or lys-8p::gfp in 4-day-old worms fed E. coli K12 strain (Figure 7A, B). Finally, we found that the mRNA levels of lys-7 and lys-8 were significantly down-regulated in worms subjected to neuronal specific (Figure 7—figure supplement 2A), but not intestinal specific, knockdown of trpa-1 by RNAi on day 4 (Figure 7—figure supplement 2B). However, supplementation with indole only up-regulated the expression of lys-7 and lys-8 in worms subjected to intestinal specific (Figure 7—figure supplement 2C), but not neuronal specific, RNAi of trpa-1 (Figure 7—figure supplement 2D). These results suggest that LYS-7 and LYS-8 function as downstream molecules of DAF-16 to maintain normal lifespan by inhibiting bacterial proliferation in worms.

Figure 7 with 2 supplements see all
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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

Discussion

Using C. elegans and its dietary bacterium as a host–microbe model, we provide a striking example of how a host responds to microbial dysbiosis in the gut (Figure 8). As worms age, E. coli proliferates in the lumen, thereby producing and secreting more indole. When its concentration crosses a threshold value, the bacterially produced compound is perceived by TRPA-1 in worms. The TRPA-1 signaling triggers DAF-16 nuclear translocation, leading to up-regulation of lysozyme genes. These antimicrobial peptides help worms to maintain normal lifespan by limiting the bacterial proliferation.

Figure 8
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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.

A complication in understanding the impact of bacterial diets on the traits of the worm is the fact that initially bacteria are a source of food, but later become pathogenic (Garigan et al., 2002; Tan and Shapira, 2011). Thus, the accumulation of E. coli in the gut is harmful for organismal fitness of worms during the course of life. As a well-known regulator of longevity and innate immunity (Garsin et al., 2003; Zou et al., 2013), DAF-16 is activated in aged C. elegans (Li et al., 2019). This transcription factor is involved in both maintaining normal lifespan and limiting proliferation of E. coli in worms (Garigan et al., 2002; Portal-Celhay and Blaser, 2012; Portal-Celhay et al., 2012). In this study, our data demonstrate that activation of DAF-16 requires contact with live bacterial cells in the gut of worms as dead E. coli fails to activate DAF-16. Thus, the accumulation of E. coli during aging, but not aging itself, results in the activation of DAF-16. Furthermore, DAF-16 mutation does not influence the lifespan in worms fed dead E. coli. Taken together, these findings clearly demonstrate that DAF-16 acts to maintain homeostasis by inhibiting bacterial proliferation in worms with age.

Indole is produced from tryptophan by tryptophanase in a large number of bacterial species. As a well-known signaling molecule, indole is involved in regulation of a variety of physiological processes in bacteria, such as cell division, biofilm formation, virulence, spore formation, and antibiotic resistance (Lee et al., 2015; Zarkan et al., 2020). Although animals cannot synthesize indole, they can sense and modify this metabolite (Lee et al., 2015). Indole and its derivatives can influence insect behaviors and human diseases, such as intestinal inflammation and diabetes (Agus et al., 2018; Lee et al., 2015). Thus, indole may function as an interspecies and interkingdom signaling molecule to influence the microbe–host interaction (Bansal et al., 2010; Oh et al., 2012; Ye et al., 2021). For instance, enteric delivery of indole (1 mM) increases the intestinal motility by inducing 5-hydroxytryptamine secretion in zebrafish larvae (Ye et al., 2021). Supplementation with 0.2 mM indole enhances the resistance of C. elegans to infection with C. albicans by reducing fungal colonization in the intestine (Oh et al., 2012). In addition, treatment of HCT-8 intestinal epithelial cells with 1 mM indole inhibits tumor necrosis factor α (TNF-α) mediated activation of nuclear factor-kappa B (NF-κB) and up-regulation of the inflammatory factor IL-8, thus improving intestinal epithelial barrier function (Bansal et al., 2010). Our data show that indole limits the bacterial proliferation in the gut of worms by driving intestinal defense gene expression via DAF-16. These findings suggest that the bacteria-derived metabolite may serve as a pathogen-associated molecular pattern that is recognized by metazoans. Although indole at a higher concentration (5 mM) is capable of inhibiting cell division in E. coli K12 strain (Chimerel et al., 2012), exogenous indole has little effect on the growth of E. coli K12 strain up to 3 mM (Chant and Summers, 2007). In general, extracellular indole concentrations detected in stationary phase LB cultures are typically 0.5–1 mM depending on the specific E. coli strain (Zarkan et al., 2020). Our data show that indole is sufficient to inhibit the accumulation of E. coli BW25113 at the concentration range from 0.05 to 0.2 mM. Thus, these results suggest that direct causal involvement of cell cycle arrest in E. coli by indole is unlikely.

Our data demonstrate that the cold-sensitive TRP channel TRPA-1 in neurons is involved in indole-mediated nuclear translocation of DAF-16 in the intestine, which is required for lifespan extension in C. elegans. Recently, Ye et al., 2021 have shown that both indole and indole-3-carboxaldehyde (IAld) are TRPA-1 agonists in vertebrates. trpa-1 is widely expressed in a variety of sensory neurons in worms (Kindt et al., 2007). Low temperature also activates TRPA-1, which in turn acts to promote longevity via the PKC-2–SGK-1–DAF-16 pathway (Xiao et al., 2013). It should be noted that unlike indole, activation of TPRA-1 by low temperature promotes the transcription activity, but not the nuclear translocation, of DAF-16 (Xiao et al., 2013). Furthermore, our data show that knockdown of sgk-1 dose not influence the nuclear translocation of DAF-16 induced by indole. Finally, indole extends lifespan via TRPA-1 only in nervous system, whereas low temperature extends lifespan via TRPA-1 both in the intestine and neurons. Thus, indole-mediated lifespan extension is essentially different from low temperature-mediated lifespan extension, although TRPA-1 and DAF-16 are involved in both processes. One possibility is that only neurons with activated TRPA-1 may release a neurotransmitter, which in turn triggers a signaling pathway to extend the lifespan of worms via activating DAF-16 in a non-cell autonomous manner, while the intestinal activated TRPA-1 is unable to release the neurotransmitter. Indeed, TRPA-1 induces the releasing of calcitonin gene-related peptide in perivascular sensory nerves, which in turn binds corresponding G-protein-coupled receptor, and causes membrane hyperpolarization and arterial dilation on smooth muscle cells (Talavera et al., 2020). Recently, Ye et al. have shown that both indole and indole-3-carboxaldehyde (IAld) are TRPA-1 agonists in vertebrates (Ye et al., 2021). As an agonist of TRPA-1, podocarpic acid can activate SKN-1 via TRPA-1 in worms (Chaudhuri et al., 2016). However, podocarpic acid fails to trigger DAF-16 translocation. Unlike podocarpic acid, indole cannot activate SKN-1 in worms. Although both indole and podocarpic acid are non-electrophilic agonists of TAPR-1, the structures of podocarpic acid and indole are totally different. These results suggest that TRPA-1 activation by podocarpic acid, low temperature, and indole occurs largely through distinct mechanisms.

In this study, our data demonstrate that DAF-16 inhibits bacterial proliferation by up-regulating expression of two lysozyme genes, lys-7 and lys-8. As a group of digestive enzymes with antimicrobial properties, lysozymes play an important role in the innate immunity in both vertebrate and invertebrate animals (O’Rourke et al., 2006). As a target gene of DAF-16, lys-7 has been proven to play an important role in resistance against a variety of pathogens, such as Microbacterium nematophilum (O’Rourke et al., 2006), Pseudomonas aeruginosa (Nandakumar and Tan, 2008), the pathogenic E. coli LF82 (Simonsen et al., 2011), Bacillus thuringiensis (Boehnisch et al., 2011), and Cryptococcus neoformans (Marsh et al., 2011). A previous study has demonstrated that a mutation in lys-7 significantly reduces the lifespan, but does not influence the accumulation of E. coli OP50 in the intestine of 2-day-old worms (Portal-Celhay et al., 2012). In the current study, the lys-7 mutants exhibit reduced the lifespan and elevated bacterial loads in the intestine of worms on days 4 and 7. This discrepancy may be due to the worms at different ages. Actually, our data also show that the lys-7;lys-8 double mutants exhibit a comparable accumulation of E. coli OP50 in worms on day 1. Although lys-8 mutation dose not influence either the lifespan or bacterial loads, it enhances the effect of lys-7. These results suggest that lys-8 acts in synergy with lys-7 to limit bacterial accumulation in the gut of worms.

It has been well established that bacterial dysbiosis is significantly associated with IBDs (Manichanh et al., 2012; Sommer and Bäckhed, 2013). Interestingly, reduced levels of indole and its derivative indole-3-propionic acid (IPA) are observed in serum of mice with dextran sulfate sodium-induced colitis and patients with IBD (Alexeev et al., 2018). Oral administration of IPA significantly ameliorates disease symptoms and promotes intestinal homeostasis by up-regulating colonic epithelial IL-10R1 in the chemically induced murine colitis model. Thus, characterization of the role for indole and its derivatives in host–microbiota interactions within the mucosa may provide new therapeutic avenues for inflammatory intestinal diseases.

Materials and methods

Nematode strains

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daf-16(mu86), lys-7(ok1384), lys-8(ok3504), LD1[skn-1b/c::gfp+rol-6(su1006)], the nematode strain for neuronal-specific RNAi, TU3401 (sid-1(pk3321); uIs69 [pCFJ90 (myo-2p:: mCherry)+unc-119p::sid-1]), TJ356 (zIs356 [daf-16p::daf-16a/b::GFP+rol-6 (su1006)]), and SAL105 (denEx2 [lys-7::GFP+pha-1(+)]) were kindly provided by the Caenorhabditis Genetics Center (CGC; http://www.cbs.umn.edu/CGC), funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The nematode strain for intestinal-specific RNAi, MGH170 (sid-1(qt9); Is[vha-6pr::sid-1]; Is[sur-5pr::GFPNLS]), was kindly provided by Dr. Gary Ruvkun (Massachusetts General Hospital, Harvard Medical School). The strain MQD1586 (476[hsp-16.2p::nCherry; dod-3p::gfp; mtl-1::bfp, unc-119(+)]) was kindly provided by Dr. Mengqiu Dong (Beijing Institute of Life Sciences). The strain trpa-1(ok999) was kindly provided by Dr. Jianke Gong (Huazhong University of Science and Technology). Mutants were backcrossed three times into the N2 strain used in the laboratory. All strains were maintained on NGM and fed with E. coli OP50 at 20°C.

Study design for age-related dysbiosis study

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Synchronized L1 larvae were grown on NGM agar plates seeded with E. coli OP50 at 20°C until they reached the young adult stage. All the experiments started from the young adult stage, which was considered day 0. From days 1 to 10, the worms were transferred to new NGM plates containing E. coli strains at 20°C daily for further experiments. For preparing for HK E. coli OP50, the bacterium cultured overnight in liquid LB medium was treated with 75°C water bath for 90 min (Qi et al., 2017). For preparing for ampicillin-killed E. coli OP50, the bacterium cultured overnight in liquid LB medium was seeded onto NGM plates containing 200 μg/ml ampicillin. Then the OP50 strain was periodically streaked onto ampicillin-containing and ampicillin-free LB plates to confirm that it could not proliferate (Garigan et al., 2002). BW25113 (E. coli K-12 WT) and E. coli K-12 ΔtnaA strain were obtained from the Keio collection (Baba et al., 2006).

RNA interference

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RNAi bacterial strains containing targeting genes were obtained from the Ahringer RNAi library (Kamath and Ahringer, 2003). All clones used in this study were verified by sequencing. Briefly, E. coli strain HT115 (DE3) expressing dsRNA was grown in LB (Luria-Bertani) containing 100 μg/ml ampicillin at 37°C for overnight, and then spread onto NGM plates containing 100 μg/ml ampicillin and 5 mM isopropyl 1-thio-β-D-galactopyranoside. The RNAi-expressing bacteria were then grown at 25°C overnight. Synchronized L1 larvae were placed on the plates at 20°C until they reached maturity. Young adult worms were used for further experiments.

Construction of transgenic strains

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The vector expressing lys-8p::gfp was generated by subcloning a 2011-bp promoter fragment of lys-8 into an expression vector (pPD95.75). The vector was injected into the syncytial gonads of WT worms with 50 ng/ml pRF4 as a transformation marker (Mello and Fire, 1995). The transgenic worms carrying were confirmed before assay.

DAF-16 nuclear localization assay

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For the effect of aging on DAF‐16::GFP localization, worms expressing daf-16p::daf-16::gfp were cultured on standard NGM plates at 20°C for 1, 4, and 7 days, respectively. For indole treatment, young adults were transferred to NGM plates containing 50–200 μM indole (Macklin, Shanghai, China) dissolved in dimethylsulfoxide (DMSO) for 24 hr at 20°C. NGM plates with equal amount of DMSO served as a control. After taken from incubation, worms were immediately mounted in M9 onto microscope slides. The slides were viewed using a Zeiss Axioskop 2 plus fluorescence microscope (Carl Zeiss, Jena, Germany) with a digit camera. The status of DAF-16 localization was categorized as cytosolic localization, nuclear localization when localization is observed throughout the entire body, or intermediate localization when nuclear localization is visible, but not completely throughout the body (Oh et al., 2005). At least 35 nematodes were counted per assay in three independent experiments.

Fluorescence microscopic analysis

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For imaging fluorescence, worms expressing hsp-16.2p::nCherry, dod-3p::gfp, lys-7p::gfp, and lys-8p::gfp were mounted in M9 onto microscope slides. The slides were imaged using a Zeiss Axioskop 2 Plus fluorescence microscope. The intensities of nCherry and GFP were analyzed using the ImageJ software (NIH). Three plates of at least 35 animals per plate were tested per assay, and all experiments were performed three times independently.

Lifespan analysis

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Synchronized L1 larvae were grown on NGM agar plates seeded with E. coli OP50 at 20°C until they reached the young adult stage. All the lifespan assays started from the young adult stage at 20°C. The first day of adulthood was recorded as day 1. From days 1 to 10, the worms were transferred to new NGM plates containing E. coli strains at 20°C daily. After that, worms were transferred every third day. The number of worms was counted every day. Worms that did not move when gently prodded and displayed no pharyngeal pumping were marked as dead. Every lifespan detection had three independent experiments (n > 35 worms per experiment).

Age-related phenotypic marker assays

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The following two age-related phenotypes were scored in 1- and 7-day-old worms (Chen et al., 2019). Pharyngeal pumping was measured by counting the number of contractions in the terminal bulb of pharynx in 30-s intervals. Body bending was measured by counting the number of body bends in 30-s intervals. At least 20 animals were determined per assay in five independent experiments.

Intestinal barrier function assay in worms

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Intestinal barrier function was determined according to the method described previously (Ma et al., 2020). Briefly, synchronized young adult animals were cultured on standard NGM plates at 20°C for 1, 4, 7, and 10 days. After removed from the NGM plates, these animals were suspended in M9 liquid medium containing E. coli OP50 (OD = 0.5–0.6), 5% food dye FD&C Blue No. 1 (Bis[4-(N-ethyl-N-3-sulfophenylmethyl) aminophenyl]-2-sulfophenylmethylium disodium salt) (AccuStandard, New Haven, CT), and incubated for 6 hr. After collected and washed with M9 buffer four times, the worms were mounted in M9 onto microscope slides. The slides were viewed using a Zeiss Axioskop 2 Plus fluorescence microscope (Carl Zeiss, Jena, Germany) to measure the leakage of the dyes in the body cavity of animals. The rate of body-cavity leakage was calculated as a percentage by dividing the number of animals with dye leakage by the number of total animals. For each time point, five independent experiments were carried out. In each experiment, at least 20 of worms were calculated.

Detection of bacterial accumulation in worms

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For detection of E. coli accumulation, worms were grown on NGM plates with E. coli expressing mCherry (the plasmid of PMF440 purchased from addgene) for 1, 4, and 7 days at 20°C. Then animals were collected and soaked in M9 buffer containing 25 mM levamisole hydrochloride (Sangon Biotech Co), 50 μg/ml kanamycin (Sangon Biotech Co), and 100 μg/ml ampicillin (Sangon Biotech Co) for 30 min at room temperature. Then worms were washed three times with M9 buffer. Some of animals were mounted in M9 onto microscope slides. The slides were viewed using a Zeiss Axioskop 2 plus fluorescence microscope (Carl Zeiss, Jena, Germany) with a digital camera. At least 35 worms were examined per assay in three independent experiments. Meanwhile, at least 30 of nematodes were transferred into 50 μl PBS plus 0.1% Triton and ground. The lysates were diluted by 10-fold serial dilutions in sterilized water and spread over LB agar plates with 100 μg/ml ampicillin. After incubation overnight at 37°C, the E. coli CFU was counted. For each group, three to five independent experiments were carried out.

Isolation and identification of the active compound

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Ten liters of the E. coli OP50 culture supernatants were collected by centrifugation, and freeze-dried in a VirTis freeze dryer. The powders were then dissolved with 10 ml of methanol. The crude extract was loaded on to an Ultimate 3000 HPLC (Thermo Fisher, Waltham, MA) coupled with automated fraction collector in batches through a continuous gradient on an Agilent ZORBAX SB-C18 column (Agilent, 5 μm, 4.6 × 250 mm) at a column temperature of 40°C to yield 45 fractions based on retention time and one fraction was collected per minute. The total flow rate was 1 ml/min; mobile phase A was 0.1% formic acid in water; and mobile phase B was 0.1% formic acid in acetonitrile. The HPLC conditions were manually optimized on the basis of separation patterns with the following gradient: 0–2 min, 10% B; 10 min, 25% B; 30 min, 35% B; 35 min, 90% B; 36 min, 95% B; 40 min, 90% B; 40.1 min, 10% B; and 45 min, 10% B. UV spectra were recorded at 204–400 nm. The injection volume for the extracts was 50 μl. After freeze-drying each fraction, 1 mg metabolites were dissolved in DMSO for DAF-16 nuclear localization assay (Supplementary file 2). The 26th fraction with DAF-16 nuclear translocation-inducing activity was further separated on silica gel column (200–300 mesh) with a continuous gradient of decreasing polarity (100%, 70%, 50%, 30%, petroleum ether/acetone) to yield four fractions (26a, 26b, 26c, and 26d). The fraction 26b with inducing DAF-16 nuclear translocation-inducing activity was further purified using a Sephadex LH-20 column to yield 32 fractions. The fraction 26b-11 with DAF-16 nuclear translocation-inducing activity contained a high-purity compound through thin layer chromatography detection. Then 26b-11 was structurally elucidated with NMR and MS data. NMR experiments were carried out on a Bruker DRX-500 spectrometer (Bruker Corp, Madison, WI) with solvent as internal standard. High-resolution MS data were performed on Q Exactive Focus UPLC‐MS (Thermo Fisher) with a PDA detector and an Obitrap mass detector using positive mode electrospray ionization.

Quantitative analysis of indole in worms

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For quantitation of indole in C. elegans, worms were collected, washed three to five times with M9 buffer, and lyophilized for 4–6 hr using a VirTis freeze dryer. Dried pellets were weighed, and transferred to a 1.5-ml centrifuge tube. After grinded for 10 min in a tissue grinder, the samples were dissolved in 300 µl solvent (methanol:water, 20:80 vol/vol). The mixtures were then grinded for another 10 min, and centrifuged at 12,000 × g for 5  min. The supernatants were collected, and filtered through 0.22 µm membranes for further analysis using LC–MS. LC–MS analyses were performed on a Q Exactive Focus UPLC-MS (Thermo Fisher) with an atmospheric pressure chemical ionization (APCI) source and operated with positive mode and coupled with Atlabtis dC18 column (Waters, 3 µm, 2.1 × 150 mm). Five microliters of samples were injected to the LC–MS system for analysis. The total flow rate was 0.3 ml/min; mobile phase A was 0.1% formic acid in water; and mobile phase B was 0.1% formic acid in acetonitrile. A/B gradient started at 5% B for 3 min after injection and increased linearly to 95% B at 13 min, then back to 5% B over 0.1 min and finally held at 5% B for an additional 1.9 min to re-equilibrate the column. Quantitation of indole was then achieved using standard curves generated using indole standard. Standard curve concentrations at 10, 50, 100, 300, 500, 700, and 9000 pmol/l in the solvent (methanol:water, 20:80 vol/vol). The data were analyzed and processed using Xcalibur software (Thermo Fisher).

Construction of K12△tnaA::tnaA strain

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All the PCR products were amplified using GXL high-fidelity DNA Polymerase (TaKaRa Biotechnology Co Ltd, Dalian, China). A 1846-bp fragment encompassing the tnaA gene transcription unit was amplified from the genomic DNA of E. coli K12 (Abdelaal and Yazdani, 2021). The transcription unit fragment and vector fragment were ligated by In-fusion Kit. The products were transformed into DH5ɑ competent cells. Then the cells were harvested, and spread onto selective medium containing 50 μg/ml of aburamycin. The plate was incubated overnight at 37°C until colonies were observed. After transformed colonies were screened by colony PCR, the correct positive transformant was transferred into an LB liquid medium (50 μg/ml aburamycin), and incubated overnight. Positive plasmids were extracted, and verified by DNA sequencing. The sequenced tnaA rescued plasmids were transformed into tnaA strain by electroporation method. The tnaA rescued strains were cultured for contents and DAF-16 nuclear translocation assay.

Quantitative real time PCR analysis

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Total RNA from worms was isolated using Trizol reagent (Invitrogen, Carlsbad, CA). Random-primed cDNAs were generated by reverse transcription of the total RNA samples using a standard protocol. A quantitative real-time PCR analysis was performed with a Roche Light Cycler 480 System (Roche Applied Science, Penzberg, Germany) using SYBR Premix (Takara, Dalian, China). The relative amount of lys-7 or lys-8 mRNA to act-1 mRNA (an internal control) was calculated using the method described previously (Pfaffl, 2001). The primers used for PCR were as follows: act-1: 5′-CGT GTT CCC ATC CAT TGT CG-3′ (F), 5′-AAG GTG TGA TGC CAG ATC TTC-3′ (R); lys-7: 5′-GTC TCC AGA GCC AGA CAA TCC-3′ (F), 5′-CCA GTG ACT CCA CCG CTG TA-3′ (R); lys-8: 5′-GCT TCA GTC TCC GTC AAG GTC-3′ (F), 5′-TGA AGC TGG CTC AAT GAA AC-3′ (R).

Statistics

Differences in survival rates were analyzed using the log-rank test. Differences in mRNA levels, the percentage of body-cavity leakage, fluorescence intensity, indole contents, and CFU were assessed by performing one-way ANOVA followed by the Student–Newman–Keuls test or by performing a two-tailed t-test. Differences in DAF-16::GFP nuclear accumulation were analyzed using the Chi-square test. Data were analyzed using GraphPad Prism 8.0.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting source data file.

References

    1. Mello C
    2. Fire A
    (1995)
    DNA transformation
    Methods in Cell Biology 48:451–482.

Decision letter

  1. Martin Sebastian Denzel
    Reviewing Editor; Altos Labs, United Kingdom
  2. Wendy S Garrett
    Senior Editor; Harvard T.H. Chan School of Public Health, United States
  3. Martin Sebastian Denzel
    Reviewer; Altos Labs, United Kingdom

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article “Indole produced during dysbiosis mediates host-microorganism chemical” for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Martin Sebastian Denzel as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Wendy Garrett as the Senior Editor.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) How was indole identified as a biologically relevant molecule?

2) What is the role of daf-2 in the context of their work?

3) How is indole sensed? Does it signal to or interact with TRPA1?

4) Is indole taken up by the intact gut or only after leakage occurs? How might it affect DAF-16?

Reviewer #1 (Recommendations for the authors):

In all, this is a very convincing paper that beautifully delineates the microbiome/host cross-talk in C. elegans. I have a few thoughts that, if addressed, would further strengthen the paper.

How was indole identified to be the relevant molecule? How was "activity-guided isolation" done? I see in the methods that fraction 26 was identified – it would be nice to provide the data in the paper that lead to this important finding in the paper.

Dysbiosis for the purpose of this paper is defined as the aberrant growth of normal bacteria. I wonder about the daf-16 response to pathogenic bacteria. Do pathogens produce more (or less) indole? Is indole a "marker" for the presence of live bacteria in the gut, or would it be a specific response to certain bacterial strains?

The text refers to "endogenous indole": this meant indole produced by bacteria in the gut? If so, it would help to word this more specifically.

Survival statistics: I was unable to access the source data – would it be possible to specify the survival experiments in the legends or in the methods? How many survival assays were done per experiment and how many animals were used?

Reviewer #2 (Recommendations for the authors):

Below I detail the main issues:

1. It looks like the authors are aware of the complications of using heat-killed bacteria as a control (given the added controls in supplementary figures). Indeed, adult worms don't do well on heat-killed E. coli and were suggested to be starved, either energetically, or for certain essential nutrients. All experiments would have been easier to interpret if paraformaldehyde-killed bacteria were used as controls (see Beydoun et al., 2021). Nevertheless, the authors did include controls with antibiotic-killed bacteria, and with starvation per se, to demonstrate that starvation is not involved, and that baseline established for comparisons is similar in worms raised on bacteria killed not by heat (method describing how ampicillin was employed to kill bacteria should be included, as well as a note on whether bacteria were dead or only metabolically inactive). The additional control experiments, currently presented in Figure 1- supp. 1 and 2, should be incorporated into the main Figure 1.

2. Throughout the paper, the authors used SEMs to describe the variance in their data. SDs are more appropriate for this.

3. The authors seem to suggest that the sensing of indole is inside the gut, released by accumulating bacteria. This leaves the question of how such sensing occurs unanswered. As far as I know, there is no knowledge about neuronal sensing of gut conditions. This should be addressed. The authors demonstrate tight correlation between gut bacteria CFUs and indole levels (Figure 2- supp. 3), but given the ability of supplemented external indole (more info is required about how this is carried out) to increase internal indole concentrations, I don't see why should such correlation exist – there are two sources for gut indole levels – colonizing bacteria and external bacteria, so excess environmental availability should drown any correlation.

Perhaps modulating bacterial accumulation (as in grinder defective tnt-3 mutants), could help clarify that.

4. The importance of TRPA-1 for indole-induced activation of DAF-16 is central to the paper. To provide stronger support, the authors may want to replicate the phenotypes, shown in Figure 4 with trpa-1 RNAi, also in trpa-1 null mutants.

Also, are the effects of TRPA-1 and DAF-16 KO/KD additive or not? This is important to establish the axis suggested here.

5. Lack of SKN-1 nuclear localization does not strike me as the most conclusive evidence for lack of p38 activation by indole. For one, p38 also activates ATF-7. The authors should also run qRT-PCR for p38 targets, for example T24B8.5, or use available GFP reporter strains for such targets.

6. The authors should also examine the outcome of activating TRPA-1 with its previously described agonist podacarpic acid (which they mention). Does it activate DAF-16 nuclear localization? Seems to be simple enough.

For discussion: do the authors consider indole as TRPA-1 agonist? Is TRPA-1 a ligand-activated channel? Have homologs been shown in other organisms to have more than one agonist, with different outcomes? How similar are the structures of podacarpic acid and indole?

7. Given the many targets of DAF-16, and the redundant nature of innate immune protection, how do the authors explain the ability of lys-7 and lys-8 to account for the full contribution of DAF-16 to lifespan and OP50 control?

https://doi.org/10.7554/eLife.85362.sa1

Author response

Essential revisions:

1) How was indole identified as a biologically relevant molecule?

We appreciate the concerns of the reviewing editor. Activity-guided isolation was

performed as follows: The crude extract of E. coli supernatant metabolites was divided into 45 fractions according to polarity using Ultimate 3000 HPLC (Thermofisher, Waltham, MA) coupled with automated fraction collector. After freeze-drying each fraction, 1 mg of metabolites were dissolved in DMSO for DAF-16 nuclear localization assay in worms (Please see new Supplementary Table S2). The 26th fraction with DAF-16 nuclear translocation-inducing activity was then separated on silica gel column (200-300 mesh) with a continuous gradient of decreasing polarity (100%, 70%, 50%, 30%, petroleum ether/acetone) to yield four fractions (26a-d). Only the fraction of 26b could induce DAF-16 nuclear translocation. Then the fraction was further separated using a Sephadex LH-20 column to yield 32 fractions. The 26b-11th fraction with DAF-16 nuclear translocation-inducing activity contained a single compound identified by thin layer chromatography, mass spectrometry and nuclear magnetic resonance (NMR). The compound exhibited a quasimolecular ion peak at m/z 181.0782 [M+H]+ in the positive APCI-MS, and was assigned to a molecular formula of C8H7N. A comparison of these 1H NMR and 13C NMR spectra with the data reported in the literature revealed that the compound was indole (Yagudaev, 1986). The detailed information was provided in our revised version.

2) What is the role of daf-2 in the context of their work?

We appreciate the concerns of the reviewing editor. It has been shown that DAF-2

initiates a kinase cascade that leads to the phosphorylation and cytoplasmic retention of DAF-16. By contrast, a reduction in the DAF-2 signaling leads to the dephosphorylation of DAF-16, allowing its nuclear translocation. We found that the mRNA levels of daf-2 were significantly increased in worms on days 4 and 7 in the presence of either live or dead E. coli OP50, compared with those in worms on day 1 (Author response image 1A). The nuclear translocation of DAF-16 in the intestine was increased in worms fed live E. coli OP50 on days 4 and 7, but not in age-matched WT worms fed heat-killed (HK) E. coli OP50 or ampicillin-killed E. coli OP50 (Figure 1A-1C). In addition, supplementation with indole significantly induced the nuclear translocation of DAF-16 (Figure 2B), but did not alter the mRNA levels of daf-2 in young adult worms (Author response image 1B). To conclude, the activation of DAF-16 is independent of DAF-2 during C. elegans aging.

Author response image 1
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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.

3) How is indole sensed? Does it signal to or interact with TRPA1?

We thank the reviewing editor for raising this important issue. The transporter of

indole has not been identified in C. elegans. In Arabidopsis, ATP-binding cassette (ABC) transporter G family 37(ABCG37) has been reported to transport a range of indole derivatives (Ruzicka et al., 2010). However, all fifteen C. elegans ABC transporters share less than 30% sequence identity with ABCG37, making it difficult to determine which one is the transporter for indole and indole derivatives (Author response image 2). Recently, Ye et al. have demonstrated that indole and indole-3-carboxaldehyde (IAld) are agonists of TRPA1, which is conserved in vertebrates (Ye et al., 2021). Thus, it is mostly likely that indole acts as an agonist of TRPA-1 in C. elegans by directly binding to TRPA-1.

Author response image 2
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Sequence alignment between Arabidopsis ABCG37 and 15 C. elegans ABC transporters.

4) Is indole taken up by the intact gut or only after leakage occurs? How might it affect DAF-16?

We appreciate the concerns of the reviewing editor. Although the transporter of indole in C. elegans remains to be characterized, we exclude a possible mechanism by which an increase in the indole levels in worms is associated with gut leakage. We found that supplementation with indole significantly increased the indole levels in young adult worms on day 1 (Figure 2—figure supplement 3B), and induced the nuclear translocation of DAF-16 in worms (Figure 2B). The intestine barrier is intact in 1-day-old worms (Figure 1I). Thus, the indole is taken up by the intact gut.

We speculate that the activation of TRPA-1 by indole in neurons could induce a pathway that may release a neurotransmitter, thereby leading to the activation of DAF-16 in the intestine.

Reviewer #1 (Recommendations for the authors):

In all, this is a very convincing paper that beautifully delineates the microbiome/host cross-talk in C. elegans. I have a few thoughts that, if addressed, would further strengthen the paper.

How was indole identified to be the relevant molecule? How was "activity-guided isolation" done? I see in the methods that fraction 26 was identified – it would be nice to provide the data in the paper that lead to this important finding in the paper.

We appreciate the concerns of the reviewer. Activity-guided isolation was performed as follows: The crude extract of E. coli supernatant metabolites was divided into 45 fractions according to polarity using Ultimate 3000 HPLC (Thermofisher, Waltham, MA) coupled with automated fraction collector. After freeze-drying each fraction, 1 mg of metabolites were dissolved in DMSO for DAF-16 nuclear localization assay in worms (Please see new Supplementary Table S2). The 26th fraction with DAF-16 nuclear translocation-inducing activity was then separated on silica gel column (200-300 mesh) with a continuous gradient of decreasing polarity (100%, 70%, 50%, 30%, petroleum ether/acetone) to yield four fractions (26a-d). Only the fraction of 26b could induce DAF-16 nuclear translocation. Then the fraction was further separated using a Sephadex LH-20 column to yield 32 fractions. The 26b-11th fraction with DAF-16 nuclear translocation-inducing activity contained a single compound identified by thin layer chromatography, mass spectrometry and nuclear magnetic resonance (NMR). The compound exhibited a quasimolecular ion peak at m/z 181.0782 [M+H]+ in the positive APCI-MS, and was assigned to a molecular formula of C8H7N. A comparison of these 1H NMR and 13C NMR spectra with the data reported in the literature revealed that the compound was indole (Yagudaev, 1986). The detailed information was provided in our revised version.

Dysbiosis for the purpose of this paper is defined as the aberrant growth of normal bacteria. I wonder about the daf-16 response to pathogenic bacteria. Do pathogens produce more (or less) indole? Is indole a "marker" for the presence of live bacteria in the gut, or would it be a specific response to certain bacterial strains?

In response to the reviewer’s suggestion, we tested three pathogenic bacteria (Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, and Staphylococcus aureus). We found that the nuclear translocation of DAF-16 was also increased in the intestine of worms fed S. Typhimurium 468, but not P. aeruginosa PA14 and S. aureus ATCC 25923 (Author response image 3A). Furthermore, we measured the levels of indole in the worms fed with each of these pathogenic bacteria. We found that the levels of indole were 80.4, 25.5, 22.8, and 23.1 nmol/g dry weight in worms fed live E. coli OP50, S. Typhimurium 468, P. aeruginosa PA14, and S. aureus ATCC 25923 on day 4, respectively (Author response image 3B). In addition, the levels of indole were 426.2, 0.7, 1.1, and 21.9 μmol/L in the culture

supernatants of E. coli, S. typhimurium, P. aeruginosa, and S. aureus, respectively (Author response image 3C). Based on these observations, we conclude that indole-induced DAF-16 activation is a specific response to E. coli.

Author response image 3
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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.

The text refers to "endogenous indole": this meant indole produced by bacteria in the gut? If so, it would help to word this more specifically.

We appreciate the concerns of the reviewer. Yes, endogenous indole we described here means “indole produced by bacteria in the gut”. We have changed the “endogenous indole” to “indole produced by bacteria in the gut” in the revised manuscript.

Survival statistics: I was unable to access the source data – would it be possible to specify the survival experiments in the legends or in the methods? How many survival assays were done per experiment and how many animals were used?

Lifespan data are collected in source data.

Reviewer #2 (Recommendations for the authors):

Below I detail the main issues:

1. It looks like the authors are aware of the complications of using heat-killed bacteria as a control (given the added controls in supplementary figures). Indeed, adult worms don't do well on heat-killed E. coli and were suggested to be starved, either energetically, or for certain essential nutrients. All experiments would have been easier to interpret if paraformaldehyde-killed bacteria were used as controls (see Beydoun et al., 2021). Nevertheless, the authors did include controls with antibiotic-killed bacteria, and with starvation per se, to demonstrate that starvation is not involved, and that baseline established for comparisons is similar in worms raised on bacteria killed not by heat (method describing how ampicillin was employed to kill bacteria should be included, as well as a note on whether bacteria were dead or only metabolically inactive). The additional control experiments, currently presented in Figure 1- supp. 1 and 2, should be incorporated into the main Figure 1.

In response to the reviewer’s suggestion, the details of ampicillin was employed to

kill bacteria were provided in the section of Methods. Meanwhile, we incorporated the previous Figure 1—figure supplement 1 and 2 into Figure 1.

2. Throughout the paper, the authors used SEMs to describe the variance in their data. SDs are more appropriate for this.

Amended.

3. The authors seem to suggest that the sensing of indole is inside the gut, released by accumulating bacteria. This leaves the question of how such sensing occurs unanswered. As far as I know, there is no knowledge about neuronal sensing of gut conditions. This should be addressed. The authors demonstrate tight correlation between gut bacteria CFUs and indole levels (Figure 2- supp. 3), but given the ability of supplemented external indole (more info is required about how this is carried out) to increase internal indole concentrations, I don't see why should such correlation exist – there are two sources for gut indole levels – colonizing bacteria and external bacteria, so excess environmental availability should drown any correlation.

Perhaps modulating bacterial accumulation (as in grinder defective tnt-3 mutants), could help clarify that.

We appreciate the concerns of the reviewer. Reviewer #3 also pointed out this problem. In this study, our data showed that the levels of indole were 30.9, 71.9, and 105.9 nmol/g dry weight in worms fed live E. coli OP50 on days 1, 4, and 7, respectively (Figure 2C). This increase in the levels of indole in worms was accompanied by an increase in CFU of live E. coli OP50 in the intestine of worms with age (Figure 2C). In addition, we determined the levels of indole in worms fed HK E. coli OP50, and found that the levels of indole were 28.2, 31.6, and 36.1 nmol/g dry weight in worms fed HK E. coli OP50 on days 1, 4, and 7, respectively (Figure 2—figure supplement 3A). It should be noted that the levels of indole in worms fed dead E. coli OP50 on day 1 were comparable of those in worms fed live E. coli OP50 on day 1 (30.9 vs 28.2 nmol/g dry weight). However, the levels of indole were not increased in worms fed HK E. coli OP50 on days 4 and 7. Furthermore, the observation that DAF-16 was retained in the cytoplasm of the intestine in worms fed live E. coli OP50 on day 1 (Figure 1A and 1B) also indicates that indole produced by E. coli OP50 on the NGM plates is not enough to induce DAF-16 nuclear translocation. By contrast, supplementation with indole (50-200 μM) significantly increased the indole levels in worms on day 1 (Figure 2—figure supplement 3B), which could induce the nuclear translocation of DAF-16 in worms (Figure 2B). Thus, the increase in the levels of indole in worms with age results from intestinal accumulation of live E. coli OP50, rather than indole produced by E. coli OP50 on the NGM plates.

4. The importance of TRPA-1 for indole-induced activation of DAF-16 is central to the paper. To provide stronger support, the authors may want to replicate the phenotypes, shown in Figure 4 with trpa-1 RNAi, also in trpa-1 null mutants.

Also, are the effects of TRPA-1 and DAF-16 KO/KD additive or not? This is important to establish the axis suggested here.

We appreciate the concerns of the reviewer and have performed the suggested experiments. We found that supplementation with indole no longer extended the lifespan of trpa-1(ok999) mutants (new Figure 4—figure supplement 2A).

Furthermore, knockdown of daf-16 by RNAi did not further shorten the lifespan of trpa-1(ok999) mutants (new Figure 4—figure supplement 2A). Moreover, supplementation with indole failed to suppress the accumulation of E. coli K-12 strain in trpa-1(ok999) mutants subjected to either empty vector or daf-16 RNAi (new Figure 4—figure supplement 2B). These results suggest that indole exhibits its function in extending the lifespan of worms and inhibiting the bacterial accumulation primarily via TRPA-1-DAF-16 axis.

5. Lack of SKN-1 nuclear localization does not strike me as the most conclusive evidence for lack of p38 activation by indole. For one, p38 also activates ATF-7. The authors should also run qRT-PCR for p38 targets, for example T24B8.5, or use available GFP reporter strains for such targets.

We appreciate the concerns of the reviewer and have performed the suggested experiments. We tested the effect of indole on five p38 target genes (T24B8.5, F35e12.5, nlp-29, Y37a1a.2, and F08g5.6). We found that supplementation with indole only increased the mRNA level of Y37a1a.2, but not the other four genes (Author response image 4). These results suggest that indole treatment does not activate the PMK-1/p38 MAPK pathway.

Author response image 4
Download asset Open asset
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.

6. The authors should also examine the outcome of activating TRPA-1 with its previously described agonist podacarpic acid (which they mention). Does it activate DAF-16 nuclear localization? Seems to be simple enough.

For discussion: do the authors consider indole as TRPA-1 agonist? Is TRPA-1 a ligand-activated channel? Have homologs been shown in other organisms to have more than one agonist, with different outcomes? How similar are the structures of podacarpic acid and indole?

We appreciate the concerns of the reviewer and have performed the suggested

experiments. It has been shown that podocarpic acid can activate SKN-1 via TRPA-1 (Chaudhuri et al., 2016). We found that supplementation with 0.02 mM podocarpic acid failed to induce the nuclear translocation of DAF-16 in worms on day 1 (new Figure 4-figure supplement 3C). Xiao et al. have demonstrated that TRPA-1 activated by low temperatures mediates calcium influx, which in turn stimulates the PKC-2-SGK-1 signaling to promote the transcription activity of DAF-16 (Xiao et al., 2013). This suggests that TRPA-1 activation by podocarpic acid, low temperature and indole occurs largely through distinct mechanisms.

Recently, Ye et al. have shown that both indole and indole-3-carboxaldehyde (IAld) are TRPA1 agonists in vertebrates (Ye et al., 2021). The agonists of mammalian TRPA1 include both non-electrophilic and electrophilic agonists, such as icilin, carvacrol, docosahexaenoic acid (DHA), isoflurane, allyl isothiocyanate (AITC), acrolein, umbellulone, and etodolac (Nishida, Kuwahara, Kozai, Sakaguchi, and Mori, 2015). Activated TRPA1 have different effects in mammals. For example, TRPA1 induces the releasing of calcitonin gene-related peptide in perivascular sensory nerves, which in turn binds corresponding G protein-coupled receptor, and causes membrane hyperpolarization and arterial dilation on smooth muscle cells (Talavera et al., 2020). Additionally, the activation of TRPA1 by indole and IAld induces the secretion of the neurotransmitter serotonin in zebrafish (Ye et al., 2021).

Both Indole and podocarpic acid are non-electrophilic agonists of TAPR-1, the structures of podocarpic acid and indole are totally different. Podocarpic acid is an abietane diterpenoid lacking the isopropyl substituent with an aromatic C-ring and a hydroxy group at the 12-position (W.Campbell, 1942). Indole is an electron-rich aromatic system containing enamine embedded C2-C3 π bond and strong nucleophilic C3 carbon (Zhang et al., 2021).

In response to the reviewer’s suggestion, we added sentences “Recently, Ye et al. have shown that both indole and indole-3-carboxaldehyde (IAld) are TRPA1 agonists in vertebrates (Ye et al., 2021). As the agonist of TRPA-1, podocarpic acid can activate SKN-1 via TRPA-1 in worms (Chaudhuri et al., 2016). However, podocarpic acid fails to trigger DAF-16 translocation. Unlike podocarpic acid, indole cannot activate SKN-1 in worms. Although both Indole and podocarpic acid are non-electrophilic agonists of TAPR-1, the structures of podocarpic acid and indole are totally different. These results suggest that TRPA-1 activation by podocarpic acid, low temperature, and indole occurs largely through distinct mechanisms.” in the section of Discussion.

7. Given the many targets of DAF-16, and the redundant nature of innate immune protection, how do the authors explain the ability of lys-7 and lys-8 to account for the full contribution of DAF-16 to lifespan and OP50 control?

We appreciate the concerns of the reviewer. C. elegans possesses a variety of putative antimicrobial proteins, such as lysozymes, defensin-like peptides, neuropeptide-like proteins, and caenacins (Dierking, Yang, and Schulenburg, 2016), Of these antimicrobial proteins, the expressions of lysozyme genes, such as lys-2, lys-7, and lys-8, are markedly up-regulated in 4-day-old worms, which is dependent on DAF-16 (Li et al., 2019). Although we have demonstrated that LYS-7 and LYS-8 are required for normal lifespan, we do not exclude the role for other targets of DAF-16 in maintaining normal lifespan in worms.

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in Reactive α-Dicarbonyl Detoxification. Curr Biol, 26(22), 3014-3025.

Dierking, K., Yang, W., and Schulenburg, H. (2016). Antimicrobial effectors in the nematode Caenorhabditis elegans: an outgroup to the Arthropoda. Philos Trans R Soc Lond B Biol Sci, 371(1695).

Lee, R. Y., Hench, J., and Ruvkun, G. (2001). Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the daf-2 insulin-like signaling pathway. Curr Biol, 11(24), 1950-1957.

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https://doi.org/10.7554/eLife.85362.sa2

Article and author information

Author details

  1. Rui-Qiu Yang

    State key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, Kunming, China
    Contribution
    Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology
    Contributed equally with
    Yong-Hong Chen, Qin-yi Wu and Jie Tang
    Competing interests
    No competing interests declared

  2. Yong-Hong Chen

    State key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, Kunming, China
    Contribution
    Data curation, Software, Formal analysis, Investigation, Visualization, Methodology
    Contributed equally with
    Rui-Qiu Yang, Qin-yi Wu and Jie Tang
    Competing interests
    No competing interests declared

  3. Qin-yi Wu

    Yunnan Provincial Key Laboratory of Molecular Biology for Sinomedicine, Yunnan University of Traditional Chinese Medicine, Kunming, China
    Contribution
    Data curation, Software, Formal analysis, Investigation
    Contributed equally with
    Rui-Qiu Yang, Yong-Hong Chen and Jie Tang
    Competing interests
    No competing interests declared

  4. Jie Tang

    1. State key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, Kunming, China
    2. Yunnan Key Laboratory of Vaccine Research Development on Severe Infectious Disease, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming, China
    Contribution
    Software, Formal analysis, Investigation, Methodology
    Contributed equally with
    Rui-Qiu Yang, Yong-Hong Chen and Qin-yi Wu
    Competing interests
    No competing interests declared

  5. Shan-Zhuang Niu

    State key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, Kunming, China
    Contribution
    Software, Investigation
    Competing interests
    No competing interests declared

  6. Qiu Zhao

    State key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, Kunming, China
    Contribution
    Software, Formal analysis
    Competing interests
    No competing interests declared

  7. Yi-Cheng Ma

    State key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, Kunming, China
    Contribution
    Conceptualization, Resources, Funding acquisition, Visualization, Writing - original draft, Writing - review and editing
    For correspondence
    mayc@ynu.edu.cn
    Competing interests
    No competing interests declared

  8. Cheng-Gang Zou

    State key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, Kunming, China
    Contribution
    Conceptualization, Funding acquisition, Writing - original draft, Writing - review and editing
    For correspondence
    chgzou@ynu.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5519-4402

Funding

Ministry of Science and Technology of the People's Republic of China (2022YFD1400700)

  • Yi-Cheng Ma

Foundation for Innovative Research Groups of the National Natural Science Foundation of China (U1802233)

  • Cheng-Gang Zou

Yunnan Characteristic Plant Extraction Laboratory (2022YKZY006)

  • Cheng-Gang Zou

Foundation for Innovative Research Groups of the National Natural Science Foundation of China (31900420)

  • Yi-Cheng Ma

Foundation for Innovative Research Groups of the National Natural Science Foundation of China (32260021)

  • Yi-Cheng Ma

Yunnan Province of China (202001BC070004)

  • Cheng-Gang Zou

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank the Caenorhabditis Genetics Center, Dr. Gary Ruvkun, Dr. Mengqiu Dong, and Dr. Jianke Gong for nematode strains. This work was supported by a grant from the National Key R&D Program of China (2022YFD1400700), the National Natural Science Foundation of China (U1802233, 31900420, and 32260021), the independent research fund of Yunnan Characteristic Plant Extraction Laboratory (2022YKZY006), and the Major Science and Technology Project in Yunnan Province of China (202001BC070004).

Senior Editor

  1. Wendy S Garrett, Harvard T.H. Chan School of Public Health, United States

Reviewing Editor

  1. Martin Sebastian Denzel, Altos Labs, United Kingdom

Reviewer

  1. Martin Sebastian Denzel, Altos Labs, United Kingdom

Version history

  1. Received: December 5, 2022
  2. Preprint posted: December 19, 2022 (view preprint)
  3. Accepted: November 3, 2023
  4. Accepted Manuscript published: November 21, 2023 (version 1)
  5. Version of Record published: December 1, 2023 (version 2)

Copyright

© 2023, Yang, Chen, Wu et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Rui-Qiu Yang
  2. Yong-Hong Chen
  3. Qin-yi Wu
  4. Jie Tang
  5. Shan-Zhuang Niu
  6. Qiu Zhao
  7. Yi-Cheng Ma
  8. Cheng-Gang Zou
(2023)
Indole produced during dysbiosis mediates host–microorganism chemical communication
eLife 12:e85362.
https://doi.org/10.7554/eLife.85362

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    1. Biochemistry and Chemical Biology
    2. Evolutionary Biology

    Evolution towards simplicity in bacterial small heat shock protein system

    Piotr Karaś, Klaudia Kochanowicz ... Krzysztof Liberek
    Research Article

    Evolution can tinker with multi-protein machines and replace them with simpler single-protein systems performing equivalent functions in an equally efficient manner. It is unclear how, on a molecular level, such simplification can arise. With ancestral reconstruction and biochemical analysis, we have traced the evolution of bacterial small heat shock proteins (sHsp), which help to refold proteins from aggregates using either two proteins with different functions (IbpA and IbpB) or a secondarily single sHsp that performs both functions in an equally efficient way. Secondarily single sHsp evolved from IbpA, an ancestor specialized in strong substrate binding. Evolution of an intermolecular binding site drove the alteration of substrate binding properties, as well as the formation of higher-order oligomers. Upon two mutations in the α-crystallin domain, secondarily single sHsp interacts with aggregated substrates less tightly. Paradoxically, less efficient binding positively influences the ability of sHsp to stimulate substrate refolding, since the dissociation of sHps from aggregates is required to initiate Hsp70-Hsp100-dependent substrate refolding. After the loss of a partner, IbpA took over its role in facilitating the sHsp dissociation from an aggregate by weakening the interaction with the substrate, which became beneficial for the refolding process. We show that the same two amino acids introduced in modern-day systems define whether the IbpA acts as a single sHsp or obligatorily cooperates with an IbpB partner. Our discoveries illuminate how one sequence has evolved to encode functions previously performed by two distinct proteins.

    1. Biochemistry and Chemical Biology
    2. Computational and Systems Biology

    Death by a thousand cuts through kinase inhibitor combinations that maximize selectivity and enable rational multitargeting

    Ian R Outhwaite, Sukrit Singh ... Markus A Seeliger
    Research Article

    Kinase inhibitors are successful therapeutics in the treatment of cancers and autoimmune diseases and are useful tools in biomedical research. However, the high sequence and structural conservation of the catalytic kinase domain complicates the development of selective kinase inhibitors. Inhibition of off-target kinases makes it difficult to study the mechanism of inhibitors in biological systems. Current efforts focus on the development of inhibitors with improved selectivity. Here, we present an alternative solution to this problem by combining inhibitors with divergent off-target effects. We develop a multicompound-multitarget scoring (MMS) method that combines inhibitors to maximize target inhibition and to minimize off-target inhibition. Additionally, this framework enables optimization of inhibitor combinations for multiple on-targets. Using MMS with published kinase inhibitor datasets we determine potent inhibitor combinations for target kinases with better selectivity than the most selective single inhibitor and validate the predicted effect and selectivity of inhibitor combinations using in vitro and in cellulo techniques. MMS greatly enhances selectivity in rational multitargeting applications. The MMS framework is generalizable to other non-kinase biological targets where compound selectivity is a challenge and diverse compound libraries are available.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Ga chaperone and GEF Ric-8A

    Daniel Muñoz-Reyes, Levi J McClelland ... Maria Jose Sanchez-Barrena
    Research Article

    The Neuronal Calcium Sensor 1, an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Ga have revealed how Ric-8A phosphorylation promotes Ga recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Ga subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Ga. Our data show that the binding of NCS-1 and Ga to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to Casein Kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A GEF activity towards Ga when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.