1. Volume 20, Issue 2, Pages 479-490 (July 2017)
Acute Dietary Restriction Acts via TOR, PP2A, and Myc Signaling to Boost Innate Immunity in Drosophila 
Jung-Eun Lee, Morsi Rayyan, Allison Liao, Isaac Edery, Scott D. Pletcher 
Cell Reports 
Volume 20, Issue 2, Pages (July 2017)
DOI: /j.celrep
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2. Figure 1
Yeast Restriction Improves Survival of Drosophila over Pathogenic Bacteria
(A) Survival rates of mated female wild-type Canton-S flies fed diets ranging from 1% yeast/sucrose to 9% yeast/sucrose and infected with P. aeruginosa (PA14 plcs). Two-way ANOVA indicates a significant effect of dietary yeast (p < 1 × 10−5) but no effect of dietary sugar (p = 0.18) or an interaction between the two (p = 0.56). p values indicate results of pairwise t tests for the 1% yeast groups compared to the 9%. Results reflect the average of up to 12 independent replicates per group (total 720 flies). Flies were acclimated to the specified food for 2 days prior to infection with P. aeruginosa and transferred to fresh food of the same type after infection.
(B) P. aeruginosa titers in individual flies at 24 hr post-infection. p values indicate results of pairwise t tests for the 1% yeast groups compared to the 9% groups (n = 20–50 flies for each group). Results reflect the average of at least two independent experiments.
(C) Survivorship following infection for yeast-restricted (YR; 1% yeast/9% sucrose) and fully fed (FF; 9% yeast/9% sucrose) flies infected with P. aeruginosa. The p value was determined by Cox regression with replicate experiments used for stratification (n = 5 replicate experiments and 668 flies total). Mock injury groups are also displayed.
(D) Survivorship following infection for yeast-restricted (1% yeast/9% sucrose) and FF (9% yeast/9% sucrose) flies infected with S. aureus. The p value was determined by Cox regression with replicate experiments used for stratification (n = 3 replicate experiments and 358 flies total). Mock injury groups are also displayed.
See also Figure S1.
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3. Figure 2
Humoral Immunity Contributes to the Yeast-Restriction-Mediated Enhancement of Host Survival over Pathogenic Bacteria
(A–C) phagoless flies devoid of hemocytes (Defaye et al., 2009) and their control siblings were yeast-restricted or FF for 2 days prior to being infected with P. aeruginosa or S. aureus. (A) Survivorship of phagoless and control flies following infection with P. aeruginosa was significantly affected by diet (p = 1 × 10−5 and p = 2 × 10−5, respectively). The “diet × genotype” interaction term of a Cox regression model was not significant (p = 0.86), indicating that flies without hemocytes experience an increase in survival following yeast restriction similar to control animals. (B) Survivorship of phagoless and control flies following infection with S. aureus was significantly affected by diet (p < 1 × 10−5 in both cases). The diet × genotype interaction term of a Cox regression model was not significant (p = 0.13). phagoless flies did not survive S. aureus infection as well as their control flies, which is consistent with hemocytes playing an important role in S. aureus infection (Defaye et al., 2009). Together, these results suggest that hemocytes do not play an important role in the benefits of yeast restriction on survival following pathogenic infection. (C) S. aureus titer in individual flies at 22 hr post-infection. Yeast restriction suppressed plcs growth in control flies (p = 0.02), whereas phagoless flies exhibited much higher overall S. aureus titer without such diet effects (p = 0.2), indicating that yeast-restricted phagoless flies were able to survive S. aureus infection more favorably without clearing more of those invading S. aureus (28 ≤ n ≤ 37 per group).
(D and E) prophenoloxidase1 prophenoloxidase 2 double mutant (PPO1ΔPPO2Δ; also designated as PPO null) that cannot activate phenoloxidase (Binggeli et al., 2014) and their genetic background control w1118 flies were yeast-restricted or FF for 3 days prior to being infected with P. aeruginosa. (D) Survivorship of control flies was significantly affected by diet (log-rank test, p = 0.003), whereas PPO null (PPO1ΔPPO2Δ) flies survived poorly irrespective of diet (log-rank test, p = 0.8). A representative result from two independent experiments is shown here. (E) Average ultimate survival rates of yeast-restricted or FF PPO1ΔPPO2Δ null and their control flies, following infection with P. aeruginosa. PPO1ΔPPO2Δ null mutations debilitated the ability of yeast-restricted flies to survive (p < 1 × 10−4) while abolishing yeast-restriction effects on the post-infection survivorship (p = 0.99). A significant diet × genotype interaction term supports a model in which PPO1ΔPPO2Δ null mutations mimic a FF diet in yeast-restricted animals (two-way ANOVA with experiments as a block factor, p = 0.004). Pairwise p values were obtained by one-way ANOVA followed by Tukey’s post hoc analysis. n = 4 independent experiments (1,000 flies in total), which include experiments shown in Figure S2. Of note, the post-infection time used to generate this plot was 66 hr for the experiments shown in the (D) and 100 hr for those shown in Figure S2.
(F–I) Canton-S flies were yeast-restricted or FF for 2–3 days prior to infection with P. aeruginosa. (F) Phenoloxidase (PO) activity following infection with P. aeruginosa. Yeast-restricted flies exhibited an increased PO activity only at 2 hr post-infection compared to FF flies (p = 0.02; pairwise t tests; n = 5 independent preparations). (G–I) mRNA levels of the antimicrobial peptides drosocin, diptericin, and attacin A following P. aeruginosa infection. Yeast-restricted flies exhibited a larger induction of these genes following infection than did FF animals (analysis of covariance; n = 2 preparations). All mRNA values were normalized using tubulin as an endogenous control.
See also Figure S2.
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4. Figure 3
Rapamycin Treatment Recapitulates the Beneficial Effects of Yeast Restriction on Drosophila Immunity
Canton-S flies were yeast-restricted or FF and treated with food supplemented with vehicle or rapamycin (RP or Rapa) for 2 days prior to infection with P. aeruginosa.
(A) Rapamycin treatment improved survivorship following infection of FF flies in a dose-dependent manner (RP6, RP12, RP25 = 6, 12, and 25 mM rapamycin) (p = ; Cox regression analysis of covariance with rapamycin as a continuous predictor; n = 50 flies per group).
(B) FF flies treated with 12 mM rapamycin exhibit survival outcomes following P. aeruginosa infection that are improved over vehicle-fed controls (p < 1 × 10−5) and statistically indistinguishable from yeast-restricted animals (p = 0.06). A significant p value associated with the “diet × rapamycin” interaction supports a model in which rapamycin mimics the effects of yeast restriction (Cox regression, p < 1 × 10−5). n = 5 replicate experiments and 1,091 flies total.
(C) P. aeruginosa titers from individual flies sampled 6 hr post-infection. Pairwise p values were obtained using a t test (n = 20–40 flies per group). A significant p value associated with the diet × rapamycin interaction supports a model in which rapamycin mimics the effects of yeast restriction (two-way ANOVA, p = 0.03). n = 2 replicate experiments.
(D) P. aeruginosa titers from individual flies sampled 23 hr post-infection. Pairwise p values were obtained using a t test (n = 30–60 flies per group). A significant p value associated with the diet × rapamycin interaction supports a model in which rapamycin mimics the effects of yeast restriction (two-way ANOVA, p = 0.03). n = 3 replicate experiments.
See also Figures S2 and S3.
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5. Figure 4
Reduced TOR Signaling Protects FF Flies from Pathogenic Bacterial Infection, Independent of d4EBP and dS6K
Treatment flies were fed the appropriate food supplemented with 200 or 400 μM RU486 (RU) for 2 days to induce transgene expression before infection, while control flies were fed the same food supplemented with ethanol vehicle (A, B, and D).
(A) Survivorship of yeast-restricted and FF transgenic flies with adult-specific expression of a dominant-negative form of Drosophila RagA (dRagAT16N), which suppresses TOR signaling (Tub GS > dRagADN), following infection with P. aeruginosa. dRagADN expression significantly improved survivorship of FF flies (p < 1 × 10−5) such that it was not different from that seen for yeast-restricted animals (p = 0.59). A significant “diet × RU” interaction term supports a model in which dRagADN mimics yeast restriction in FF animals (p = ; Cox regression; n = 6 replicate experiments and a total of 1,407 flies).
(B) P. aeruginosa titers from individual flies expressing dRagADN and vehicle control, measured at 21 hr post-infection. Pairwise p values obtained using t test (n = 8–10 samples per group). dRagADN expression significantly reduced bacterial titer in FF flies (p = 0.01). A significant diet × RU interaction supports a model in which dRagADN expression mimics the effects of yeast restriction (two-way ANOVA, p = 0.04).
(C) Survivorship of yeast-restricted and FF flies carrying a thor (d4EBP) loss-of-function allele together with their appropriate background control strain following infection with P. aeruginosa. There was a strong effect of diet in both strains (p < 1 × 10−5, Cox regression). Mutant and control flies responded similarly to diet (p = 0.67 for diet × genotype interaction; Cox regression; n = 2 replicate experiments and a total of 464 flies).
(D) Survivorship of yeast-restricted and FF transgenic flies with adult-specific expression of a constitutively active form of Drosophila S6K (Tub GS > dS6KCA) following infection with P. aeruginosa. There was a strong effect of diet in both treatments (p < 1 × 10−5, Cox regression). Transgenic and control flies responded similarly to diet (p = 0.72 for diet × RU interaction; Cox regression; n = 2 replicate experiments and a total of 414 flies).
See also Figures S3 and S4.
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6. Figure 5
Yeast Restriction Improves Drosophila Immunity through Post-transcriptional Regulation of Myc
Whole fly protein extracts were used for western blot analysis. All mRNA values were normalized using either tubulin or rp49 as endogenous controls. Transgenic flies were used to overexpress dmyc ubiquitously upon exposure to RU486 (RU) feeding (Tub GS > myc).
(A) Myc protein abundance in Canton-S flies was increased by yeast restriction, and this yeast-restriction effect was abolished by rapamycin feeding. Of note, Myc is mostly phosphorylated (“P”; see Figure S5).
(B) Quantitation of western blot results showing that rapamycin treatment markedly increased Myc abundance in FF flies and reduced differences in its protein levels between the yeast-restricted and FF group (p values obtained by one-way ANOVA followed by Tukey’s post hoc analysis). A significant p value associated with the diet × rapamycin interaction supports a model in which rapamycin mimics the effects of yeast restriction (two-way ANOVA, p = 0.02). Myc signals were normalized by total protein as estimated by Ponceau-S stain. n = 9 replicate experiments.
(C) Survivorship of yeast-restricted and FF transgenic flies with adult-specific overexpression of myc (Tub GS > myc). FF transgenic flies treated with RU486 exhibit survival outcomes following P. aeruginosa infection that are improved over vehicle-fed controls (p < 1 × 10−5) and statistically indistinguishable from yeast-restricted animals (p = 0.77). A significant p value associated with the diet × RU interaction supports a model in which myc overexpression mimics the effects of yeast-restriction (Cox regression, p < 1 × 10−5). N = 2 replicate experiments and 479 flies total.
(D) P. aeruginosa titers measured at 21 hr post-infection. Pairwise p values obtained using one-way ANOVA followed by Tukey’s post hoc analysis (n = 8–10 flies per group). myc overexpression significantly reduced bacterial titer in FF flies (p = 0.001). A significant p value associated with the diet × RU interaction supports a model in which myc overexpression mimics the effects of yeast restriction (two-way ANOVA, p = 0.01).
(E–G) mRNA levels of the antimicrobial peptides drosocin (E), diptericin (F), and attacin A (G) observed in the transgenic flies with adult-specific overexpression of myc following P. aeruginosa infection. Yeast-restricted flies exhibited a larger induction of these genes following infection than did FF animals by 9 hr post-infection (analysis of covariance). myc overexpression modestly potentiated response dynamics in FF animals (analysis of covariance). n = 4 preparations. All mRNA values were normalized using tubulin as an endogenous control.
(H) Real-time qPCR analysis of myc mRNA abundance in transgenic flies that overexpress myc upon exposure to RU486. p values obtained by one-way ANOVA followed by Tukey’s post hoc analysis from five independent preparations. Yeast-restricted and FF flies responded similarly to RU486 (two-way ANOVA, p = 0.14 for diet × RU interaction).
(I) Overexpression of myc increased Myc protein abundance in the yeast-restricted group (p = 2 × 10−7) more strongly than in the FF group (p = 0.01) (two-way ANOVA, p = for diet × RU interaction). Overexpressed Myc levels in the FF animals were statistically indistinguishable from the basal Myc levels in the yeast-restricted animals (p = 0.9). Pairwise p values were obtained by one-way ANOVA followed by Tukey’s post hoc analysis. n = 5 replicate experiments. “P” denotes phosphorylated forms of Myc.
See also Figures S4, S5, and S7.
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7. Figure 6
Reducing the Activity of Protein Phosphatase 2A Increases Myc Abundance in FF Flies and Protects Them from Pathogenic Bacterial Infection
Transgenic flies were used to overexpress a dominant negative form of Drosophila protein phosphatase 2A catalytic subunit (mtsDN) ubiquitously upon exposure to RU486 (RU) feeding (Da GS > mtsDN).
(A) Protein phosphatase 2A (PP2A) activity of transgenic flies with adult-specific expression of a dominant-negative form of Drosophila RagA (dRagAT16N), which suppresses TOR signaling (Tub GS > dRagADN). Yeast-restricted flies exhibited a decreased PP2A activity (p < 1 × 10−5), whereas dRagADN expression significantly reduced the PP2A activity of FF flies (p = ) such that it was not different from that seen for yeast-restricted animals (p = 0.22). A significant diet × RU interaction term supports a model in which dRagADN mimics yeast restriction in FF animals (two-way ANOVA, p = 0.008). Pairwise p values were obtained by one-way ANOVA followed by Tukey’s post hoc analysis. n = 6 independent preparations.
(B) Flies fed 75 μM okadaic acid (OA) increased Myc protein abundance in the yeast-restricted group (p = 0.001) and in the FF group (p = 0.001). Myc levels in the OA-fed FF animals were statistically indistinguishable from the basal Myc levels in the yeast-restricted animals (p = 0.5). Pairwise p values were obtained by one-way ANOVA followed by Tukey’s post hoc analysis. n = 3 replicate experiments. “P” denotes phosphorylated forms of Myc.
(C) FF flies treated with 75 μM OA exhibit survival outcomes following P. aeruginosa infection that are improved over vehicle-fed controls (p = 0.02; Cox regression) and statistically indistinguishable from yeast-restricted animals (p = 0.93; Cox regression). n = 3 independent experiments (523 flies in total).
(D) PP2A activity of transgenic flies with adult-specific expression of a dominant-negative form of Drosophila PP2A catalytic subunit (Da GS > mtsDN). mtsDN expression significantly reduced PP2A activity of FF flies (p = 0.03) such that it was not different from that seen for yeast-restricted animals (p = 0.99). Pairwise p values were obtained by one-way ANOVA followed by Tukey’s post hoc analysis. n = 4 independent preparations.
(E) Adult-specific expression of a dominant-negative form of Mts increased Myc protein abundance in the FF group (p < 6 × 10−6) without affecting its level in the yeast-restricted group (p = 0.8). Overexpressed Myc levels in the FF animals were statistically indistinguishable from the basal Myc levels in the yeast-restricted animals (p = 0.3). A significant diet × RU interaction term supports a model in which MtsDN mimics yeast restriction in FF animals (two-way ANOVA, p < 1 × 10−5). Pairwise p values were obtained by one-way ANOVA followed by Tukey’s post hoc analysis. n = 3 replicate experiments. “P” denotes phosphorylated forms of Myc.
(F) Survivorship of yeast-restricted and FF transgenic flies expressing mtsDN and those treated with vehicle control, following infection with P. aeruginosa. mtsDN expression significantly improved survivorship of FF flies (log-rank test, p < 1 × 10−4). A representative result from one experiment is shown here due to a wide range of survivorship values among five independent experiments.
(G) Average ultimate survival rates of yeast-restricted and FF transgenic flies expressing mtsDN and vehicle control following infection with P. aeruginosa. mtsDN expression significantly improved survivorship of FF flies (p = 0.003). A significant diet × RU interaction term supports a model in which MtsDN mimics yeast restriction in FF animals (two-way ANOVA, p = 0.02). Pairwise p values were obtained by one-way ANOVA followed by Tukey’s post hoc analysis. n = 5 replicate experiments (1,200 flies in total).
(H) P. aeruginosa titers from individual flies expressing mtsDN and vehicle control, measured at 28 hr post-infection. mtsDN expression significantly reduced bacterial titer in FF flies (p = 0.04). Pairwise p values were obtained using a t test (n = 26–30 samples per group).
See also Figures S4–S6.
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8. Figure 7 Model of This Study
Yeast restriction decreases the availability of amino acids in flies. Low levels of intracellular amino acids are sensed by RagA, inhibiting TOR signaling. When TOR becomes inactive, PP2A activity decreases, which in turn stabilizes Myc. Given that overexpressed Myc protein only correlates with the induced levels of AMP mRNAs and not their basal levels, Myc may partner with other factor(s) that becomes available following Pseudomonas infection to modestly increase the transcription of select antimicrobial peptide genes. Yeast restriction also triggers an early burst of melanization, which is executed by active PO, as well as improving tolerance of host animals. It is currently unknown whether Myc is involved in boosting melanization and/or tolerance of yeast-restricted flies to improve host survival following Pseudomonas infection. Of note, this yeast-restriction-TOR-PP2A-Myc signaling does not affect cellular immunity.
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9. Cell Reports 2017 20, 479-490DOI: (10.1016/j.celrep.2017.06.052)
Copyright © 2017 The Author(s) Terms and Conditions