Microbes Promote Amino Acid Harvest to Rescue Undernutrition in Drosophila

Abstract

Microbes play an important role in the pathogenesis of nutritional disorders such as protein-specific malnutrition. However, the precise contribution of microbes to host energy balance during undernutrition is unclear. Here, we show that Issatchenkia orientalis, a fungal microbe isolated from field-caught Drosophila melanogaster, promotes amino acid harvest to rescue the lifespan of undernourished flies. Using radioisotope-labeled dietary components (amino acids, nucleotides, and sucrose) to quantify nutrient transfer from food to microbe to fly, we demonstrate that I. orientalis extracts amino acids directly from nutrient-poor diets and increases protein flux to the fly. This microbial association restores body mass, protein, glycerol, and ATP levels and phenocopies the metabolic profile of adequately fed flies. Our study uncovers amino acid harvest as a fundamental mechanism linking microbial and host metabolism, and highlights Drosophila as a platform for quantitative studies of host-microbe relationships.

Figure 1. I. orientalis extends fly lifespan on undernutrition diet

(A) Survival of axenic or I. orientalis-associated monoxenic flies on 0.1% yeast extract (YE) diet. I. orientalis association increases survival compared to the axenic control (p < 10⁻¹⁰, log-rank test). (B) Survival on 0.5% YE diet. Survival curves of I. orientalis-associated and axenic flies do not differ (p > 0.090, log-rank test). Monoxenic flies were inoculated once as adults with I. orientalis. N = 59–61 flies for each condition. See also Figure S1.

Figure 2. I. orientalis increases amino acid harvest in Drosophila

(A) Radioisotope-labeled feeding assay for examining nutrient accumulation in the fly. Flies feed for 24 h on radiolabeled food that is pre-inoculated with I. orientalis. Radioisotope abundance in the fly is then measured to reveal the effect of microbial association on nutrient accumulation. (B) Effect of I. orientalis association on fly accumulation of radiolabeled nutrients on 0.1% or 0.5% YE medium. Results (average ± s.d.) are normalized to the axenic control (dashed line) for each radioactive tracer. Significant differences between each tracer and its axenic control are shown (Mann-Whitney rank-sum test: *, p < 0.05). N = 4 vials of 10 flies for each condition. Met = [³⁵S]-methionine; Leu = [¹⁴C]-leucine; dCTP = [α-³²P]-dCTP; Suc = [¹⁴C]-sucrose. (C) Distribution of accumulated [³⁵S]-methionine in flies. After 24 h feeding on [³⁵S]-methionine-labeled 0.1% YE medium that is pre-inoculated with I. orientalis, the entire fly digestive tract is dissected and ³⁵S is quantitated from the gut and remaining fly carcass. Results (average ± s.d.) are shown as a percentage of [³⁵S]-methionine accumulation. Student's t-test: ***, p < 0.001. N = 4 samples of 2 flies each. See also Figure S2.

Figure 3. Principal component analysis of metabolic parameters from axenic and I. orientalis-associated flies maintained on 0.1% YE and 0.5% YE diets

(A) Plot of PC1 and PC2 scores, which account for 45% and 28% of the total variance, respectively. The metabolic state of I. orientalis-associated flies on 0.1% YE medium more closely resembles that of animals on higher YE diet (0.5% YE) than that of undernourished axenic flies. Loadings for PC1 and PC2, respectively: body mass, −0.452 and −0.146; protein, −0.419 and 0.233; glucose, 0.452 and 0.255; glycogen, 0.066 and 0.592; trehalose, 0.103 and 0.556; glycerol, −0.323 and 0.423; TAG, 0.274 and −0.145; ATP, −0.471 and 0.008. (B) PC1 scores (average ± s.d.) from (A). Significant differences are shown (one-way ANOVA followed by Tukey post-test for multiple comparisons: ***, p < 0.001). N = 6 for each condition. Abbreviations: Ax, axenic; I. ori, I. orientalis. See also Figure S3.

Figure 4. Heat-killedI. orientalisextends lifespan on an undernutrition diet

(A) Survival of axenic or I. orientalis-associated flies on 0.1% YE undernutrition diet. Live microbes were supplied once in early adulthood. The indicated quantity of heat-killed (HK) microbes was provided once in early adulthood (single dose) or at every food change (twice/week) throughout life (recurring). Microbial association (live or HK) extends survival in all experiments compared to the axenic control (p < 5 × 10⁻⁹ for all comparisons, log-rank test) except in the single dose trial (p = 0.134, log-rank test). N = 57–64 flies for each condition. (B) Body mass increases with microbe supplementation (average of 3 vials). Body mass of axenic flies was significantly lower than that of microbe-associated flies on days 20 and 27 (Kruskal-Wallis followed by Student-Newman-Keuls post-test for multiple comparisons: *, p < 0.05). (C) Accumulation of [³⁵S]-methionine (average ± s.d.) in the presence of live or HK I. orientalis on 0.1% YE medium. While live and HK microbes both extend fly lifespan, only live I. orientalis increases accumulation of the diet-supplemented radiolabel (Kruskal-Wallis followed by Tukey's post-test for multiple comparisons: *, p < 0.05). N = 4 vials of 10 flies for each condition. (D) Model of microbe-mediated amino acid harvest. I. orientalis increases amino acid/protein flux, resulting in improved nutrition and longevity in the fly host. The size of the arrows and radiation symbols represent the amount of amino acid flux and radiolabeled amino acid levels, respectively. See also Figure S4.