Diet significantly influences the health and well-being of all organisms, including humans. Essential amino acids (EAAs), vital for protein synthesis, must be obtained through diet. Imbalances in EAA intake affect lifespan and healthspan. Organisms regulate their feeding behavior to compensate for EAA deficiencies or excesses. In Drosophila melanogaster females, both EAA deprivation and mating trigger changes in neuronal circuits, increasing protein appetite. While the physiological and neuronal mechanisms controlling bulk food intake are understood, the regulation of nutrient-specific appetites remains less clear. The gut microbiome is a crucial modulator of physiology and behavior, impacting feeding behavior, food choice, and reproduction. However, understanding the mechanisms underlying microbiome influence remains a challenge, as microbes often act as communities rather than isolated entities. This study aims to identify mechanisms by which bacterial communities affect the host, particularly focusing on metabolic interactions and their role in host resilience to dietary changes. Drosophila melanogaster, with its simple, experimentally tractable microbiome, is used as a model system. This model allows investigation of the relationship between dietary AAs and the gut microbiome, and its impact on protein appetite and reproduction. The study focuses on the interaction between Acetobacter pomorum and Lactobacillus plantarum, two dominant species within the Drosophila microbiome.

The literature highlights the critical role of diet in shaping gut microbiota composition and its effects on host physiology. Rapid changes in gut microbiome composition occur in response to dietary shifts, such as transitioning from plant-based to animal-based diets or altering protein-to-carbohydrate intake. The impact of diet on the microbiome also demonstrates high personal variability. In vitro studies have begun to investigate the nutritional preferences of individual human gut bacteria; however, a comprehensive mechanistic understanding of how bacterial dietary needs shape the microbiome and its influence on the host is lacking. The gut microbiome's resilience to dietary perturbations is emphasized as a key factor in maintaining its beneficial contribution to host physiology. Microbial communities utilize diverse metabolic interactions, including syntrophy, to overcome nutrient limitations imposed by dietary changes. Drosophila melanogaster is identified as a powerful model organism to study host-microbe interactions due to its simple, easily manipulated microbiome and the ability to generate germ-free flies. This allows for detailed investigations of microbial communities and their influence on reproduction, growth, and behavior.

This study used an interdisciplinary approach combining chemically defined (holidic) diets with gnotobiotic Drosophila melanogaster. The researchers focused on two amino acids, isoleucine (Ile) and histidine (His), to investigate the interaction between dietary AAs and the gut microbiome. The impact of different dietary conditions and community compositions on bacterial titers was assessed both in vivo and in vitro using high-throughput methods. In vitro studies involved growing bacteria in liquid holidic medium (HM) with and without Ile, both as monocultures and co-cultures of Acetobacter pomorum (Ap) and Lactobacillus plantarum (Lp). Isotope-resolved metabolomics, using 13C-labeled glucose and lactate, was employed to track the synthesis and flow of amino acids within the microbial communities. The researchers measured bacterial growth using colony-forming units (CFUs) and assessed the fly's protein appetite using the flyPAD assay, a quantitative method for measuring food preference. Egg-laying assays were conducted to evaluate the impact on reproduction. To distinguish between the effects of bacterial metabolites and bacterial biomass, experiments were performed using heat-killed bacteria. Finally, metabolomics analysis was performed on fly heads to assess free amino acid levels in germ-free and gnotobiotic flies. Statistical analyses included Dunn’s multiple comparison test, unpaired two-tailed Student’s t-test, Kruskal-Wallis test, and others as appropriate.

The study revealed a synergistic relationship between Ap and Lp in suppressing protein appetite in flies deprived of EAAs. Neither bacterium alone could achieve this effect. In vitro experiments demonstrated that Ap supports Lp growth in the absence of Ile by synthesizing and secreting Ile. Lp contributes lactate, which serves as a precursor for Ap's synthesis of Ile and other amino acids. This creates a 'circular economy' where Lp provides lactate, Ap produces EAAs, and these EAAs are used by Lp. Lactate was both necessary and sufficient for Ap to modify the fly's protein appetite. While Ap produces some EAAs, the amounts secreted are insufficient to directly suppress protein appetite. However, the Ap/Lp community significantly improved egg laying in EAA-deprived flies, primarily due to the production of EAAs which are used in egg development. This effect was also observed with heat-killed bacteria, indicating that the bacterial biomass itself contributed to increased egg laying. Metabolomics analysis did not reveal increased free EAAs in the flies' heads due to the presence of the bacteria, suggesting that the produced EAAs were utilized for egg production rather than influencing appetite. The researchers propose that a lactate-dependent mechanism in Ap, distinct from the provision of amino acids, is responsible for altering feeding behavior. The study identifies a metabolic pathway in Ap, using lactate derived from the TCA cycle, for amino acid synthesis, potentially via an aspartate-derived pathway.

This study demonstrates the significance of metabolic cross-feeding within gut microbial communities in mediating host responses to dietary imbalances. The synergistic relationship between Ap and Lp shows how bacterial interactions can buffer nutritional stress, maintaining microbiome stability and positively influencing host reproduction. The finding that lactate is both necessary and sufficient for Ap to alter feeding behavior highlights the importance of specific bacterial metabolites in regulating host behavior. The study distinguishes between the contributions of bacterial metabolites (EAAs for egg laying) and bacterial biomass (potentially through other metabolites) on host physiology. The identified metabolic pathway in Ap provides a mechanism for EAA synthesis from lactate, which is crucial for the symbiotic relationship and its impact on the host. While EAAs are not the direct cause of altered feeding behavior, they are necessary for egg laying. The findings challenge the simplistic model where bacteria solely supply EAAs to compensate for dietary deficiencies and suggest other host-mediated mechanisms are involved. Future research should focus on identifying those mechanisms, along with the potential role of other metabolites produced by the bacterial community and their interactions with the host.

This research unravels a complex interplay between gut microbes and host physiology, demonstrating the critical role of metabolic cross-feeding in maintaining microbiome resilience and impacting host behavior and reproduction. The identification of the Ap/Lp syntrophic relationship and the lactate-dependent mechanism for regulating feeding behavior provides significant insights into host-microbe interactions. Future research should investigate the specific metabolites involved in mediating behavioral changes and explore the broader implications of these findings for understanding the complex relationship between diet, microbiome, and host health across different organisms.

The study focused on a specific pair of bacterial species in a model organism, and the results may not be directly generalizable to other bacterial communities or host species. The study primarily used in vitro experiments, and future studies need to investigate more thoroughly the in vivo mechanisms of Ap's impact on feeding behavior. The specific mechanisms by which bacterial metabolites modulate host neuronal circuits remain unclear and warrant further investigation. Further research may also be needed to clarify the precise role of bacterial biomass in enhanced egg laying.