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. Author manuscript; available in PMC: 2017 Aug 17.
Published in final edited form as: Cell Rep. 2017 Jul 11;20(2):479–490. doi: 10.1016/j.celrep.2017.06.052

Acute dietary restriction acts via TOR, PP2A, and Myc signaling to boost innate immunity in Drosophila

Jung-Eun Lee 1,*, Morsi Rayyan 1,2,5, Allison Liao 1,3,5, Isaac Edery 4, Scott D Pletcher 1,6,*
PMCID: PMC5560489  NIHMSID: NIHMS888098  PMID: 28700947

Summary

Dietary restriction promotes health and longevity across taxa through mechanisms that are largely unknown. Here we show that acute yeast-restriction significantly improves the ability of adult female Drosophila melanogaster to resist pathogenic bacterial infections through an immune pathway involving down-regulation of Target of Rapamycin (TOR) signaling, which stabilized the transcription factor Myc by increasing the steady state level of its phosphorylated forms through decreased activity of protein phosphatase 2A. Upregulation of Myc through genetic and pharmacological means mimicked the effects of yeast-restriction in fully-fed flies, identifying Myc as a pro-immune molecule. Short-term dietary or pharmacological interventions that modulate TOR-PP2A-Myc signaling may provide an effective method to enhance immunity in vulnerable human populations.

Keywords: pathogenic bacterial infection, innate immunity, tolerance, Myc, protein phosphatase 2A, Target of Rapamycin, melanization, phenol oxidase, yeast-restriction, dietary restriction

eTOC blurb

Lee et al. reveal how dietary restriction boosts innate immune response against pathogenic bacterial infection, via a signaling mechanism in which reduced TOR signaling results in stabilization of Myc through its suppressor protein phosphatase 2A.

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Introduction

Studies of dietary restriction–a reduction in nutrient intake without malnutrition–in a diverse array of organisms have revealed it to be an effective way to extend lifespan and promote broad-spectrum improvement in health during aging (Fontana et al., 2010, McCay et al., 1935). Early work focused on total caloric intake as the driving force behind these beneficial effects, but studies that have comprehensively examined the effects of individual macronutrients on lifespan underscore the importance of protein-to-carbohydrate ratio. In the fruit fly, Drosophila melanogaster, yeast restriction, however, has been used as an alternative to wholesale dilution of the diet to effectively extend female fly lifespan (Chippindale et al., 1993, Hwangbo et al., 2004, Kapahi et al., 2004, Min and Tatar, 2006, Skorupa et al., 2008). These effects have also been observed in mammals, where protein restriction increased rodent lifespan (Goodrick, 1978, Leto et al., 1976, McCay et al., 1935), and ad libitum feeding of low protein-high carbohydrate diets (but not high protein-low carbohydrate diets) extended lifespan in mice despite long-lived animals consuming more calories (Solon-Biet et al., 2014). Together, these studies establish that the life-extending benefits associated with dietary restriction can be achieved without reducing total caloric intake when the relative consumption of protein to carbohydrates is low.

A striking feature of the effects of dietary restriction is its acute nature, yielding beneficial outcomes with short-term application. In Drosophila, a switch to a restricted diet reduced short-term mortality risk within 48 hours (Mair et al., 2003), and in mice, one week of protein starvation decreased tissue damage caused by temporary blockage of blood flow during surgical operation, greatly improving survival following renal ischemic injury (Peng et al., 2012). Furthermore, a protein-free diet or modest protein restriction for one week protected mice from mortality associated with malaria infection (Ariyasinghe et al., 2006). Even ad libitum feeding of low protein-high carbohydrate diets for eight weeks resulted in metabolic improvement of mice, compared to those fed high protein-low carbohydrate (Solon-Biet et al., 2015).

A significant threat to global health is infectious diseases. In 2009, there were 89,000 deaths caused by pneumonia, septicemia, and influenza virus in the US alone (2012). Opportunistic infections, primarily due to growing resistance to existing antibiotics (2013), increase the risk of secondary infection that is associated with many, often standard, procedures such as organ transplantation, chemotherapy, dialysis, and elective surgery (2013, Carratala and Garcia-Vidal, 2008). Acute preventative strategies that strengthen immunity prior to such procedures are therefore of strong interest.

To answer the questions of whether, similar to general health and aging, innate immune function is acutely modulated by individual nutrients, we executed a comprehensive analysis of the effects of dietary composition on survival following pathogenic infection in Drosophila, a valuable model to study innate immunity (Kimbrell and Beutler, 2001). Although lacking adaptive immunity, insects are equipped with innate immunity, which is an ancient first-line defense mechanism that recognizes the pattern of invading microorganisms as well as their virulence factors (Gottar et al., 2006). Drosophila innate immunity has humoral and cellular components: the synthesis of antimicrobial peptides (AMPs) by the fat body (analogous to the mammalian liver and adipocytes), a deposition of melanin upon injury (melanization), and phagocytosis performed by hemocytes. This innate immune response is highly conserved between Drosophila and mammals (Hoffmann, 2003, Kimbrell and Beutler, 2001).

Here we present evidence that yeast restriction, but not carbohydrate restriction, substantially improves fly survival following bacterial infection through several components of innate immunity. We find that yeast restriction-mediated enhancement of innate immunity is orchestrated by components of the TOR signaling network, in which reduced TOR signaling results in a stabilization of the transcription factor Myc through its suppressor protein phosphatase 2A. Myc in turn mediates a sustained induction of genes that encode antimicrobial peptides, which are effective bacterial killers. These results implicate a function for PP2A and Myc as signaling molecules that serve to potentiate the immune response in yeast-restricted animals following pathogenic infection.

Results

Yeast-restriction improves survival of Drosophila over pathogenic bacteria through humoral immunity

We evaluated the effects of dietary composition on mortality from pathogenic infections in Drosophila. The nutritional content of the fly diet derives primarily from two ingredients: sucrose and brewer’s yeast. By independently manipulating the concentration of each of these macronutrients from 1% to 9% (w/v), we found that yeast levels, but not sucrose levels, influenced the ability of mated female flies to survive infection from PA14 plcs, a mutant strain of a human opportunistic pathogen, Pseudomonas aeruginosa, in which virulence has been attenuated (Lau et al., 2003). Flies fed a 1% yeast diet survived P. aeruginosa infection markedly better and resisted pathogen growth more effectively than did flies fed a 9% yeast diet regardless of carbohydrate levels (Figures 1A and 1B). Given that sucrose and brewer’s yeast are essentially isocaloric (Mair et al., 2005) and that survival correlates with dietary yeast content, the caloric value of the food cannot be the cause for our results. The largest and most consistent differences were observed between flies fed 1% vs. 9% yeast diets, both containing 9% sucrose. We termed flies fed the 1% yeast diet as yeast-restricted (YR) and those with the 9% value as fully-fed (FF), and we focused our efforts using these two diets. The beneficial health effects of yeast-restriction manifested rapidly (after only three days of feeding), leading to increased survival of both young and aged animals following infection (Figure S1). Resistance to a second human pathogen, Staphylococcus aureus, was also potentiated (Figures 1C, 1D, and 2B).

Figure 1. Yeast-restriction improves survival of Drosophila over pathogenic bacteria. See also Figure S1.

Figure 1

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(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 Pseudomonas 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 twelve 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 24h 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 Pseudomonas 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 (YR; 1% yeast/9% sucrose) and fully-fed (FF; 9% yeast/9% sucrose) flies infected with Staphylococcus 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.

Figure 2. Humoral immunity contributes to the yeast restriction-mediated enhancement of host survival over pathogenic bacteria. See also Figure S2.

Figure 2

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(AC) phagoless flies devoid of hemocytes (Defaye et al., 2009) and their control siblings were yeast-restricted or fully-fed 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 x genotype” interaction term of a Cox regression model was not significant (P=0.86), indicating that flies without hemocytes experience a similar increase in survival following yeast-restriction as do 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 x genotype” interaction term of a Cox regression model was not significant (P=0.13), indicating that flies without hemocytes experience a similar increase in survival following yeast-restriction as do control animals. 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 h 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-E) prophenoloxidase1 prophenoloxidase 2 double mutant (PPO1ΔPPO2Δ; also designated as PPO null) that cannot activate phenol oxidase (Binggeli et al., 2014) and their genetic background control w1118 flies were yeast-restricted or fully-fed 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 fully-fed 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 x genotype” interaction term supports a model where PPO1ΔPPO2Δ null mutations mimic a fully-fed 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 (1000 flies in total), which include experiments shown in Appendix Figure S3. Of note, the post-infection time used to generate this plot was 66 h for the experiments shown in the panel D and 100 h for those shown in Appendix Figure S3. (F-I) Canton-S flies were yeast-restricted or fully-fed 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 h post-infection, compared to fully-fed 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 fully-fed animals (analysis of covariance; N=2 preparations). All mRNA values were normalized using tubulin as an endogenous control.

We first tested whether cellular immunity is required for the improved survivorship that we observed in yeast-restricted flies. We generated phagoless transgenic flies (Defaye et al., 2009) that mostly lack plasmatocytes, which correspond to mammalian macrophages, monocytes, and neutrophils and are the only professional immune cells in adult flies (Lavine and Strand, 2002). While more susceptible to Staphylococcus aureus, as expected (Defaye et al., 2009), phagoless flies nevertheless exhibited significant improvement in survival following infection with either P. aeruginosa or S. aureus when yeast-restricted, compared to those fully-fed (Figures 2A and 2B). The beneficial effects of yeast-restriction on pathogenic survival are therefore independent of cellular immunity.

We next asked whether humoral immunity is required for the improved survivorship that we observed in yeast-restricted flies. The first arm of Drosophila humoral immunity consists of the melanization cascade (De Gregorio et al., 2001), which is triggered by cuticular injury and promotes melanin deposition on the injury site to confine and kill the invading microorganism. We found that prophenoloxidase1 prophenoloxidase 2 double mutant flies (PPO1ΔPPO2Δ), which are unable to initiate melanization, succumbed to Pseudomonas infection equally fast in both yeast-restricted and fully-fed conditions (Figures 2D, 2E, S2A, and S2B). Furthermore, activation of phenol oxidase, which is a key enzyme that executes melanization, was potentiated by yeast-restriction in a wild-type strain (Figure 2F). The second arm of Drosophila humoral immunity consists of the antimicrobial peptides (AMP), which are potent, broad-spectrum antibiotics that are rapidly induced following microbial infection (Hoffmann, 2003). The expression of drosocin, diptericin, and attacin A genes were higher during the first 4 to 6 h post-infection in yeast-restricted flies (Figures 2G–2I) whereas the expression of defensin was higher only during the first 2 hours following infection (Figure S2F).

To gain insight into whether mechanisms outside of humoral immunity influence fly survival under yeast-restriction, we took advantage of Dif1key1 null mutants, where antibacterial AMP expression is largely diminished due to the key1 mutation (Rutschmann et al., 2000) and phenoloxidase activity does not increase in response to bacterial infection (Ligoxygakis et al., 2002). Because these flies are severely immune-compromised, we used Enterobacter cloacae β12, non-pathogenic gram-negative bacteria that cause a low mortality of wild-type flies only in high doses (Hedengren et al., 1999). Yeast-restriction improved the post-infection survival of Dif1key1 mutants (Figures S2G–S2H) without affecting the growth of Enterobacter cloacae β12 (Figure S2I), suggesting that tolerance, defined as the ability to withstand a given pathogen load (Medzhitov et al., 2012, Schneider and Ayres, 2008), plays a role in protecting yeast-restricted flies from E. cloacae infection. Yeast-restricted phagoless flies also did not clear S. aureus more effectively than those fully-fed, despite their favorable survival outcome (Figures 2B and 2C; YR vs. FF phagoless flies). A similar trend was observed with old flies infected with P. aeruginosa (Figures S1A and S1B; YR vs. FF 7-week old flies).

TOR signaling mediates the beneficial effects of yeast-restriction on Drosophila immunity

Yeasts provide essential amino acids to wild living Drosophila (Vega and Dowd, 2005) and serve as the sole source of amino acids in the laboratory fly diet. The Target of Rapamycin (TOR) pathway is sensitive to the intracellular levels of amino acids and has a broad impact on health (Wullschleger et al., 2006). To determine whether TOR is responsible for the effects of yeast-restriction in our study, we first measured Drosophila TOR (dTOR) signaling in yeast-restricted and fully-fed flies using ATG1 abundance as a dTOR readout. ATG1 is a target of dTOR, dTOR strongly binds to ATG1 in fed but not in starved larvae, and the ATG1-dTOR interaction inversely correlates with the ATG1 abundance (Chang and Neufeld, 2009). We observed a similar relationship between ATG1 abundance and dTOR/nutritional status in adult flies (Figures S3A–S3D). As a second, independent measure of dTOR signaling, we measured the proportion of S6K phosphorylated on threonine 398, which correlates positively with dTOR-dependent activity of Drosophila S6K (Radimerski et al., 2002) (Figures S3E and S3F). Although total S6K was less abundant in fully-fed flies (Figure S3E), a higher proportion of S6K was phosphorylated in fully-fed flies (Figure S3F), confirming that yeast-restriction reduces dTOR signaling.

Given that yeast-restriction and rapamycin feeding suppress dTOR signaling, we next examined whether pharmacological downregulation of dTOR with rapamycin would improve immune function in fully-fed flies. Rapamycin treatment increased post-infection survival of fully-fed animals in a dose-dependent manner (Figure 3A); fully-fed flies exposed to 12 mM of rapamycin exhibited survival outcomes that were statistically indistinguishable from yeast-restricted animals (Figure 3B). Rapamycin also enhanced the ability of fully-fed flies to suppress pathogen growth measured at both 6 h and 23 h after infection (Figures 3C and 3D), which coincided with its potentiation of select AMP gene expression (Figures S2C–S2F). Pro-survival effects of rapamycin were not due to bactericidal effects of the drug because it did not enhance suppression of P. aeruginosa growth in yeast-restricted flies (Figures 3C and 3D) nor did it affect P. aeruginosa growth in vitro (Figure S3G).

Figure 3. Rapamycin treatment recapitulates the beneficial effects of yeast-restriction on Drosophila immunity. See also Figures S2 and S3.

Figure 3

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Canton-S flies were yeast-restricted or fully-fed 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 fully-fed flies in a dose-dependent manner (RP6, RP12, RP25=6, 12, and 25 mM of rapamycin) (P=0.0002; Cox regression analysis of covariance with rapamycin as a continuous predictor; N=50 flies per group). (B) Fully-fed flies treated with 12 mM of 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 x rapamycin” interaction supports a model where rapamycin mimics the effects of yeast-restriction (Cox regression, P<1×10−5). N=5 replicate experiments and 1091 flies total. (C) P. aeruginosa titers from individual flies sampled 6 h post-infection. Pairwise P-values obtained using t-test (N=20–40 flies per group). A significant P-value associated with the “diet x rapamycin” interaction supports a model where 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 h post infection. Pairwise P-values obtained using t-test (N=30–60 flies per group). A significant P-value associated with the “diet x rapamycin” interaction supports a model where rapamycin mimics the effects of yeast-restriction (two-way ANOVA, P=0.03). N=3 replicate experiments.

We next took a genetic approach to confirm a direct role for dTOR in fly immunity. Manipulations that suppress TOR during development are lethal (Oldham et al., 2000, Zhang et al., 2000).leading us to ask whether acute suppression of TOR signaling in adult flies is sufficient to recapitulate the effects of yeast-restriction on immunity. To this end, the Gene-Switch system was used in which UAS-dependent gene expression is induced by feeding flies food containing mifepristone (RU486) (Osterwalder et al., 2001, Roman et al., 2001). Ubiquitous and adult-specific overexpression of a dominant negative version of Drosophila RagA, a major signaling molecule in regulating amino acid-induced TORC1 activation (Kim et al., 2008), two to three days prior to infection improved survival of fully-fed flies over P. aeruginosa (Figure 4A) and reduced their bacterial load (Figure 4B) without affecting their feeding (Figure S4A).

Figure 4. Reduced TOR signaling protects fully-fed flies from pathogenic bacterial infection, independent of d4EBP and dS6K. See also Figures S3 and S4.

Figure 4

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Treatment flies were fed the appropriate food supplemented with 200 or 400 μM of RU486 (RU) for 2 days to induce transgene expression before infection, while control flies were fed the same food supplemented with ethanol vehicle. (Figures 4A, 4B, and 4D). (A) Survivorship of yeast-restricted and fully-fed 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 fully-fed flies (P<1×10−5) such that it was not different from that seen for yeast-restricted animals (P=0.59). A significant “diet x RU” interaction term supports a model where dRagADN mimics yeast- restriction in fully fed animals (P=0.0003; Cox regression; N=6 replicate experiments and a total of 1407 flies). (B) P. aeruginosa titers from individual flies expressing dRagADN and vehicle control, measured at 21 h post infection. Pairwise P-values obtained using t-test (N=8–10 samples per group). dRagADN expression significantly reduced bacterial titer in fully-fed flies (P=0.01). A significant “diet x RU” interaction supports a model where dRagADN expression mimics the effects of yeast-restriction (two-way ANOVA, P=0.04). (C) Survivorship of yeast-restricted and fully-fed 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 x genotype” interaction; Cox regression; N=2 replicate experiments and a total of 464 flies). (D) Survivorship of yeast-restricted and fully-fed 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 x RU” interaction; Cox regression; N=2 replicate experiments and a total of 414 flies).

Yeast-restriction boosts Drosophila immunity through TOR-mediated upregulation of Myc

To better elucidate the pathway(s) linking yeast availability with innate immune function, we first examined canonical effector molecules of TOR signaling (Wullschleger et al., 2006). We found that neither thor (Drosophila 4EBP) loss of function nor overexpression of constitutively active Drosophila S6K (dS6K), both of which are expected to mimic enhanced TOR signaling (Hay and Sonenberg, 2004), impaired the ability of yeast-restricted flies to survive pathogenic infection (Figures 4C and 4D). Conversely, overexpression of constitutively active Thor or dominant negative dS6K, both of which are expected to mimic reduced TOR signaling (Hay and Sonenberg, 2004), did not improve the ability of fully-fed flies to survive pathogenic infection (Figures S4D and S4E). Together, these data suggest that neither Thor nor dS6K is sufficient to affect the pro-immune pathway mediated by yeast-restriction.

Recent work has identified Drosophila Myc as a target of the TOR pathway (Teleman et al., 2008), in which dTOR signaling positively correlated with Myc protein abundance in S2 cell culture (Parisi et al., 2011, Teleman et al., 2008). If Myc mediates the effects of yeast-restriction through dTOR, we predicted that its level would decrease in response to yeast-restriction. Contrary to our initial hypothesis, we found that Myc was more abundant in yeast-restricted adult flies (Figure 5A). Furthermore, rapamycin increased Myc levels overall and eliminated differences between yeast-restricted and fully-fed flies (Figures 5A and 5B). Overexpression of myc phenocopied both yeast-restriction and rapamycin feeding by greatly improving survivorship following infection with P. aeruginosa (Figure 5C), retarding bacterial growth (Figure 5D), and modestly potentiating the expression of drosocin, diptericin, and attacin A but not defensin in fully-fed flies following infection (Figures 5E–5G and S3F) without reducing their feeding (Figure S4B).

Figure 5. Yeast-restriction improves Drosophila immunity through post-transcriptional regulation of Myc. See also Figures S3, S4, and S7.

Figure 5

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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) (Figures 5C-5I). (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 S14). (B) Quantitation of Western blot results showing that rapamycin treatment markedly increased Myc abundance in fully-fed flies and reduced differences in its protein levels between the YR 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 x rapamycin” interaction supports a model where 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 fully-fed transgenic flies with adult-specific overexpression of myc (Tub GS > myc). Fully-fed 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 x RU” interaction supports a model where 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 h 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 fully-fed flies (P=0.001). A significant P-value associated with the “diet x RU” interaction supports a model where myc overexpression mimics the effects of yeast-restriction (two-way ANOVA, P=0.01). (E-G) mRNA levels of the antimicrobial peptides, drosocin, diptericin, and attacin A, 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 fully-fed animals by 9 h post infection (analysis of covariance). myc overexpression modestly potentiated response dynamics in fully-fed animals (analysis of covariance). N=4 preparations. All mRNA values were normalized using tubulin as an endogenous control. (H) Real time quantitative PCR 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 fully-fed flies responded similarly to RU486 (two-way ANOVA, P=0.14 for “diet x RU” interaction). (I) Overexpression of myc increased Myc protein abundance in the YR group (P=2×10−7) more strongly than in the FF group (P=0.01) (two-way ANOVA, P=0.0004 for “diet xRU” 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.

Our data suggest a complex relationship between dietary yeast/TOR signaling, Myc regulation, and immune function. For example, the increased levels of Myc protein in yeast-restricted flies compared to fully fed controls (Figures 5A and 5B) were not reflected in endogenous myc mRNA levels, which were similar (Figure 5H). Furthermore, when myc was overexpressed in yeast-restricted animals, Myc protein abundance was significantly increased over similarly fed control animals, but post-infection survival was unaffected (Figures 5C and 5I). On the other hand, modest increases in Myc levels were sufficient to significantly improve immune function of fully-fed flies (Figures 5C, 5D, and 5I), suggesting a threshold model whereby Myc abundance beyond that observed in yeast-restricted flies has little effect. Taken together, these data indicate that yeast-restriction increases Myc protein abundance through post-transcriptional regulation via reduced TOR signaling, which enhances humoral innate immunity.

TOR downregulates Myc through its suppressor protein phosphatase 2A

In mammals, the stability of c-Myc protein is reduced by its dephosphorylation of serine 62 via protein phosphatase 2A (PP2A) and phosphorylation of threonine 58 by Glycogen Synthase Kinase 3. Shaggy (Sgg, the Drosophila orthologue of Glycogen Synthase Kinase 3) primes Myc for proteosomal degradation by the ubiquitin ligase Archipelago (Ago) (Galletti et al., 2009, Moberg et al., 2004). We therefore asked whether PP2A, Sgg, or Ago are required for differential Myc levels in fully-fed flies. Ubiquitous RNAi-mediated knockdown of Ago or Sgg increased Myc protein levels in yeast-restricted adult flies but had little effect in fully-fed animals (Figures S5A and S5B), suggesting that they are not rate-limiting for regulating Myc abundance in fully-fed conditions. A potent inhibition of PP2A, however, via feeding of okadaic acid (OA), increased Myc levels in fully-fed flies in a dose-dependent manner (Figure S5C), due to an increase in phosphorylated Myc (Figure S5D). These data suggest that PP2A serves as a primary mechanism to reduce Myc levels in fully-fed conditions. If TOR signaling were a key molecular link between yeast-restriction and PP2A activity, we would predict higher PP2A activity in fully-fed conditions, which would be abrogated upon TOR inhibition. This is, indeed, what we observed (Figure 6A).

Figure 6. Reducing the activity of protein phosphatase 2A increases Myc abundance in fully-fed flies and protect them from pathogenic bacterial infection. See also Figures S4, S5, and S6.

Figure 6

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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) (Figures 6D-6H). (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 PP2A activity of fully-fed flies (P=0.0006) such that it was not different from that seen for yeast-restricted animals (P=0.22). A significant “diet x RU” interaction term supports a model where dRagADN mimics yeast-restriction in fully fed 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 YR 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) Fully-fed flies treated with 75 μM okadaic acid (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 protein phosphatase 2A catalytic subunit (Da GS > mtsDN). mtsDN expression significantly reduced PP2A activity of fully-fed 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 YR 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 x RU” interaction term supports a model where MtsDN mimics yeast-restriction in fully-fed 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 fully-fed transgenic flies expressing mtsDN and those treated with vehicle control, following infection with P. aeruginosa. mtsDN expression significantly improved survivorship of fully-fed 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 fully-fed transgenic flies expressing mtsDN and vehicle control, following infection with P. aeruginosa. mtsDN expression significantly improved survivorship of fully-fed flies (P=0.003). A significant “diet x RU” interaction term supports a model where MtsDN mimics yeast-restriction in fully fed 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 (1200 flies in total). (H) P. aeruginosa titers from individual flies expressing mtsDN and vehicle control, measured at 28 h post infection. mtsDN expression significantly reduced bacterial titer in fully-fed flies (P=0.04). Pairwise P-values were obtained using t-test (N=26–30 samples per group).

We used pharmacological and genetic approaches to downregulate PP2A activity acutely prior to infection in fully-fed flies. These animals treated with 75 μM OA for three days exhibited increased levels of Myc and higher rates of survival to P. aeruginosa infection, both of which were comparable to those seen in yeast-restricted flies treated with vehicle (Figures 6B and 6C). OA did not affect P. aeruginosa growth in vitro (Figure S5E), demonstrating that OA does not improve fly survival by directly killing the pathogen. Given that high concentrations of OA can inhibit type 1 protein phosphatase as well as PP2A (Haeseleer et al., 2013), we targeted PP2A activity directly using adult-specific overexpression of a dominant negative allele of the Drosophila PP2A catalytic subunit (mtsDN) (Hannus et al., 2002). Overexpression of mtsDN reduced PP2A activity in fully-fed flies (Figure 6D), elevated Myc abundance, and improved survivorship following infection to levels seen in yeast-restricted flies (Figures 6E–6G) without reducing their feeding (Figure S4C). This manipulation also suppressed bacterial growth in the fully-fed group (Figure 6H), suggesting that greater PP2A activity is responsible for the diminished immune response in yeast-rich conditions, likely due to the inhibitory role of PP2A on Myc abundance.

Currently we cannot exclude the possibility that PP2A targets in addition to Myc contribute to boosting immunity in yeast-restricted animals due to technical challenges associated with manipulating this pathway. myc null mutant adult animals are inviable (Pierce et al., 2004), and adult-specific RNAi-mediated knockdown of myc failed to limit Myc protein levels in yeast-restricted flies, mostly likely due to low PP2A activity in these conditions (Figures 6A, 6D, and S6E). On the other hand, overexpression of mts substantially increased levels of Mts β isoforms without affecting Mts α isoforms (Figures S6A, S6C, and S6D), and it only modestly increased PP2A activity in yeast-restricted flies (Figure S6E). This manipulation led to no change in Myc abundance or survivorship of flies following infection (Figures S6B, S6F, and S6G). An understanding of the molecular details as to how dTOR signaling boosts PP2A activity and how PP2A decreases Myc abundance in fully-fed flies would shed light on these issues.

Insulin-like signaling complements the TOR pathway in mediating health-benefits of dietary restriction (Fontana et al., 2010), and it promotes transcriptional activity of Myc through Drosophila FOXO (dFOXO) in S2 cell culture (Demontis and Perrimon, 2009). We tested whether insulin-like signaling also contributes to the pro-survival effects of yeast-restriction by comparing survivorship of foxo null mutants (Slack et al., 2011) and their backcrossed control flies on yeast-restricted and fully-fed diets. We found that survival following P. aeruginosa infection was similarly affected by diet in both mutant and control animals (Figures S7A and S7B), suggesting that dFOXO does not influence Myc in adult flies. Furthermore, unlike dFOXO activated under starvation (Becker et al., 2010), yeast-restriction does not increase the basal mRNA levels of antimicrobial peptides (Figures S7C–S7E). We therefore find no evidence that dFOXO is mediating the effects of yeast restriction on Drosophila immunity.

Discussion

We found that yeast-restriction increases the chance of fly survival following bacterial infection by boosting humoral immunity and tolerance. We also identified functions for PP2A and Myc as signaling molecules that potentiate innate immunity in response to yeast-restriction and improve survival following pathogenic infection. Our data suggest that the beneficial effects of yeast-restriction on post-infection survival are governed by reduced TOR signaling, which diminishes PP2A activity. Reduced PP2A activity in turn increases the level of phosphorylated Myc, markedly raising its steady state level in yeast-restricted flies. We propose a model where acute yeast-restriction reduces the activity of PP2A through inhibition of TOR signaling, which in turn stabilizes Myc and improves humoral immunity following pathogenic infection (Figure 7).

Figure 7. Model of this study.

Figure 7

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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 phenoloxidase (PO), as well as improving tolerance of host animals. It is currently unknown whether Myc is involved klin 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.

The mechanism underlying the ability of Myc to potentiate the immune response is currently unknown, but our data suggest some possibilities. Overexpression of myc does strengthen resistance, which is the ability to suppress pathogen growth: it strongly reduced pathogen growth of fully-fed flies and improved their survival. Increased Myc abundance did not impact the basal levels of AMP gene expression, but it modestly increased its induction following infection, suggesting the possibility that Myc requires other factor(s) to increase the transcription of a subset of AMPs in yeast-restricted flies during Pseudomonas infection. On the other hand, PPO1ΔPPO2Δ mutants survived Pseudomonas infection poorly irrespective of diet, suggesting that an early burst of melanization, rather than potentiated AMP expression, may contribute to improved survival of yeast-restricted host animals. Given the impact of yeast-restriction on multiple aspects of humoral immunity, it warrants further investigation whether Myc causes a preferential activation of phenol oxidase in yeast-restricted flies during early infection.

Tolerance phenotypes can be observed when hosts exhibit differential survival outcomes with the same pathogen load, which is consistent with the increased survival of yeast-restricted flies in the absence of changes in pathogen load under malnutrition (i.e. 1% sucrose 1% yeast, Figures 1A and 1B), in advanced age (Figures S1A and S1B), in the absence of hemocytes (Figures 2B and 2C; control vs. phagoless flies), and with compromised humoral immunity (Figures S2G–S2I). These results suggest that yeast-restriction can improve tolerance as well as resistance, and it will be important to determine whether Myc underlies these effects as well.

In contrast with our finding that yeast-restriction improved the ability of female flies to survive P. aeruginosa or S. aureus delivered underneath the cuticle, Allen et al. recently reported that high nutrient diets improve tolerance of male flies injected with Burkholderia cepacia via TOR complex, although the detailed mechanisms underlying these effects were not identified (Allen et al., 2016). Yeast-restriction did not affect the post-infection survival of male flies in our experimental setting (Figure S7F), perhaps due to the use of different infection routes. Allen et al. injected B. cepacia directly into the hemolymph, thus causing a near instantaneous systemic infection, while our needle-prick procedure was designed to mimic a localized infection. Regardless, our collective findings suggest that nutrients impact several different components of host defense mechanisms during pathogenic bacterial infection through TOR signaling.

Rapamycin protects susceptible mice from Listeria monocytogenes infection (Weichhart et al., 2008), and mycobacterial infection increases the level of c-Myc in primary human blood macrophages (Yim et al.), suggesting that the TOR-Myc axis may also be important to protect mammals against bacterial infection. Given the strong evolutionary conservation of TOR, PP2A, and Myc, our results provide a mechanistic framework that focuses on protein restriction as a means of enhancing host survival following pathogenic bacterial infection, which may be translated into practical and inexpensive interventions that reduce mortality risk in susceptible human populations.

Materials and Methods

Fly diet

Flies were allowed to develop and age as adults on a standard yeast-sugar-cornmeal diet (‘CT’ food as described in Table 1), which is similar to the 9% sucrose 3% yeast food. For treatment with rapamycin (LC Laboratories) or okadaic acid (LC Laboratories), 40 μl of 12.5 mM of rapamycin or 75 μM of okadaic acid was aliquoted onto the surface of 11 ml food in a standard wide vial (Teleman et al., 2005). Ethanol was used as a vehicle for both. Treated food was evaporated under a fume hood for 2–4 hours before use. For RU486 treatment, a thin layer of food containing 200 or 400 μM of RU486 was added to the top of the same base food (We initially used 400 μM of RU486 but switched to 200uM when we observed that it was sufficient to induce transgene expression to a comparable magnitude).

Survival assays

Bacterial infection and measurement of survival rates were performed as described (Lee and Edery, 2008) with the following modification. Larval density was strictly controlled by dispensing 20 μl of eggs to a standard Drosophila culture bottle (Linford et al., 2013). For each infection experiment, 20 young adult female flies (2–6 days old) of the same genotype were placed in a vial with experimental food and then entrained under 12 h light: 12 h dark cycles at 25°C for 2–3 days. Infection experiments were performed on either the third or fourth day of entrainment; flies were always infected during the light phase to account for circadian effects on pathogen survival (Lee and Edery, 2008)analys. The bacterial dose was adjusted to target a survival rate of minimum 30% to the most resistant treatment (often yeast-restricted flies). Marginal survival probabilities from several replicate experiments were calculated and significance values were obtained using survival analysis techniques (see below). A voice-activated data recording system (DLife) developed in the Pletcher laboratory was used to record the number of survivors post-injury/infection (Linford et al., 2013).

Bacterial growth assay

At 0, 6, and 23 h post-infection, individual flies were sampled and placed in a standard microfuge tube that contained 100 μl of ice-cold LB/gentamycin media and four stainless steel balls of 2.3 mm diameter (BioSpec Products). Flies were then individually homogenized with an automatic tissue homogenizer at 30 Hz for 30 seconds (TissueLyser, Qiagen) at 4°C. For measurement of bacterial titer at 0 and 6h post infection, 60–70 μl of each fly extract was plated onto an LB/gentamycin plate manually by shaking glass plating beads (ColiRollers™, Novagen) inside the plate. For measurement of bacterial titer at 23h post infection, 20 μl of each fly extract was spirally plated onto an LB/gentamycin plate, using an automatic plater (AutoPlate, Advanced Instruments, INC.). This mode of spiral plating dilutes each fly extract exponentially during plating, which allows accurate measurement of the bacterial titer up to 10,000 bacteria per plate with a diameter of 100 mm. Plates were incubated at 37°C overnight, and visible colonies were photographed, and enumerated with an automated bacterial counter developed in the Pletcher laboratory. At least 8 replicate plates were averaged to estimate the bacterial titer in each treatment group.

Hemolymph Extraction

Ten flies were loaded into a Zymo Spin IIIC spin column (Zymo Research, C1006) housed by a reservoir Eppendorf tube that contained 9 μl of hemolymph extraction buffer (5 mM CaCl2, 1X Complete EDTA-free protease inhibitor [Roche], 1X PBS). These flies were covered with glass beads of 2.5 mm diameter (BioSpec Products, #11079125) in the spin column and centrifuged at 9000 g for 5 minutes (4°C). Roughly 0.5 to 1 μl of hemolymph was obtained and diluted into 9 μl of extraction buffer during this spin and snap-frozen until ready to use. Of note, spin columns and collection tubes were kept on ice and hemolymph was extracted in the presence of Complete EDTA-free protease inhibitor (Binggeli et al., 2014, Nam et al., 2012) to prevent an artificial activation of phenoloxidase, which is triggered by physical injury during the spin.

Phenoloxidase assay

Total protein concentration of hemolymph was determined by the bicinchoninic acid assay, using 1 μl of diluted hemolymph for 25 μl of assay in a half-bottom 96-well plate. 2 mg/ml of L-DOPA was freshly made in 35% ethanol at 37°C for at least 10 minutes with constant shaking in a thermomixer (Qiagen, 1400 rpm) right before used in phenoloxidase assay. Hemolymph that contains 1 μg of total protein was mixed with 50 μl of 2 mg/ml of L-DOPA in a well of a half-bottom 96-well plate. This assay plate was incubated at 30°C for 30 minutes an d phenoloxidase activity was determined based on the A490.

Statistical analysis

Survivorship distributions following pathogenic infection were estimated using the Kaplan-Meier estimator, with right censoring applied for animals that remained alive at the end of the experiment. Treatment effects on survival and their associated significance values were determined using proportional hazards models (i.e., Cox regression) or general linear models together with a probit link. Tests for proportionality among treatments were applied using weighted residuals and the cox.zph function in R. When appropriate, multiple experiments were incorporated into a stratified analysis by defining separate experiments as strata (Cox regression) or as a blocking factor (probit analysis). For simple tests that compare two treatments (e.g., yeast-restriction vs. fully-fed), main effect P-values are reported. For tests involving epistasis, which address the impact of a drug or genetic manipulation on the effect of diet, the interaction P-value for the two treatments is reported. Least-squared regression was used to quantify the patterns of antimicrobial peptide expression following infection. Analyses of covariance were applied to determine significance values for the effects of diet and for the interaction between diet and rapamycin treatment. For pair-wise comparisons involving PP2A activity or ultimate survival rates, data analysis focused on the effects of diet or RU486 treatment within each block of experiment (complete block design; (Kuehl, 1994)) and significance values for the interaction between diet and RU486 treatment were determined accordingly (two-way ANOVA with experiments as a block factor). For pair-wise comparisons involving protein abundances or bacterial titer, comparison among groups was carried out using ANOVA or Student’s t-test as appropriate. All error bars indicate standard error of means. Statistical analysis described above was carried out with the free statistical software package R or OriginPro 8 software (OriginLab Corporation).

Supplementary Material

1
2
3

Highlights.

  • Adult flies survive bacterial infections much better under acute yeast-restriction.

  • Melanization play a key role in yeast restriction-mediated immune protection.

  • Target of Rapamycin (TOR) regulates yeast restriction-mediated immune protection

  • TOR reduces Myc protein abundance through protein phosphatase 2A.

Acknowledgments

We are grateful to J. Park and G. Garcia for technical advice and N. Linford for valuable feedback. T. Neufeld, F. Leulier, C. Kocks, D. Ferrandon, P. Kapahi, and JH. Lee generously shared their fly stocks or antibody with us. J. Eakin, L. Johnson, D. Paris, and A. Zhang provided fly food and N. Juliar, G. Costello, and G. Hwang assisted us with experiments. This study was supported by a National Institute of Health Career Transition Award (K99AG046370) to J-E. Lee, a National Institute of Health grant (R01 NS-042088) to I. Edery and by National Institutes of Health grants (R01AG030593, TR01AG043972, and R01AG023166), and the Glenn Foundation (to S. D. Pletcher).

Footnotes

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Author contributions:

J-E. Lee conceived this study and designed experiments. J-E. Lee, M. Rayyan, and A. Liao performed experiments. J-E. Lee, M. Rayyan, A. Liao, I. Edery, and S. D. Pletcher analyzed data. J-E. Lee, I. Edery, and S. D. Pletcher drafted this manuscript.

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Supplementary Materials

1
NIHMS888098-supplement-1.pdf (1.8MB, pdf)
2
NIHMS888098-supplement-2.docx (48.7KB, docx)
3
NIHMS888098-supplement-3.pdf (1.9MB, pdf)

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