Drosophila-associated bacteria differentially shape the nutritional requirements of their host during juvenile growth

Abstract

The interplay between nutrition and the microbial communities colonizing the gastrointestinal tract (i.e., gut microbiota) determines juvenile growth trajectory. Nutritional deficiencies trigger developmental delays, and an immature gut microbiota is a hallmark of pathologies related to childhood undernutrition. However, how host-associated bacteria modulate the impact of nutrition on juvenile growth remains elusive. Here, using gnotobiotic Drosophila melanogaster larvae independently associated with Acetobacter pomorumWJL (ApWJL) and Lactobacillus plantarumNC8 (LpNC8), 2 model Drosophila-associated bacteria, we performed a large-scale, systematic nutritional screen based on larval growth in 40 different and precisely controlled nutritional environments. We combined these results with genome-based metabolic network reconstruction to define the biosynthetic capacities of Drosophila germ-free (GF) larvae and its 2 bacterial partners. We first established that ApWJL and LpNC8 differentially fulfill the nutritional requirements of the ex-GF larvae and parsed such difference down to individual amino acids, vitamins, other micronutrients, and trace metals. We found that Drosophila-associated bacteria not only fortify the host's diet with essential nutrients but, in specific instances, functionally compensate for host auxotrophies by either providing a metabolic intermediate or nutrient derivative to the host or by uptaking, concentrating, and delivering contaminant traces of micronutrients. Our systematic work reveals that beyond the molecular dialogue engaged between the host and its bacterial partners, Drosophila and its associated bacteria establish an integrated nutritional network relying on nutrient provision and utilization.

Fig 1. Expert automated genome annotation and metabolic network reconstruction of Drosophila, Ap^WJL, and Lp^NC8.

(A) Amino acid biosynthetic pathways. (B) Vitamins and cofactors biosynthetic pathways. Left panels, D. melanogaster. Central panels, Ap^WJL. Right panels, Lp^NC8. Color code: blue, biosynthesized amino acids or vitamins; brown, limited amino acid or vitamin biosynthesis (biosynthesis of the metabolite may be possible, but it is limited and/or requires secondary metabolic pathways); black, nonbiosynthesized amino acids or vitamins; gray, pathway intermediary metabolites. Red cross: nonfunctional pathway (lack of key enzyme[s]). Orange nods, major metabolic pathways. α-cglu, α-keto-glutarate; AceCoA, Acetyl-CoA; Ant, Antranilate; Ap^WJL, A. pomorum^WJL; Aro, Arogenate; Cho, chorismate; Cit, Citrate; Cysta, Cystathionine; Dihyn-P3, 7,8-Dihydroneopterin-3′-P3; Dm-ribi, 6,7-Dimethyl-8-ribityllumazine; Ery-4P, Erythrose-4P; FAD, Flavin Adenine Dinucleotide; FMN, Flavin mononucleotide; Fum, Fumarate; Glc, Glucose; Gly-3P, Glycerate-3P; Homocys, Homocysteine; Homoser, Homoserine; Ind, Indole; Lp^NC8, L. plantarum^NC8; Orn, Ornithine; Oxa, Oxaloacetate; P-ra-imi, 1-(5′-Phospho-ribosyl)-5-aminoimidazole; Phoser, Phosphoserine; Pre, Prephenate; Pyn-P, Pyridoxine phosphate; Pyr, Pyruvate; Rib-5P, Ribose-5P; TCA, Tricarboxylic acid Cycle; [ThiS]-COSH, [ThiS]-thiocarboxylate.

Fig 2. Drosophila, Ap^WJL, and Lp^NC8 have differential biosynthetic capacities of nutrients contained in the HD.

Venn diagram represents the number of nutrients present in the FLYAA HD that can be synthesized by each organism. The list of corresponding metabolites is provided. Dotted circles: biosynthesis of this metabolite by Lp^NC8 (green) may be possible but might be limiting. Ap^WJL, A. pomorum^WJL; FLYAA, fly exome-matched amino acid ratio; HD, Holidic Diet; Lp^NC8, L. plantarum^NC8.

Fig 3. Ap^WJL and Lp^NC8 auxotrophies detected in liquid fly HD.

(A) Heat map representing the mean OD_Max reached by Ap^WJL or Lp^NC8 after 72 h of culture. Each line shows growth in a different version of the liquid HD: complete HD (first line) or HD lacking nutrient X (ΔX, lines below). Cultures were made in 96-well plates under agitation. Asterisks (*) pinpoint contradictions with our metabolic pathway automated annotations, which are explained in panel B. (B) Growth of Lp^NC8 in 4 versions of liquid HD: complete HD, HDΔThr, HDΔAla, and HDΔAsp in static conditions. Plot shows means with standard error based on 3 replicates by assay. Each dot represents an independent replicate. The dashed line represents the level of inoculation at t = 0 h (10⁴ CFUs per mL). Ap^WJL, A. pomorum^WJL; CFU, colony-forming unit; EAA^Fly, fly essential amino acid; HD, Holidic Diet; Lp^NC8, L. plantarum^NC8; NALs, nucleic acids and lipids; NEAA^FLY, fly nonessential amino acid; OD_Max, maximal optical density.

Fig 4. Ap^WJL and Lp^NC8 can differentially fulfill their host’s nutritional requirements in HDs.

(A) Heat map representing the mean D₅₀ of GF larvae (first column) and larvae associated with Ap^WJL, Lp^NC8, Ap^WJL_HK, and Lp^NC8_HK (columns 2, 3, 4, 5 respectively). Each line shows D₅₀ in a different version of HD: complete HD (first line) or HDs lacking nutrient X (ΔX, lines below). White means larvae did not reach pupariation in these conditions. Means, standard errors of the mean and statistical tests (Dunn test of multiple comparisons) are detailed in S4 Table. (B–D) Absence of correlation between time of development and quantity of bacteria. Y axis shows D₅₀, and X axis shows quantity of bacteria (Log₁₀ CFUs) in the larval gut (B), in the diet in presence of larvae 3 days after inoculation (C), and in the diet in presence of larvae 6 days after inoculation (D). Each dot shows a different condition. Complete HD: on complete HD. ΔX: on HDs lacking nutrient X. Black dots: in monoassociation with Ap^WJL, green dots: in monoassociation with Lp^NC8. For each bacterium, we tested Pearson’s product–moment correlation between D₅₀ and quantity of bacteria. Ap^WJL, A. pomorum^WJL; CFU, colony-forming unit; cor, Pearson correlation coefficient for each bacterium; D₅₀, day when 50% of larvae population has entered metamorphosis; EAA^Fly, fly essential amino acid; GF, germ-free; HD, Holidic Diet; HK, heat-killed; Lp^NC8, L. plantarum^NC8; NALs, nucleic acids and lipids; NEAA^Fly, fly nonessential amino acid.

Fig 5. Evaluation of HDΔAsn, HDΔPhe, and HDΔCys contexts.

(A) D₅₀ of yw, DGRP_25210, and w¹¹¹⁸ larvae on HDΔAsn. Boxplots show minimum, maximum, and median. Each dot shows an independent replicate. GF yw larvae did not reach pupariation. For the other 2 lines, we performed a Kruskal–Wallis test followed by post hoc Dunn tests to compare each gnotobiotic condition to GF. **p-value < 0.005, ***p-value < 0.0005, ****p-value < 0.0001. (B) Growth of Lp^NC8 in liquid HDΔPhe and liquid HDΔCys, in static conditions, 3 days after inoculation. Plot shows mean with standard error. Each dot shows an independent replicate. The dashed line represents the level of inoculation at t = 0 h (10⁴ CFUs per mL). (C) Growth of Lp^NC8 on solid HDΔCys, in absence and in presence of larvae, 3 days and 6 days after inoculation. Plot shows mean with standard error. Each dot represents an independent replicate. The dashed line represents the level of inoculation at t = 0 h (10⁴ CFUs per tube). We performed two-way ANOVA followed by post hoc Sidak test. **p-value < 0.005. Ap^WJL, A. pomorum^WJL; CFU, colony-forming unit; DGRP, XXX; D₅₀, day when 50% of larvae population has entered metamorphosis; EAA^Fly, fly essential amino acid; GF, germ-free; HD, Holidic Diet; HK, heat-killed; Lp^NC8, L. plantarum^NC8; NEAA^Fly, fly nonessential amino acid; ns, nonsignificant; yw, XXX.

Fig 6. Ap^WJL and Lp^NC8 can produce and release EAAs^Fly during growth.

(A) HPLC measured concentration of Arg, His, Ile, Leu, Phe, Thr, and Val in the supernatant of an Ap^WJL culture in HDΔArg, HDΔHis, HDΔIle, HDΔLeu, HDΔPhe, HDΔThr, and HDΔVal, respectively, 72 h after inoculation. Plot shows mean with standard error. Each dot shows an independent replicate. Each amino acid was not detected prior to microbial growth (S1 Data). (B) HPLC measured concentration of His in the supernatant of a Lp^NC8 culture in HDΔHis, 72 h after inoculation. Plot shows mean with standard error. Each dot shows an independent replicate (53.08 μM, 52.82 μM, and 52.99 μM). Ap^WJL, A. pomorum^WJL; EAA^Fly, fly essential amino acid; HD, Holidic Diet; HPLC, High-Performance Liquid Chromatography; Lp^NC8, L. plantarum^NC8.

Fig 7. Ap^WJL and Lp^NC8 differentially shape the nutritional requirements of their juvenile host.

For each gnotobiotic condition, essential nutrients are represented in black and nonessential nutrients in color. Color code: blue, this nutrient can be synthesized by the bacteria; red, this nutrient cannot be synthesized by the bacteria, suggesting a mechanism of functional compensation. In purple: lack of this nutrient may be compensated by an intermediate metabolite or a derivative produced by the bacteria. AA, amino acid; Ap^WJL, A. pomorum^WJL; GF, germ-free; Lp^NC8, L. plantarum^NC8; NAL, nucleic acid and lipid.