Host-microbiome metabolism of a plant toxin in bees
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eLife assessment
The manuscript makes an important contribution to understanding the roles of the bee host and microbiome in degrading amygdalin, a dietary secondary metabolite. Several bacterial strains and their enzymes responsible for the deglycosylation of amygdalin are identified. Conclusions are reached convincingly through a comprehensive combination of in vitro and in vivo experiments including gene-expression analysis, proteomics, HPLC-MS, and the use of recombinant E. coli to test enzyme function. As the consequences of microbial-derived amygdalin metabolisation on host health remain uncertain from the experiments conducted, the manuscript could be improved through a clearer discussion of future work needed and in parts more careful wording to not prematurely suggest benefits to the host.
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Abstract
While foraging for nectar and pollen, bees are exposed to a myriad of xenobiotics, including plant metabolites, which may exert a wide range of effects on their health. Although the bee genome encodes enzymes that help in the metabolism of xenobiotics, it has lower detoxification gene diversity than the genomes of other insects. Therefore, bees may rely on other components that shape their physiology, such as the microbiota, to degrade potentially toxic molecules. In this study, we show that amygdalin, a cyanogenic glycoside found in honey bee-pollinated almond trees, can be metabolized by both bees and members of the gut microbiota. In microbiota-deprived bees, amygdalin is degraded into prunasin, leading to prunasin accumulation in the midgut and hindgut. In microbiota-colonized bees, on the other hand, amygdalin is degraded even further, and prunasin does not accumulate in the gut, suggesting that the microbiota contribute to the full degradation of amygdalin into hydrogen cyanide. In vitro experiments demonstrated that amygdalin degradation by bee gut bacteria is strain-specific and not characteristic of a particular genus or species. We found strains of Bifidobacterium , Bombilactobacillus, and Gilliamella that can degrade amygdalin. The degradation mechanism appears to vary since only some strains produce prunasin as an intermediate. Finally, we investigated the basis of degradation in Bifidobacterium wkB204, a strain that fully degrades amygdalin. We found overexpression and secretion of several carbohydrate-degrading enzymes, including one in glycoside hydrolase family 3 (GH3). We expressed this GH3 in Escherichia coli and detected prunasin as a byproduct when cell lysates were cultured with amygdalin, supporting its contribution to amygdalin degradation. These findings demonstrate that both host and microbiota can act together to metabolize dietary plant metabolites.
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eLife assessment
The manuscript makes an important contribution to understanding the roles of the bee host and microbiome in degrading amygdalin, a dietary secondary metabolite. Several bacterial strains and their enzymes responsible for the deglycosylation of amygdalin are identified. Conclusions are reached convincingly through a comprehensive combination of in vitro and in vivo experiments including gene-expression analysis, proteomics, HPLC-MS, and the use of recombinant E. coli to test enzyme function. As the consequences of microbial-derived amygdalin metabolisation on host health remain uncertain from the experiments conducted, the manuscript could be improved through a clearer discussion of future work needed and in parts more careful wording to not prematurely suggest benefits to the host.
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Reviewer #1 (Public Review):
The authors investigate the relative importance of the bee host and bacterial microbiome in processing the nectar secondary metabolite amygdalin, with a focus on understanding the contributions of the different members of the microbiome, and the enzymatic basis for metabolic transformations. The manuscript clearly describes the experimental procedures, presents the results in graphically appealing figures and clear text, and puts the work into a broader context in the discussion. The conclusions are backed by sophisticated in vitro and in vivo experimental data. A particular strength of the manuscript is the combined use of genomic, gene expression, proteomic, and small metabolite analyses to pin down the mechanistic basis of the degradation of amygdalin. While at this stage the authors cannot infer the …
Reviewer #1 (Public Review):
The authors investigate the relative importance of the bee host and bacterial microbiome in processing the nectar secondary metabolite amygdalin, with a focus on understanding the contributions of the different members of the microbiome, and the enzymatic basis for metabolic transformations. The manuscript clearly describes the experimental procedures, presents the results in graphically appealing figures and clear text, and puts the work into a broader context in the discussion. The conclusions are backed by sophisticated in vitro and in vivo experimental data. A particular strength of the manuscript is the combined use of genomic, gene expression, proteomic, and small metabolite analyses to pin down the mechanistic basis of the degradation of amygdalin. While at this stage the authors cannot infer the importance of their findings for bee health, their insights and methods should stimulate additional experiments into the role of microbial conversion of dietary metabolites for bee health.
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Reviewer #2 (Public Review):
In this communication by Motta and colleagues, the authors address the emerging role of the gut microbiome in degrading and detoxifying plant metabolites, using bees as a study system. The experiments are elegantly controlled, spanning in vitro and in vivo work that leverages the increasing tractability of bees and their microbial symbionts. This is evident in the extensive screening of Bifidobacterium, Bombilactobacillus, Lactobacillus, and Gilliamella relative to their susceptibility to amygdalin. This provided a foundation to pinpoint which strains can degrade the cyanogenic glycoside, the potential pathways underlying that process, and the key enzymes involved. The strain Bifidobacterium wkB204 displayed elevated expression of GH3, correlating to the ability of this microbe to degrade amygdalin in vitro. …
Reviewer #2 (Public Review):
In this communication by Motta and colleagues, the authors address the emerging role of the gut microbiome in degrading and detoxifying plant metabolites, using bees as a study system. The experiments are elegantly controlled, spanning in vitro and in vivo work that leverages the increasing tractability of bees and their microbial symbionts. This is evident in the extensive screening of Bifidobacterium, Bombilactobacillus, Lactobacillus, and Gilliamella relative to their susceptibility to amygdalin. This provided a foundation to pinpoint which strains can degrade the cyanogenic glycoside, the potential pathways underlying that process, and the key enzymes involved. The strain Bifidobacterium wkB204 displayed elevated expression of GH3, correlating to the ability of this microbe to degrade amygdalin in vitro. Expression of the GH3 in E. coli corroborated its putative role in the transformation of amygdalin to prunasin, consistent with the single inoculation effects of Bifidobacterium wkB204 into microbiota-deprived bees. These experiments collectively point to the importance of the bee microbiota for the consistent degradation of amygdalin. The findings are nicely contextualized relative to prior work on the gut microbiome and the metabolism of the cyanogenic glycoside, including efforts on bees and rats.
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Reviewer #3 (Public Review):
Motta, Erick et al. investigated the role of members of the bacterial gut microbiota of honey and bumble bees in the degradation of amygdalin, a plant cyanogenic glycoside found in almond trees and other plants. The role of the microbiota in contributing to secondary plant compounds in this system is of interest because it has been demonstrated that the genomes of these bees are depauperate in genes of detoxification enzymes relative to other insects. Using in vitro assays across a range of honey and bumble bee-derived strains of the bacterial species Bifidobacterium, Bombilactobacillus, Gilliamella, and Lactobacillus nr. melliventris the authors demonstrate strain-specific growth on amygdalin as a carbon source, clearly showing amygdalin metabolism by particular strains. The data strongly support that …
Reviewer #3 (Public Review):
Motta, Erick et al. investigated the role of members of the bacterial gut microbiota of honey and bumble bees in the degradation of amygdalin, a plant cyanogenic glycoside found in almond trees and other plants. The role of the microbiota in contributing to secondary plant compounds in this system is of interest because it has been demonstrated that the genomes of these bees are depauperate in genes of detoxification enzymes relative to other insects. Using in vitro assays across a range of honey and bumble bee-derived strains of the bacterial species Bifidobacterium, Bombilactobacillus, Gilliamella, and Lactobacillus nr. melliventris the authors demonstrate strain-specific growth on amygdalin as a carbon source, clearly showing amygdalin metabolism by particular strains. The data strongly support that amygdalin degradation occurrence is not a pan-species trait, but rather strain-specific, and also that even within a bacterial species the strains metabolizing amygdalin achieve this through different pathways, with some strains producing the metabolite prunasin, but others not. Subsequent proteomics analysis suggests that a glycoside hydrolase family 3 (GH3) is likely responsible for the degradation of amygdalin. The conclusion that this GH3 is at least partially responsible for strain-specific degradation is supported by gene expression analysis of the enzyme and experiments with E. Coli transformed with the gene. Further in vivo studies demonstrate that the honey bee microbiota contributes to amygdalin metabolism, including specific strains of Bifidobacterium, but that the hosts themselves can metabolize amygdalin to prunasin in the absence of gut microbes, but not to the same degree.
The approach and evidence supporting the step-wise conclusions are comprehensive. However, further extension is required to gain a full appreciation for what the importance and relevance of the results for conclusions relating to cooperation between hosts and microbiota and particularly the consequences for host health.
Although the authors rightly do not directly interpret the attributed breakdown of amygdalin and its metabolites by specific bacterial strains as a benefit, this is alluded to in the title and parts of the discussion. Following the degradation of amygdalin through intermediates, hydrogen cyanide is produced. Hydrogen cyanide is generally considered to be detrimental. As such, it could be argued that is not appropriate to consider the production of such a compound as cooperative between host and microbiota, given that cooperation is usually to a beneficial end. Experiments exposing hosts with microbiota absent and present to amygdalin and relevant breakdown products and subsequently measuring relevant health outcomes would be an important step in aiding in the interpretation of the otherwise clear experimental outcomes. Especially given the relatively limited number of strains tested showing the ability to degrade amygdalin, it is possible that there is limited adaptive value, and/or the ability could be due to either chance or selection for the metabolism of other compounds. This is especially relevant when considering further work that may look at how health-related outcomes such as parasite resistance are affected.
This being said, the work adds to demonstrations of different functions of host gut microbiota, how they can mediate the environment encountered by hosts, and the increasing appreciation that effects derived from the microbiota can be not only dependent upon the bacterial species present but frequently the specific strains.
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