Peer review of: Metabolic cross-feeding in imbalanced diets allows gut microbes to improve reproduction (2020)

Reviewers' comments:  

 

Reviewer #1 (Remarks to the Author):  

 

Silivia et al. utilized isotope tracing based metabolomics to discover a syntrophic relationship 
between Acetobacter pomorum (Ap) and Lactobacillus plantarum (Lp). In general, it is found that Lp 
provided lactate for Ap as a precursor to synthesize and secrete AAs. These AAs could allow Lp to 
overcome the imbalanced diary. The story is potentially interesting. I was asked to carefully look at 
the metabolomics technical part. And I found there are severe technical issues with isotope tracing 
based metabolomics.  

 

1. The authors did not mention in Methods whether they have corrected nature isotope abundances 
in the final isotopologue (not isotopomer) results. Some of the unlabeled results are not easy to 
explain. For example, in Figure 3 of 12C-glucose labeled samples, isoleucine has an observable M+4 
peak but no M+3/M+5 peaks. If nature isotope abundances were corrected, M+1 to M+6 peaks of 
isoleucine should be close to 0. I would suspect the M+4 peak is a contamination of other 
metabolites. The authors should provide the raw MS peaks and details on this. The similar issues 
were also observed in Figure 5 and other figures.  

 

2. For each metabolite, M+0 to M+n (n=6 for isoleucine) isotopologues should be demonstrated. But 
the authors only demonstrated M+3 to M+5 without explanations. I would like to see all 
isotopologue results with the correction of nature isotope abundances. It is common practice in 
isotope-tracing experiments.  

 

3. The metabolic pathways from glucose or lactate to isoleucine are not explained or proposed. 
Since lactate has only 3 carbons, while isoleucine has 6 carbons. In general, in U-13C-lactate labeling 
experiment, it is reasonable that isoleucine has 3 carbons labelled from lactate. But in Figure 5, M+4 
is the major isotopologue peak. How is lactate used for synthesize isoleucine (M+4)? It might be 
possible, but a metabolic pathway and explanation is required.  

 

4. Similar question for U-13C-glucose labeling experiment, glucose has 6 carbons, but the authors 
only demonstrated isoleucine (M+5)? How is glucose used for synthesize isoleucine? Converted to 
lactate first? Then the same question repeated as comment 3.  

 

5. In Figure 3, for the third and the fourth bars, the summed peak areas should be equal to each 
other (since they have the same experimental condition), but with different distributions on 
isotopologues. How to explain the differences on summed peak areas???  

 

6. In addition, the MID pattern of isoleucine in figure 3 (second bar) and figure 5 (third bar) are quite 
different. How to explain the difference?  

 

7. Page 11, line 269, “the LpWCFS1∆ldh mutant strain failed to suppress yeast appetite (Fig. 4a)”. But 
actually, as the result showed, compared with the LpWCFS result, the LpWCFS1∆ldh did not have 
significant changes for increased yeast appetite.  

 

8. Figure 5, the authors should provide a scheme to show how M+3, M+4 and M+5 isoleucine were 
produced.  

 

9. In the isotope-tracer experiments, could author use mole percentage enrichment (MPE) to 
quantify the new synthesized metabolites (isoleucine and other AAs)? It would help to clearly 
demonstrate the percentage of new synthesized metabolites.  

 

10. It is very challenging to read supplementary Figure S4, please change to a color scheme. I will re-
evaluate this figure in revision.  

 

11. The details for identification of metabolite should be given.  

 

 

 

Reviewer #2 (Remarks to the Author):  

 

This well-written manuscript by Henriques et all examines uses a multiprong approach to 
understand how two bacteria, Acetobacter Pomorum (Ap) and Lactobacillus plantarum (Lp) 
metabolically interact within the context of Drosophila melanogaster host diet. By using an liquid 
chemically defined diet that mimics the Drosophila holidic diet, the authors examined how these 
two bacterial species grow in the absence of two essential amino acids – isoleucine and histidine. 
These experiments revealed that although Lp is an isoleucine auxotroph, coculture of Lp with Ap 

allows Lp to grow in the absence of isoleucine. Using stable isotope tracers, the authors then 
demonstrate that in the presence of Lp, Ap synthesized and secreted labeled isoleucine. Moreover, 
the authors demonstrate the Lp synthesizes isoleucine using lactate provided by Ap, thus revealing 
an elegant symbiosis between the two bacteria. Finally, the authors provide evidence that this 
symbiotic relationship is likely not responsible for the ability of these two bacteria to jointly regulate 
Drosophila feeding behavior.  

 

Overall, I found this a clear, logical, and important study. While the mechanism by which these two 
bacteria jointly regulate feeding behavior remains elusive, it doesn’t diminish my interest in this 
story. In fact, I found it refreshing that the authors pointedly demonstrated that isoleucine 
production is essential for Lp growth but not fly behavior – a breath of fresh air in a field where 
these sort of metabolic interactions are usually the basis for wild speculation. While I can think of 
additional experiments that could be conducted by the authors, none would improve the 
manuscript.  

 

 

 

Reviewer #3 (Remarks to the Author):  

 

In this manuscript, Henriques et al. showed an intriguing metabolic connection between two 
dominant commensal bacterial species in Drosophila gut, Acetobacter pomorum (Ap) and 
Lactobacillus plantarum (Lp). Given their previous finding that these two gut microbes are required 
for controlling the appetite of the host, the authors attempted to identify the underlying 
mechanism. The careful gnotobiotic and metabolic experiments demonstrated the clear metabolic 
relay to produce essential amino acids from glucose via lactate. The in vitro data of bacterial culture 
using liquid holidic medium (prepared based on the fly diet) was convincing. Isotopic tracer 
experiments have beautifully demonstrated lactate is a product of glucose in Lp, and amino acids are 
produced from lactate in Ap. Lp is therefore required for the provision of lactate. The authors 
successfully showed that adding lactate is sufficient for Ap to influence the host behaviour. Amino 
acid production in Ap might also help Lp to grow under detrimental medium that lacks isoleucine. 
Overall, the manuscript is written well, addressing the essential question of "community-wise" 
bacterial metabolism and its effect on host physiology. However, it has still too many questions 
unanswered.  

 

 

Major concerns:  

 

1, Lack of identification of the factor regulating host appetite, as it is mentioned in the manuscript, is 
critical especially when the authors' take-home message seemed to be "host behaviour" altered by 
the microbial metabolic interaction shown in the title as well as in the abstract. What is only 
revealed in the study is that lactate mediates Lp's effect on the behaviour. This reviewer assumes 
that the rescue of increased appetite with Ile(-) diet by Ap/Lp would be because it promotes the 
growth of Ap. If it is the case, high abundance of Ap (~10^9 CFU) but not Lp, during gnotobiotic 
preparation may rescue the behaviour.  

 

2, Strikingly, the study did not reveal the mechanism of synergistic growth of the two bacterial 
species in vivo (fly gut/vial). Liquid culture nicely showed the unidirectional effect of Ap on Lp 
growth on Ile(-) medium. However, the authors did not demonstrate that this was because of the 
provision of Ile by Ap, especially given that the level of Ile production by Ap is very low. If possible, 
they need to use the loss of function mutant of Ap, which cannot utilise lactate or synthesise Ile. 
Besides, if lactate from Lp can also "feed" Ap, the growth of Ap in the liquid culture should be faster, 
as it was evident in the fly gut. Is it possible that lactate can increase Ap's growth in the fly gut, as 
well as in the liquid? The growth measurement in Fig.2 needs more time course, especially for the 
first 24 hours; one day of bacterial culture is too long to observe the upregulation of the growth 
speed. If this is not the case, then the bacterial growth in liquid is irrelevant to that in solid diet or in 
fly gut.  

 

3, The major finding of the present study would be the production of Ile by Ap from Lp-derived 
lactate. However, this is expecting, given that Acetobactereceae is proved to produce amino acids 
from lactate by using stable isotope labelling technique (Adler et al., Appl Environ, Microbiol 80, 
4702, 2014). Considering the study did not identify the Ap-derived factor controlling the host 
behaviour, the present may not significantly advance our understanding of microbial metabolism in 
the context of the host-microbe interaction. It should also be explained why Ap does not use glucose 
directly to produce Ile, while it produces (secretes) Ile by exogenous lactate.  

 

Other concerns:  

Fig.1a, It is possible that the quantity of bacteria, rather than quality, is important for the appetite 
control. It might be better to test the various amount of bacterial feeding on behaviour.  

Fig.1c, Lp in Ile(-) is greater in amount (~10^5) compared to that in complete medium (Lp alone 
conditions). What is the reason for this? There should be no growth at all under Ile (-) medium for 
this bacterial species, but the data suggests the opposite.  

Fig.2b, Can the authors deny the possibility that the rescue of Lp growth by Ap is due to the 
provision of Ile by dead Ap?  

Fig.3, How much did the total Ile content increased by co-culture?  

Fig.3, Is the production of Ile abolished by co-culture of Ap with the ldh mutant Lp? 

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