Intestinal GCN2 controls Drosophila systemic growth in response to Lactiplantibacillus plantarum symbiotic cues encoded by r/tRNA operons
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Evaluation Summary:
Previous studies found that a component of the microbiota, Lactobacillus plantarum, can provide support to its host Drosophila melanogaster during development. They further explore this interaction using defined diets where they find that under conditions that have low levels of some essential amino acids, the bacteria can still promote survival even though the bacteria is not synthesizing the amino acid. Through a screen of bacterial transposon insertion mutants, these authors identify bacterial transfer and ribosomal RNAs as necessary for this effect. And studies in the fly demonstrate that the host kinase GCN2, a protein known to associate with host tRNAs, in enterocytes is the mediator of this response. This manuscript links the intestinal microbiota to host protective responses.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Symbiotic bacteria interact with their host through symbiotic cues. Here, we took advantage of the mutualism between Drosophila and Lactiplantibacillus plantarum (Lp) to investigate a novel mechanism of host-symbiont interaction. Using chemically defined diets, we found that association with Lp improves the growth of larvae-fed amino acid-imbalanced diets, even though Lp cannot produce the limiting amino acid. We show that in this context Lp supports its host’s growth through a molecular dialogue that requires functional operons encoding ribosomal and transfer RNAs (r/tRNAs) in Lp and the general control nonderepressible 2 (GCN2) kinase in Drosophila ’s enterocytes. Our data indicate that Lp’s r/tRNAs are packaged in extracellular vesicles and activate GCN2 in a subset of larval enterocytes, a mechanism necessary to remodel the intestinal transcriptome and ultimately to support anabolic growth. Based on our findings, we propose a novel beneficial molecular dialogue between host and microbes, which relies on a non-canonical role of GCN2 as a mediator of non-nutritional symbiotic cues encoded by r/tRNA operons.
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Author Response
Reviewer #1 (Public Review):
This is an interesting article that uses the power of drosophila to explore how organisms work with their symbionts to adapt to a changing environment. The authors show that reducing some nonessential amino acids that cannot be produced by the "symbiont" Lactobacillus can nevertheless be rescued by the presence of this bacteria. They suggest it is not through provisioning from the bacteria using genetic screens in the bacteria, they find four bacterial strains that have a reduced ability to restore the delay. They then show that the mutants have transposon insertions in r/tRNA loci and reduced rRNA levels. These mutants and a newly generated deletion allele shows similar phenotypes (although very modest (~1day change). due to imabalance. Experiments next demonstrate that colonization with …
Author Response
Reviewer #1 (Public Review):
This is an interesting article that uses the power of drosophila to explore how organisms work with their symbionts to adapt to a changing environment. The authors show that reducing some nonessential amino acids that cannot be produced by the "symbiont" Lactobacillus can nevertheless be rescued by the presence of this bacteria. They suggest it is not through provisioning from the bacteria using genetic screens in the bacteria, they find four bacterial strains that have a reduced ability to restore the delay. They then show that the mutants have transposon insertions in r/tRNA loci and reduced rRNA levels. These mutants and a newly generated deletion allele shows similar phenotypes (although very modest (~1day change). due to imabalance. Experiments next demonstrate that colonization with Lp leads to induction of an ATF4 reporter independent of diet. But that colonization of the mutant Lp, has reduced activation during a balanced diet but not in an imbalanced diet. This was also the case for a mutant identified in the screen. Next the authors explore the role of enterocyte GCN2. They show that there are selective requirements for GNC2 depending on the diet and aa imbalance. This is very complicated. As the depletion of GCN2 by one allele does not impact GF pupation on an imbalanced diet, it does for other alleles. And they find that this activity is independent of ATF4 and 4EBP, two known members of the pathway.
Major strengths include the screen for bacterial mutants and demonstration that depletion of specific amino acids have specific dependencies (both bacterial and host). However, there is a disconnect between the bacterial mutants and the host physiology. How do the mutants impact host biology? Is it through an RNA signal? If so how does this get sensed? Is GCN2 involved, and if so by what mechanism?
We thank the reviewer for his/her evaluation. The connection between the L. plantarum (Lp) mutants and host physiology is mostly established by the following observations:
bacterial mutants for r/tRNAs failed to activate GCN2 to the same extent as WT bacteria. Although the difference on imbalanced diet is not significant (p-value=0.069, new Fig. 5A-B), there is a trend towards a decreased activation with the r/tRNA deletion mutant. We also observed this trend with the r/tRNA insertion mutant (new Fig. S4A-B). This decrease reached statistical significance when we performed short-term association (new Fig. S4E-F) or on balanced diet (new Fig. 5C-D and new Fig. S4C-D).
providing tRNAs to larvae supports activation of GCN2 in enterocytes (new Fig. 5E-F).
knocked-down of GCN2 in enterocytes using RNAi triggers a growth delay in larvae (new Fig. 6A, new Fig. S5A-B).
when we knocked-down GCN2 using RNAi, we did not observe any difference between the growth of larvae associated with Lp WT and the r/tRNA mutant (new Fig. 6H-I).
We believe these results strongly indicate that the phenotype of delayed growth upon association with r/tRNA mutant relies at least partly on a decreased GCN2 activation in enterocytes. Given the mechanism of activation of GCN2 (GCN2 is activated by structured RNA such as tRNAs or rRNAs) we propose that GCN2 is a sensor of bacterial r/tRNAs. This is supported by our new finding that Lp produces extracellular vesicles containing r/tRNAs (new Fig. 3). However, we agree that this point remains speculative. We amended our Abstract and Discussion accordingly (L30, L924-929) to clarify that direct activation of GCN2 by Lp’s r/tRNAs remains speculative.
Reviewer #2 (Public Review):
This manuscript investigates an intriguing observation, the data are strong, and the manuscript is clearly written. The authors very convincingly demonstrate that regions of the chromosome that encode L. plantarum tRNAs are also necessary for activation of D. melanogaster GCN2 and accelerated development in the setting of AA imbalance and that this effect on development is dependent on GCN2. They further provide transcriptomic data that broaden our understanding of the host intestinal response to L. plantarum in the setting of AA imbalance. In other host-microbe interactions such as the squid-Vibrio fischeri symbiosis, the bacterial RNA has been visualized in host cells, suggesting transport. Here, experimental data demonstrating bacterial RNA in host cells is lacking and then direct interaction of GCN2 with prokaryotic tRNAs is hypothesized but not proven. As a result, the basis of the observed effect of bacterial tRNAS remains vague. Open questions such how/if the bacterial tRNA enters the host enterocytes, whether these interact with GCN2, and whether other bacterial products are required for the response remain to be answered.
We thank the reviewer for his/her interest in our work. Association with LpΔopr/tRNA leads to reduced activation of GCN2 in enterocytes, and tRNAs feeding activate GCN2. Given the mechanism of activation of GCN2, we speculate that tRNAs produced by Lp directly interacts with GCN2 in enterocytes. We add new data showing that Lp produces extracellular vesicles, and these vesicles contain r/tRNAs (new Fig. 8). Since extracellular vesicles can transport molecules from bacteria to hosts (Brown et al. 2015) this observation supports our model: enterocytes may acquire Lp’s r/tRNAs from extracellular vesicles.
Reviewer #3 (Public Review):
The strength of this study relies on the use of a chemically well-defined diet of the host and of the identification of Lp mutants that fail to rescue the noxious effects of an imbalanced amino-acid regimen. Thus, the genetic approach in both host and symbiont is a major asset of this study. The results are surprising as an imbalance of one essential amino-acid in the diet, valine, can nevertheless be compensated by Lp, even though it is itself unable to synthesize this amino-acid. The experiments are well-conducted and conclusions are appropriate.
We thank the reviewer for his/her kind words and for his/her interest in our work.
This study however does not identify how GCN2 promotes growth in this context. There is just a descriptive transcriptomics approach that is however not validated at the functional level (and also not by RTqPCR experiments) as it does not provide obvious leads beyond a Gene Ontology exploitation of the data.
To answer the reviewer’s questions, we have further characterized one hit from our RNAseq analysis: Lp association causes down-regulation of the growth repressor fezzik. We show that fezzik knock-down in enterocytes improves larval growth, which suggests that Lp improves growth partly through GCN2-dependant r/tRNA-dependent repression of fezzik expression (new Fig. 8 and new Fig. S8).
The authors propose that Lp promotes a more thorough absorption of valine, a possibility that makes sense but is not backed up by any data.
We now provide new data showing that association with Lp increases the amounts of Valine in larva’s hemolymph (new Fig. 1E). Since Lp cannot produce Valine, this supports our model of increased nutrient absorption by the gut of Lp-associated larvae.
Also, how Lp releases r/tRNAs is not addressed experimentally.
We now provide new data showing that Lp produces extracellular vesicles that contain r/tRNAs (new Fig. 3).
A minor logical flaw is the use of GCN2 pathway activation read-outs that are actually not required to mediate Lp's beneficial action.
Our hypothesis is that GCN2 activation leads to both activation of ATF4, which is not required to mediate Lp’s beneficial action, and induction of other targets (e.g. fezzik repression, EGFR activation) that are required to mediate Lp’s beneficial action. We showed that ATF4 activation is a good readout of GCN2 activation (GCN2 knock-down completely suppresses the reporter’s expression in the anterior midgut, new Fig. 4C-F).
The authors claim that GCN2 action is not mediated through ATF4 or Thor based on RNA interference experiments. However, in contrast to the GCN2 case, they have not validated the RNAi lines and tested also only one for each.
To address the reviewer’s concerns, we have used two lines of 4E-BP loss-of-function alleles. These lines do not show a growth delay on imbalanced diet (new Fig. S5I). Regarding ATF4, we used the RNAseq to validate the ATF4-RNAi: the Mex>ATF4RNAi-Lp condition shows a statistically significant ~8 fold reduction in ATF4 expression compared to the control-Lp condition (N.B. ATF4 is annotated as crc in our dataset).
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Evaluation Summary:
Previous studies found that a component of the microbiota, Lactobacillus plantarum, can provide support to its host Drosophila melanogaster during development. They further explore this interaction using defined diets where they find that under conditions that have low levels of some essential amino acids, the bacteria can still promote survival even though the bacteria is not synthesizing the amino acid. Through a screen of bacterial transposon insertion mutants, these authors identify bacterial transfer and ribosomal RNAs as necessary for this effect. And studies in the fly demonstrate that the host kinase GCN2, a protein known to associate with host tRNAs, in enterocytes is the mediator of this response. This manuscript links the intestinal microbiota to host protective responses.
(This preprint has been reviewed …
Evaluation Summary:
Previous studies found that a component of the microbiota, Lactobacillus plantarum, can provide support to its host Drosophila melanogaster during development. They further explore this interaction using defined diets where they find that under conditions that have low levels of some essential amino acids, the bacteria can still promote survival even though the bacteria is not synthesizing the amino acid. Through a screen of bacterial transposon insertion mutants, these authors identify bacterial transfer and ribosomal RNAs as necessary for this effect. And studies in the fly demonstrate that the host kinase GCN2, a protein known to associate with host tRNAs, in enterocytes is the mediator of this response. This manuscript links the intestinal microbiota to host protective responses.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This is an interesting article that uses the power of drosophila to explore how organisms work with their symbionts to adapt to a changing environment. The authors show that reducing some non-essential amino acids that cannot be produced by the "symbiont" Lactobacillus can nevertheless be rescued by the presence of this bacteria. They suggest it is not through provisioning from the bacteria using genetic screens in the bacteria, they find four bacterial strains that have a reduced ability to restore the delay. They then show that the mutants have transposon insertions in r/tRNA loci and reduced rRNA levels. These mutants and a newly generated deletion allele shows similar phenotypes (although very modest (~1day change). due to imabalance. Experiments next demonstrate that colonization with Lp leads to …
Reviewer #1 (Public Review):
This is an interesting article that uses the power of drosophila to explore how organisms work with their symbionts to adapt to a changing environment. The authors show that reducing some non-essential amino acids that cannot be produced by the "symbiont" Lactobacillus can nevertheless be rescued by the presence of this bacteria. They suggest it is not through provisioning from the bacteria using genetic screens in the bacteria, they find four bacterial strains that have a reduced ability to restore the delay. They then show that the mutants have transposon insertions in r/tRNA loci and reduced rRNA levels. These mutants and a newly generated deletion allele shows similar phenotypes (although very modest (~1day change). due to imabalance. Experiments next demonstrate that colonization with Lp leads to induction of an ATF4 reporter independent of diet. But that colonization of the mutant Lp, has reduced activation during a balanced diet but not in an imbalanced diet. This was also the case for a mutant identified in the screen. Next the authors explore the role of enterocyte GCN2. They show that there are selective requirements for GNC2 depending on the diet and aa imbalance. This is very complicated. As the depletion of GCN2 by one allele does not impact GF pupation on an imbalanced diet, it does for other alleles. And they find that this activity is independent of ATF4 and 4EBP, two known members of the pathway.
Major strengths include the screen for bacterial mutants and demonstration that depletion of specific amino acids have specific dependencies (both bacterial and host). However, there is a disconnect between the bacterial mutants and the host physiology. How do the mutants impact host biology? Is it through an RNA signal? If so how does this get sensed? Is GCN2 involved, and if so by what mechanism?
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Reviewer #2 (Public Review):
This manuscript investigates an intriguing observation, the data are strong, and the manuscript is clearly written. The authors very convincingly demonstrate that regions of the chromosome that encode L. plantarum tRNAs are also necessary for activation of D. melanogaster GCN2 and accelerated development in the setting of AA imbalance and that this effect on development is dependent on GCN2. They further provide transcriptomic data that broaden our understanding of the host intestinal response to L. plantarum in the setting of AA imbalance. In other host-microbe interactions such as the squid-Vibrio fischeri symbiosis, the bacterial RNA has been visualized in host cells, suggesting transport. Here, experimental data demonstrating bacterial RNA in host cells is lacking and then direct interaction of GCN2 with …
Reviewer #2 (Public Review):
This manuscript investigates an intriguing observation, the data are strong, and the manuscript is clearly written. The authors very convincingly demonstrate that regions of the chromosome that encode L. plantarum tRNAs are also necessary for activation of D. melanogaster GCN2 and accelerated development in the setting of AA imbalance and that this effect on development is dependent on GCN2. They further provide transcriptomic data that broaden our understanding of the host intestinal response to L. plantarum in the setting of AA imbalance. In other host-microbe interactions such as the squid-Vibrio fischeri symbiosis, the bacterial RNA has been visualized in host cells, suggesting transport. Here, experimental data demonstrating bacterial RNA in host cells is lacking and then direct interaction of GCN2 with prokaryotic tRNAs is hypothesized but not proven. As a result, the basis of the observed effect of bacterial tRNAS remains vague. Open questions such how/if the bacterial tRNA enters the host enterocytes, whether these interact with GCN2, and whether other bacterial products are required for the response remain to be answered.
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Reviewer #3 (Public Review):
The strength of this study relies on the use of a chemically well-defined diet of the host and of the identification of Lp mutants that fail to rescue the noxious effects of an imbalanced amino-acid regimen. Thus, the genetic approach in both host and symbiont is a major asset of this study. The results are surprising as an imbalance of one essential amino-acid in the diet, valine, can nevertheless be compensated by Lp, even though it is itself unable to synthesize this amino-acid. The experiments are well-conducted and conclusions are appropriate.
This study however does not identify how GCN2 promotes growth in this context. There is just a descriptive transcriptomics approach that is however not validated at the functional level (and also not by RTqPCR experiments) as it does not provide obvious leads …
Reviewer #3 (Public Review):
The strength of this study relies on the use of a chemically well-defined diet of the host and of the identification of Lp mutants that fail to rescue the noxious effects of an imbalanced amino-acid regimen. Thus, the genetic approach in both host and symbiont is a major asset of this study. The results are surprising as an imbalance of one essential amino-acid in the diet, valine, can nevertheless be compensated by Lp, even though it is itself unable to synthesize this amino-acid. The experiments are well-conducted and conclusions are appropriate.
This study however does not identify how GCN2 promotes growth in this context. There is just a descriptive transcriptomics approach that is however not validated at the functional level (and also not by RTqPCR experiments) as it does not provide obvious leads beyond a Gene Ontology exploitation of the data. The authors propose that Lp promotes a more thorough absorption of valine, a possibility that makes sense but is not backed up by any data. Also, how Lp release sr/tRNAs is not addressed experimentally.
A minor logical flaw is the use of GCN2 pathway activation read-outs that are actually not required to mediate Lp's beneficial action.
The authors claim that GCN2 action is not mediated through ATF4 or Thor based on RNA interference experiments. However, in contrast to the GCN2 case, they have not validated the RNAi lines and tested also only one for each.
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