Iron-deplete diet enhances Caenorhabditis elegans lifespan via oxidative stress response pathways
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Abstract
Gut microbes play a crucial role in modulating host lifespan. However, the microbial factors that influence host longevity and their mechanisms of action remain poorly understood. Using the expression of Caenorhabditis elegans FAT-7, a stearoyl-CoA 9-desaturase, as a proxy for lifespan modulation, we conduct a genome-wide bacterial mutant screen and identify 26 Escherichia coli mutants that enhance host lifespan. Transcriptomic and biochemical analyses reveal that these mutant diets induce oxidative stress and activate the mitochondrial unfolded protein response (UPRmt). Antioxidant supplementation abolishes lifespan extension, confirming that oxidative stress drives these effects. The extension of lifespan requires the oxidative stress response regulators SKN-1, SEK-1, and HLH-30. Mechanistically, these effects are linked to reduced iron availability, as iron supplementation restores FAT-7 expression, suppresses UPRmt activation, and abolishes lifespan extension. Iron chelation mimics the pro-longevity effects of the mutant diets, highlighting dietary iron as a key modulator of aging. Our findings reveal a bacterial-host metabolic axis that links oxidative stress, iron homeostasis, and longevity in C. elegans .
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Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Summary: In this paper, the authors perform a screen by feeding C. elegans different E. coli genetic mutants and examining the effect on the expression of fat-7, a stearoyl-CoA 9-desturase, which has been associated with longevity. They identify 26 E. coli strains that decrease fat-7 expression, all of which slow development and increase lifespan. RNA sequencing of worms treated with 4 of these strains identified genes involved in defense against oxidative stress among those genes that are commonly upregulated. Feeding C. elegans these 4 bacterial strains results in increased ROS and …
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Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Summary: In this paper, the authors perform a screen by feeding C. elegans different E. coli genetic mutants and examining the effect on the expression of fat-7, a stearoyl-CoA 9-desturase, which has been associated with longevity. They identify 26 E. coli strains that decrease fat-7 expression, all of which slow development and increase lifespan. RNA sequencing of worms treated with 4 of these strains identified genes involved in defense against oxidative stress among those genes that are commonly upregulated. Feeding C. elegans these 4 bacterial strains results in increased ROS and activation of the mitochondrial unfolded protein response, which appears to contribute to lifespan extension as these bacterial strains do not increase lifespan when the mitochondrial unfolded protein response transcription factor ATFS-1 is disrupted. Finally, the authors demonstrate a role for iron levels in mediating these phenotypes: iron supplementation inhibits the phenotypes caused by the identified bacterial strains, while iron chelation mimics these phenotypes. Response: We thank the reviewer for an excellent summary of our work.
Major comments: The proposed model involves an increase in ROS levels activating the UPRmt and then leading to lifespan extension. If the elevation is ROS levels is contributing then treatment with antioxidants should prevent UPRmt activation and lifespan extension. Response: This is an excellent point. We will treat the FAT-7-suppressing diets with antioxidants and observe the effect on C. elegans UPRmt activation and lifespan.
The authors suggest that iron depletion may disrupt iron-sulfur cluster proteins. The Rieske iron-sulfur protein ISP-1 from mitochondrial electron transport chain complex III has previously been associated with lifespan. Point mutations affecting the function of ISP-1 or RNAi decreasing the levels of ISP-1 both result in increased lifespan (PMID 20346072, 11709184). Thus, iron depletion may be increasing ROS, activating UPRmt and increasing lifespan through decreasing ISP-1 levels.
Response: The reviewer has raised an intriguing possibility that the increased lifespan on the FAT-7-suppressing diets could be because of perturbation of ISP-1 function. While ISP-1 levels may not be directly affected by the mutant diets, ISP-1 function might be perturbed on these diets as ISP-1 function requires iron-sulfur clusters. Therefore, we will study the lifespan of isp-1(qm150) mutant on the FAT-7-suppressing diets to explore whether the lifespan extension on these diets is ISP-1 dependent.
All of the Kaplan-meier survival plots are missing statistical analyses. Please add p-values.
Response: The p-values for all the survival plots are included in the respective figure legends.
It would be helpful to include a model diagram of the proposed mechanisms in the main figures.
Response: We will make a model diagram after completing the experiments suggested by the reviewers.
Minor comments: Rather than "mutant diets" it would be more informative to call these "FAT-7-decreasing diets"
Response: We have changed “mutant diets” to “FAT-7-suppressing diets” throughout the manuscript.
Is it surprising that none of the bacterial strains increased FAT-7 levels? Why do you think this is?
Response: Yes, it was indeed surprising to find only bacterial strains that reduced FAT-7 levels and none that increased them. One possible explanation is that these bacterial mutants may not directly regulate fat-7 expression. Instead, they might alter the overall dietary composition, which is known to influence fat-7 levels. It appears that none of the tested mutants modified the diet in a manner that would lead to fat-7 upregulation.
Page 5. "We hypothesized that diets reducing FAT-7 might elevate oleic acid levels". Since FAT-7 converts stearic acid to oleic acid, wouldn't deceasing FAT-7 levels decrease oleic acid levels and increase stearic acid levels?
Response: FAT-7 expression is regulated by a feedback mechanism and is sensitive to the fatty acid composition within host cells; elevated levels of unsaturated fatty acids, such as oleic acid, suppress FAT-7 expression. There are two possible ways bacterial mutants could lead to reduced FAT-7 levels: (1) by directly inhibiting FAT-7 expression, which would be expected to result in increased stearic acid levels; or (2) by supplying higher amounts of oleic acid through their composition, thereby suppressing FAT-7 expression via feedback regulation. We focused on the second possibility, as elevated oleic acid levels—like those seen with FAT-7-suppressing diets—are known to promote C. elegans lifespan. To avoid confusion, we have revised the statement to: “We hypothesized that bacterial diets might reduce FAT-7 expression because they have elevated levels of oleic acid”.
Page 6. The authors cite Bennett et al. 2014 for the statement that "Activation of the UPRmt has been associated with lifespan extension". This paper reaches the opposite conclusion "Activation of the mitochondrial unfolded protein response does not predict longevity in Caenorhabditis elegans". Also, in the Bennett paper and PMID 34585931, it is shown that constitutive activation of ATFS-1 decreases lifespan. Thus, the relationship between the UPRmt and lifespan is not straightforward. These points should be mentioned.
Response: The reviewer has raised an important point. We have now included a paragraph in the discussion to highlight these points. The revised manuscript reads: “All 26 FAT-7-suppressing diets identified in our study elevated hsp-6p::GFP expression and extended C. elegans lifespan. Although UPRmt activation and lifespan extension were consistently observed across these diets, there was no strong correlation between hsp-6p::GFP levels and the degree of lifespan extension. The role of the UPRmt in promoting longevity remains controversial (Bennett et al., 2014; Soo et al., 2021; Wu et al., 2018). For instance, gain-of-function mutations in atfs-1 have been shown to reduce lifespan (Bennett et al., 2014; Soo et al., 2021). However, a recent study demonstrated that mild UPRmt activation can extend lifespan, whereas strong activation has the opposite effect (Di Pede et al., 2025). These findings suggest that UPRmt contributes to longevity only under specific conditions and at specific activation levels. In our study, lifespan extension on FAT-7-suppressing diets was dependent on ATFS-1, indicating that UPRmt activation was necessary for this effect.”
Page 6. "Our transcriptomic analysis suggested elevated ROS". Rather than refer to gene expression, it would be better to refer to the ROS measurements that were performed.
Response: We have changed it to the following sentence: “Our ROS measurement analysis suggested elevated ROS levels in worms fed FAT-7-suppressing diets.”
The long-lived mitochondrial mutants isp-1 and nuo-6 have increased ROS, UPRmt activation and increased lifespan. Multiple studies have examined gene expression in these long-lived mutant strains. How does gene expression in these mutants compare to worms treated with the FAT-7-decreasing E. coli mutants? While not necessary for this publication, it would be interesting to see whether the FAT-7-decreasing E. coli strains can increase isp-1 and nuo-6 lifespan.
Response: We will compare the gene expression changes observed in isp-1 and nuo-6 mutants with the gene expression changes observed in worms exposed to FAT-7-suppressing diets. Additionally, we will examine the lifespan of isp-1 mutants on the mutant diets. These data will be included in the revised manuscript.
SEK-1 is also involved in the p38-mediated innate immune signaling pathway, which has been shown to contribute to longevity in C. elegans. In fact, disruption of sek-1 using RNAi decreased the lifespan of several long-lived mutant strains PMID 36514863.
Response: We thank the reviewer for highlighting this point. We have now added that the role of SEK-1 in regulating lifespan on FAT-7-suppressing diets could also be because of its role in innate immunity. The revised manuscript reads: “Notably, SEK-1 also regulates innate immunity and is essential for the extended lifespan observed in several long-lived C. elegans mutants (Soo et al., 2023). Therefore, its effect on lifespan in response to FAT-7-suppressing diets may also stem from its role in innate immune regulation.”
Figure 2. Why were cyoA and ycbk chosen to show the full Kaplan-meier survival plot?
Response: These were selected randomly to show the range of the lifespan phenotype observed.
Figure 2, panel D. A better title may be "Mean Survival (Percent increase from control)"
Response: We have made this change.
While not necessary for this paper, it would be interesting to determine whether the FAT-7-decreasing E. coli strains alter resistance to oxidative stress.
Response: We will study the survival of worms on these diets upon supplementation with paraquat.
Figure 4. It may be interesting to include a correlation plot comparing hsp-6::GFP fluorescence and lifespan. It looks like the magnitudes of increase for each phenotype are not correlated.
Response: We have added a new Figure (Figure S4) to show the correlation between hsp-6::GFP fluorescence levels and percent change in mean lifespan. Indeed, there is no correlation between these phenotypes.
Reviewer #1 (Significance (Required)):
Overall, this is an interesting paper and the experiments are rigorously performed. The bacterial screen was comprehensive and was followed up by careful mechanistic experiments. This paper will be of interest to researchers studying the biology of aging. A diagram of the working model of the underlying mechanisms would enhance the paper. Response: We thank the reviewer for highlighting the significance of the study. We will include a model in the revised manuscript.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
In this manuscript, Das et al. investigate how different bacterial mutants affect the lifespan of C. elegans. The authors screened a library of E. coli mutants using a fat-7 reporter and identified 26 strains that reduce fat-7 expression, cause developmental delay, induce the mitochondrial unfolded protein response (using hsp-6 reporter), and increase worm lifespan. Among these, they focused on four strains and demonstrated that the effects of these mutants on developmental delay, fat-7 expression, and hsp-6 induction could be suppressed by iron supplementation. Furthermore, they showed that iron depletion alone is sufficient to induce fat-7 expression in worms. The lifespan extension observed in worms fed these mutant bacterial strains depends on SKN-1, SEK-1, and HLH-30. Overall, this is a well-written manuscript that highlights the role of iron in regulating fat-7 expression. However, the findings from the initial screen do not significantly expand upon what is already known in the literature. Many of the identified hits overlap with those reported by Zhang et al. (2019), which also highlighted the role of iron in developmental delay and hsp-6 induction. While the lifespan data and the role of fat-7 are novel aspects of this study, the authors have not conducted detailed mechanistic investigations to address key questions, such as: 1) How does the deletion of these bacterial genes alter the metabolic state of the diet? 2) How do these metabolic changes influence fat-7 expression in worms? 3) How does the downregulation of fat-7 contribute to longevity? Addressing these points would strengthen the mechanistic insights of the study.
Response: We thank the reviewer for a thoughtful summary of our work and for the valuable feedback provided to improve the manuscript. We would like to emphasize that the screening conditions and objectives of our study were fundamentally different from those of Zhang et al. (2019). Furthermore, Zhang et al. (2019) did not investigate the effects of the bacterial mutants identified in their screens on C. elegans lifespan. Notably, the 26 bacterial mutants identified in our screen do not overlap with those reported in previous studies that examined bacterial strains promoting C. elegans longevity. As detailed below, we will address the points raised by the reviewer that will certainly strengthen the mechanistic insights of the study.
Here are my detailed comments:
- Suppressing FAT-7 levels in C. elegans does not inherently increase lifespan. To directly attribute this effect to FAT-7, it would be important to attempt a rescue experiment to restore FAT-7 expression and assess whether the lifespan extension persists. Additionally, measuring oleic acid levels in these mutants would help determine whether a high-oleic-acid diet is suppressing FAT-7 expression. The role of oleic acid cannot be ruled out using fat-2 mutants (Fig. 3B), as fat-2 mutants accumulate oleic acid when fed WT bacteria, but this may not translate to endogenous oleic acid accumulation in conditions where FAT-7 is suppressed.
Response: We thank the reviewer for these useful suggestions. We will overexpress FAT-7 under a pan-tissue promoter (eft-3) and study lifespan on FAT-7-suppressing diets. Moreover, to explore whether oleic acid has any role in enhancing lifespan on FAT-7-suppressing diets, we will study the lifespan of worms on these diets upon supplementing with oleic acid along with wild-type bacterium control.
To understand the host-microbe interaction in this study, it is important to determine what specific changes in the bacteria contribute to the observed phenotypes in worms. Identifying these bacterial factors will provide a clearer picture of their role in influencing worms stress signaling and lifespan.
Response: The phenotypes observed in C. elegans across all the identified bacterial mutants are remarkably consistent, including increased UPRmt activation, reduced FAT-7 levels, delayed development, and extended lifespan. This consistency suggests that a common underlying factor is driving these effects. Although the bacterial mutants appear genetically diverse, gene expression data from C. elegans, along with comparisons to the findings of Zhang et al. (2019), indicate that elevated levels of reactive oxygen species (ROS) may represent this shared factor. These results suggest that bacterial ROS play a central role in mediating the host-microbe interactions underlying the observed phenotypes. To further support this hypothesis, we will directly measure ROS levels in the identified bacterial mutants. Additionally, we will test whether antioxidant treatment can suppress the C. elegans phenotypes, thereby establishing a causal role for bacterial ROS.
It is important to rule out any changes in food consumption in worms fed these bacterial mutants, as differences in feeding amount could attribute to the observed lifespan effects.
Response: We will carry out pharyngeal pumping rate measurements to study whether there is any difference in food consumption in worms fed these bacterial mutants.
In figure 5A to 5G, please include the same-day controls to help clarify how iron supplementation effects these phenotypes relative to the control. For example, in Fig. 5F, it appears that iron extends the lifespan of worms fed the control diet. It would be clearer if appropriate controls were included in all of these figures or summarized in a table to help understand the impact of iron.
Response: We will include these controls in the revised manuscript.
How does iron depletion affect the levels of fat-7, and how does this contribute to the activation of the longevity pathways discussed in the manuscript.
Response: This is an intriguing question. There are at least two possible explanations: (1) oxidative stress may directly downregulate fat-7 expression, and (2) iron depletion could reduce ferroptosis, which in turn may influence fatty acid metabolism. In the revised manuscript, we will include data on how oxidative stress affects FAT-7 expression.
Minor comments
- Please include a detailed table of the lifespan data for all replicates as a supplementary table.
Response: We have included the details of survival curves for all the data in the new Table S2.
In the Methods section, specify at what stage the worms were exposed to iron and the iron chelator for the lifespan experiments.
Response: The L1-synchronized worms were exposed to iron and iron chelator plates and allowed to develop till the late L4 stage before being transferred to lifespan assay plates that also contained the respective supplements. This information is now included in the Methods section.
Please clarify whether equal optical density (O.D.) of cells was seeded for both the WT and mutant strains, and mention if the mutants exhibit any growth defects.
Response: We have examined the growth of the bacterial mutants and found that they do not exhibit growth defects. Therefore, for all the assays, NGM plates were seeded with saturated cultures of all the bacterial strains. We have now included the growth curves data in the manuscript (Figure S4).
Reviewer #2 (Significance (Required)):
Significance General Assessment: This study by Das et al. explores the impact of bacterial mutants on C. elegans lifespan. A key strength of the study is the identification of bacterial mutants that influence the expression of the gene encoding fatty acid desaturase (fat-7) and lifespan in C. elegans. Furthermore, the study highlights the role of iron in regulating fat-7 expression, suggesting that iron imbalance may play a crucial role in modulating fatty acid metabolism. However, the study's main limitation is that it does not significantly extend the current understanding of the microbial modulation of host metabolism and aging, as many of the identified bacterial hits overlap with those previously reported in Zhang et al. (2019). The manuscript would benefit from more in-depth mechanistic exploration, especially with regard to how specific bacterial factors influence the metabolic state of the worms and how these changes ultimately modulate fat-7 expression and longevity.
Response: We thank the reviewer for highlighting the significance of our study. Once again, we would like to emphasize that the screening conditions and objectives of our study differed fundamentally from those of Zhang et al. (2019). Furthermore, Zhang et al. did not investigate the impact of the bacterial mutants identified in their screen on C. elegans lifespan. As outlined above, we will address the reviewer’s comments, which will undoubtedly strengthen the mechanistic insights of our study.
Advance: This study presents a conceptual advance by exploring the iron-dependent regulation of fat-7 expression and lifespan in C. elegans, linking bacterial mutations with key longevity pathways (SKN-1, SEK-1, and HLH-30). The novelty lies in the direct investigation of the bacterial-induced changes in fat-7 expression, though the role of iron in these mutants for development and induction of mito-UPR was previously shown in the literature. This study also adds to the growing body of work on C. elegans as a model for studying aging and host-microbe interactions, particularly in understanding how diet and microbial exposure affect metabolic processes and lifespan.
Response: We thank the reviewer for highlighting the advancement made by our study.
Audience: This research will primarily interest specialized audiences in aging research, microbiology, and metabolism, especially those focused on host-microbe interactions. Keywords of my expertise: Host-microbe interactions, metabolism, system biology, C. elegans, aging.
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #2
Evidence, reproducibility and clarity
In this manuscript, Das et al. investigate how different bacterial mutants affect the lifespan of C. elegans. The authors screened a library of E. coli mutants using a fat-7 reporter and identified 26 strains that reduce fat-7 expression, cause developmental delay, induce the mitochondrial unfolded protein response (using hsp-6 reporter), and increase worm lifespan. Among these, they focused on four strains and demonstrated that the effects of these mutants on developmental delay, fat-7 expression, and hsp-6 induction could be suppressed by iron supplementation. Furthermore, they showed that iron depletion alone is sufficient to …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
In this manuscript, Das et al. investigate how different bacterial mutants affect the lifespan of C. elegans. The authors screened a library of E. coli mutants using a fat-7 reporter and identified 26 strains that reduce fat-7 expression, cause developmental delay, induce the mitochondrial unfolded protein response (using hsp-6 reporter), and increase worm lifespan. Among these, they focused on four strains and demonstrated that the effects of these mutants on developmental delay, fat-7 expression, and hsp-6 induction could be suppressed by iron supplementation. Furthermore, they showed that iron depletion alone is sufficient to induce fat-7 expression in worms. The lifespan extension observed in worms fed these mutant bacterial strains depends on SKN-1, SEK-1, and HLH-30.
Overall, this is a well-written manuscript that highlights the role of iron in regulating fat-7 expression. However, the findings from the initial screen do not significantly expand upon what is already known in the literature. Many of the identified hits overlap with those reported by Zhang et al. (2019), which also highlighted the role of iron in developmental delay and hsp-6 induction. While the lifespan data and the role of fat-7 are novel aspects of this study, the authors have not conducted detailed mechanistic investigations to address key questions, such as: 1) How does the deletion of these bacterial genes alter the metabolic state of the diet? 2) How do these metabolic changes influence fat-7 expression in worms? 3) How does the downregulation of fat-7 contribute to longevity? Addressing these points would strengthen the mechanistic insights of the study.
Here are my detailed comments:
- Suppressing FAT-7 levels in C. elegans does not inherently increase lifespan. To directly attribute this effect to FAT-7, it would be important to attempt a rescue experiment to restore FAT-7 expression and assess whether the lifespan extension persists. Additionally, measuring oleic acid levels in these mutants would help determine whether a high-oleic-acid diet is suppressing FAT-7 expression. The role of oleic acid cannot be ruled out using fat-2 mutants (Fig. 3B), as fat-2 mutants accumulate oleic acid when fed WT bacteria, but this may not translate to endogenous oleic acid accumulation in conditions where FAT-7 is suppressed.
- To understand the host-microbe interaction in this study, it is important to determine what specific changes in the bacteria contribute to the observed phenotypes in worms. Identifying these bacterial factors will provide a clearer picture of their role in influencing worms stress signaling and lifespan.
- It is important to rule out any changes in food consumption in worms fed these bacterial mutants, as differences in feeding amount could attribute to the observed lifespan effects.
- In figure 5A to 5G, please include the same-day controls to help clarify how iron supplementation effects these phenotypes relative to the control. For example, in Fig. 5F, it appears that iron extends the lifespan of worms fed the control diet. It would be clearer if appropriate controls were included in all of these figures or summarized in a table to help understand the impact of iron.
- How does iron depletion affect the levels of fat-7, and how does this contribute to the activation of the longevity pathways discussed in the manuscript.
Minor comments
- Please include a detailed table of the lifespan data for all replicates as a supplementary table.
- In the Methods section, specify at what stage the worms were exposed to iron and the iron chelator for the lifespan experiments.
- Please clarify whether equal optical density (O.D.) of cells was seeded for both the WT and mutant strains, and mention if the mutants exhibit any growth defects.
Significance
General Assessment: This study by Das et al. explores the impact of bacterial mutants on C. elegans lifespan. A key strength of the study is the identification of bacterial mutants that influence the expression of the gene encoding fatty acid desaturase (fat-7) and lifespan in C. elegans. Furthermore, the study highlights the role of iron in regulating fat-7 expression, suggesting that iron imbalance may play a crucial role in modulating fatty acid metabolism. However, the study's main limitation is that it does not significantly extend the current understanding of the microbial modulation of host metabolism and aging, as many of the identified bacterial hits overlap with those previously reported in Zhang et al. (2019). The manuscript would benefit from more in-depth mechanistic exploration, especially with regard to how specific bacterial factors influence the metabolic state of the worms and how these changes ultimately modulate fat-7 expression and longevity.
Advance: This study presents a conceptual advance by exploring the iron-dependent regulation of fat-7 expression and lifespan in C. elegans, linking bacterial mutations with key longevity pathways (SKN-1, SEK-1, and HLH-30). The novelty lies in the direct investigation of the bacterial-induced changes in fat-7 expression, though the role of iron in these mutants for development and induction of mito-UPR was previously shown in the literature. This study also adds to the growing body of work on C. elegans as a model for studying aging and host-microbe interactions, particularly in understanding how diet and microbial exposure affect metabolic processes and lifespan.
Audience: This research will primarily interest specialized audiences in aging research, microbiology, and metabolism, especially those focused on host-microbe interactions.
Keywords of my expertise: Host-microbe interactions, metabolism, system biology, C. elegans, aging.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary:
In this paper, the authors perform a screen by feeding C. elegans different E. coli genetic mutants and examining the effect on the expression of fat-7, a stearoyl-CoA 9-desturase, which has been associated with longevity. They identify 26 E. coli strains that decrease fat-7 expression, all of which slow development and increase lifespan. RNA sequencing of worms treated with 4 of these strains identified genes involved in defense against oxidative stress among those genes that are commonly upregulated. Feeding C. elegans these 4 bacterial strains results in increased ROS and activation of the mitochondrial unfolded protein …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary:
In this paper, the authors perform a screen by feeding C. elegans different E. coli genetic mutants and examining the effect on the expression of fat-7, a stearoyl-CoA 9-desturase, which has been associated with longevity. They identify 26 E. coli strains that decrease fat-7 expression, all of which slow development and increase lifespan. RNA sequencing of worms treated with 4 of these strains identified genes involved in defense against oxidative stress among those genes that are commonly upregulated. Feeding C. elegans these 4 bacterial strains results in increased ROS and activation of the mitochondrial unfolded protein response, which appears to contribute to lifespan extension as these bacterial strains do not increase lifespan when the mitochondrial unfolded protein response transcription factor ATFS-1 is disrupted. Finally, the authors demonstrate a role for iron levels in mediating these phenotypes: iron supplementation inhibits the phenotypes caused by the identified bacterial strains, while iron chelation mimics these phenotypes.
Major comments:
The proposed model involves an increase in ROS levels activating the UPRmt and then leading to lifespan extension. If the elevation is ROS levels is contributing then treatment with antioxidants should prevent UPRmt activation and lifespan extension.
The authors suggest that iron depletion may disrupt iron-sulfur cluster proteins. The Rieske iron-sulfur protein ISP-1 from mitochondrial electron transport chain complex III has previously been associated with lifespan. Point mutations affecting the function of ISP-1 or RNAi decreasing the levels of ISP-1 both result in increased lifespan (PMID 20346072, 11709184). Thus, iron depletion may be increasing ROS, activating UPRmt and increasing lifespan through decreasing ISP-1 levels.
All of the Kaplan-meier survival plots are missing statistical analyses. Please add p-values.
It would be helpful to include a model diagram of the proposed mechanisms in the main figures.
Minor comments:
Rather than "mutant diets" it would be more informative to call these "FAT-7-decreasing diets"
Is it surprising that none of the bacterial strains increased FAT-7 levels? Why do you think this is?
Page 5. "We hypothesized that diets reducing FAT-7 might elevate oleic acid levels". Since FAT-7 converts stearic acid to oleic acid, wouldn't deceasing FAT-7 levels decrease oleic acid levels and increase stearic acid levels?
Page 6. The authors cite Bennett et al. 2014 for the statement that "Activation of the UPRmt has been associated with lifespan extension". This paper reaches the opposite conclusion "Activation of the mitochondrial unfolded protein response does not predict longevity in Caenorhabditis elegans". Also, in the Bennett paper and PMID 34585931, it is shown that constitutive activation of ATFS-1 decreases lifespan. Thus, the relationship between the UPRmt and lifespan is not straightforward. These points should be mentioned.
Page 6. "Our transcriptomic analysis suggested elevated ROS". Rather than refer to gene expression, it would be better to refer to the ROS measurements that were performed.
The long-lived mitochondrial mutants isp-1 and nuo-6 have increased ROS, UPRmt activation and increased lifespan. Multiple studies have examined gene expression in these long-lived mutant strains. How does gene expression in these mutants compare to worms treated with the FAT-7-decreasing E. coli mutants? While not necessary for this publication, it would be interesting to see whether the FAT-7-decreasing E. coli strains can increase isp-1 and nuo-6 lifespan.
SEK-1 is also involved in the p38-mediated innate immune signaling pathway, which has been shown to contribute to longevity in C. elegans. In fact, disruption of sek-1 using RNAi decreased the lifespan of several long-lived mutant strains PMID 36514863.
Figure 2. Why were cyoA and ycbk chosen to show the full Kaplan-meier survival plot?
Figure 2, panel D. A better title may be "Mean Survival (Percent increase from control)"
While not necessary for this paper, it would be interesting to determine whether the FAT-7-decreasing E. coli strains alter resistance to oxidative stress.
Figure 4. It may be interesting to include a correlation plot comparing hsp-6::GFP fluorescence and lifespan. It looks like the magnitudes of increase for each phenotype are not correlated.
Significance
Overall, this is an interesting paper and the experiments are rigorously performed. The bacterial screen was comprehensive and was followed up by careful mechanistic experiments. This paper will be of interest to researchers studying the biology of aging. A diagram of the working model of the underlying mechanisms would enhance the paper.
-
