SOJ Microbiology & Infectious Diseases

Flies naturally contain microbes in their intestines after eating microbe-rich food like decaying fruits. When ingesting microbes, insects are also exposed to their toxins. The sensitivity of insects to ingested microbial toxins and their mechanism of response to toxins has not been thoroughly studied. Transcriptional regulator c-Myc has been shown to regulate the response to some but not all microbial toxins in mammals. We tested the sensitivity of wild-type and Myc mutant Drosophila melanogaster strains to two exotoxins, Clostridium perfringens α-toxin and Vibrio cholerae toxin, and two endotoxins, lipopolysaccharides (LPS) of Salmonella minnesota and S. typhimurium. We observed that both sexes of wild-type flies were insensitive to tested toxins. Similarly, Myc mutant males were insensitive to the four toxins. In contrast, female Myc mutants were significantly more sensitive to all tested toxins than wild-type females. The median survival of female Myc mutants was shortened by at least 54 hours in the presence of bacterial toxins. The component of LPS, lipid A, shortened the median survival of Myc females by 104 hours, indicating that the toxicity of LPS is caused by lipid A. This study demonstrates a sex-specific mechanism of the response of insects to toxins and describes that Myc protects female fruit flies from the tested microbial toxins.

Keywords: bacterial toxins; sensitivity; resistance; survival; immunity; Drosophila melanogaster

Abbreviations: Immune deficiency (Imd); antimicrobial peptides (AMP); mitogen-activated protein kinase (MAPK); Bloomington Drosophila stock center (BDSC); wild type (WT); lipopolysaccharide (LPS); adenosine diphosphate (ADP); Jun N-terminal Kinase (JNK); Nuclear Factor-κB (NF-κB); absorption, distribution, metabolism, and excretion (ADME); absorption, distribution, metabolism, excretion, and toxicity (ADMET); confidence interval (CI); deoxyribonucleic acid (DNA); intestinal stem cells (ISCs); Janus kinases (JAK); signal transducer and activator of transcription (STAT); epidermal growth factor EGF; and Wingless (Wg).

Insects that eat decaying food, like rotting fruits, accumulate microbes and toxins in their intestines. Some of these microbes are mammalian pathogens, and insects can serve as their vectors (1). For example, the vectoring of toxin-producing mammalian pathogens by insects and other arthropods has been reported for Vibrio cholerae (2, 3), Escherichia coli (2, 4), Clostridium perfringens (5), and Salmonella species (6, 7).

Insects defend against microbes by targeting cell walls with antimicrobial peptides (AMP) and destroying pathogens with phagocytosing circulating hemocytes (8). To produce AMP, insects rely on Toll and the immune deficiency (Imd) signaling pathways, which are evolutionarily conserved to respond to invading pathogens. Lysine (lys)-type peptidoglycan found in the cell wall of most Gram-positive bacteria activates the Toll pathway (9). A lys derivative, diaminopimelate (dap)-type peptidoglycans found in the cell wall of all Gram-negative bacteria, Gram-positive Bacilli and most Clostridia, and components of fungal wall activate the Imd pathway (10-12).

The innate immune response to microbial toxins in insects is not well understood. In mammals, Staphylococcus aureus bacteria induce cells to secrete toxin receptor-bearing exosomes that scavenge pore-forming α-toxins as decoys (13). Moreover, once microbial toxins cause cellular damage, there exist numerous repair processes. Host cells repair toxin-induced damage to the plasma membrane by the lipogenic process and by clogging and removing toxin pores (14). The intracellular damage caused by microbial toxins is repaired by cytoskeleton remodeling and cell survival pathways such as Mitogen-Activated Protein Kinase (MAPK) pathways (14). Furthermore, host cells degrade organelles that contain microbial toxins by autophagy (14). Ingested microbial toxins may cause stress and damage to cells of the intestinal lining, which would require a mechanism of enterocyte repair. D. melanogaster midgut contains intestinal stem cells (ISCs) located adjacent to the basement membrane of the midgut epithelium (15, 16). These ISCs undergo division by which each ISC produces a renewed ISC and an enteroblast, which in turn differentiates to the absorptive enterocyte (15, 16). Thus, Drosophila midguts regenerate in response to tissue damage and undergo a constant turnover (17). Midgut damage induced by feeding flies with chemicals or bacterial infection can stimulate ISC proliferation and mount tissue regeneration (18, 19). Several signaling pathways are involved in ISC regulation, such as Hippo, JAK/STAT, JNK, EGF, and Wg pathways. These pathways are activated during bacterial infection to stimulate ISC proliferation and converge to regulate the transcription factor Myc (17, 18).

Drosophila Myc is an evolutionally conserved transcription factor that controls cell growth and survival (20). Myc was identified as an essential regulator of ISC proliferation in Drosophila: tissue damage upregulates Myc expression in ISCs. Myc is required for elevated ISC proliferation and gut regeneration in response to tissue damage (21). Myc expression is also regulated by the stress-responsive Jun N-terminal Kinase (JNK), whose activation in progenitor cells leads to the overproliferation of ISCs (22).

The human Myc family includes three members c-Myc, n-Myc, and l-Myc (23). Several previous studies have demonstrated that some but not all microbial toxins induce the expression of mammalian c-Myc. For example, it was shown that lipopolysaccharide (LPS), an endotoxin from Gram-negative bacteria, increased the expression of the human c-Myc (24- 27). Additional toxins that have also been shown to induce the expression of mammalian c-Myc are cyanobacteria microcystin (28), Bacteroides fragilis toxin (29), E. coli enterotoxin B (30), and Pasteurella haemolytica leukotoxin (31). In contrast, cholera toxin does not activate the expression of the human c-Myc (32), and c-Myc does not contribute to the pathogenicity of the C. perfringens α-toxin(33).

Drosophila has only one Myc gene, which shares the closest homology to the human c-Myc (34). The gene encoding for Myc is located on the D. melanogaster X chromosome. Since females have two and males have one X chromosome, insects of different sexes employ a mechanism by which the transcription of X chromosomal genes is equalized between females and males. Nevertheless, it has been shown that the dosage compensation process in Drosophila is not absolute and that many X-linked genes are under- or over-expressed in males compared to females (35-37). It has been observed that the expression of the D. melanogaster Myc gene is higher in females than in males (38). In contrast to mammalian Myc, the role of Myc in D. melanogaster’s response to ingested microbial toxins has not been studied.

We hypothesized that Myc contributes to the resistance to ingested microbial toxins in D. melanogaster, since Myc is involved in ISC proliferation, and ISC-derived intestinal regeneration is a likely response to toxin-induced midgut damage. To address this, we investigated the survival of wild-type and Myc mutant D. melanogaster intoxicated with various endo- and exotoxins. This study describes the role of Myc in the sex-specific response of insects to microbial toxins. Our experiments provide further

Chemicals and reagents
Toxins used for Drosophila feeding assays were purchased from List Biological Laboratories (Campbell, California). Toxins (product numbers) used for screening in Drosophila included: C. perfringens α-toxin (126A), S. typhimurium lipopolysaccharide (225), S. minnesota R595 lipopolysaccharide (304) and monophosphoryl lipid A (401), and V. cholerae cholera toxin (100B).

Drosophila rearing
Oregon-R D. melanogaster (Bloomington Drosophila Stock Center (BDSC) stock #2376) and MYC mutant (BDSC #64769) fly strains were used to conduct experiments. Fly strains were kept at 25oC with 12-hour light/dark cycles. All strains were fed a standard cornmeal-molasses-agar fly medium with yeast flakes.

Drosophila oral feeding survival assay
Oral toxin-feeding survival assays were conducted based on the Drosophila bacterial intestinal infection protocol by Alameh et al. (39). Flies were not starved before experiments. Drosophila vials were prepared by placing three 25 mm diameter circles of extra-thick Whatman blotting paper (Bio-Rad Laboratories, catalog #1703965) at the bottom of the vials and capping them with a cellulose plug. Bacterial toxins and toxin components were diluted in 50 mM sucrose solution at a concentration of 1 μg/ mL, which falls within the range of the concentration of plasmacirculating toxins in infected mammals during the late stages of infections, and as previously tested (39). This concentration is also within the range of toxins found in water used to rinse food contaminated with bacteria (40). Whatman paper was not changed during the experiment.

Depending on the experimental condition, flies were exposed to 50 mM sucrose solution alone or sucrose solution containing 1 μg/mL of bacterial toxins. Flies were anesthetized via CO₂, separated by sex, and placed in vials containing 2.5 mL sucrose solutions. Each vial contained at least 20 males or 20 females per condition. All experiments were performed with male and female flies separately to determine a potential variability in survival and immunity due to sex differences. Because the immunity of flies changes with age (8, 41-43), the tested flies were of mixed ages. Experimental vials were incubated at 30^°C and checked a minimum of 2 times per day to monitor the time of death. Insects that ingest toxins are also exposed to microbes. Other previous studies tested the immunity of D. melanogaster infected with various Gram-positive and -negative bacteria as well as fungi at 30oC. Thus, we designed our experimental intoxication model consistent with other studies (44-47). Although the optimal temperature for the flies ranges from 22 to 25^°C, the toxin-treated flies were maintained at 30^°C. The incubation temperature of 30oC after infection was also chosen because the optimal growth temperature for the pathogenic bacteria whose toxins we used in this study is 30^°C or higher.

We provided two types of quantitative analyses of our data: parametric and non-parametric. Parametric analysis was calculated as a P value, accounting for differences between the entire two Kaplan-Meier curves. We also provide non-parametric analysis, which does not make any assumptions, and the central tendency is measured as the differences in median survivals between two curves. Data analysis was conducted using GraphPad Prism software. All P values reported are products of the respective positive control to a single experimental condition using two statistical analyses: the Log-rank (Mantel-Cox) and the Gehan-Breslow-Wilcoxon tests. An alpha of 0.05 was deemed the threshold for significance. We report P values adjusted by the Bonferroni correction. A change in median survival was reported. Since the chance of dying in a small-time interval was different early in the study and late in the study, the values for the 95% CI of the ratio of median survivals were not meaningful and were not reported. Each insect experiment shown is representative of three independent experiments with 20 flies per condition. Figure 3 shows the average and standard errors of median survival of flies exposed to each toxin. Predictions of delivery, distribution, and physicochemical properties of peptides were calculated using ADMET Predictor v. 10.3 (SimulationsPlus, Lancaster, CA).

The effect of exotoxins on the survival of wild-type and Myc mutant Drosophila melanogaster We tested the response of D. melanogaster to four orally fed exotoxins and endotoxins produced by human bacterial pathogens. These toxins were chosen because they are made by various Gram-negative and Gram-positive bacteria and for their diversity in molecular structures and biochemical functions. The tested toxins are the pore-forming C. perfringens α-toxin ADP ribosylating V. cholerae toxin, and lipopolysaccharide (LPS) from Salmonella typhimurium and S. minnesota, as well as the lipid A component of S. minnesota LPS.

Since D. melanogaster Myc has been shown to play a role in the insect immunity (13), and its expression is higher in females than in males (38), we evaluated the sensitivity of Myc mutants to exo- and endotoxins in two sexes and compared it to that of the wild-type (WT) flies. WT fly survival varied when exposed to the various toxins of human bacterial pathogens. Pore-forming C. perfringens α-toxin significantly increased the median survival of both male and female flies by 84.5 and 20 hours, respectively (Fig. 1A-B). In contrast, V. cholerae (cholera) toxin did not affect the survival of male or female WT flies (Fig. 1C-D).

To determine if Myc is required for the response of Drosophila to microbial exotoxins, we measured the survival of MYC mutant males and females in the presence of toxins tested in the WT. This mutant strain is a null Myc expression mutant because the transcription starts and the first two exons of the Myc gene were deleted (48). In contrast to the extended survival observed in male WT flies, the survival of male MYC flies treated with α-toxin was insignificantly reduced by 15 hours compared to untreated male MYC flies (Fig. 1A). This effect was more striking in female MYC flies, in which α-toxin treatment reduced survival by 54 hours (Fig. 1B).

As with male WT flies, we observed that male MYC flies were insensitive to cholera toxin, as their survival was unaffected by the toxin (Fig. 1C). In contrast, cholera toxin shortened the survival of female MYC flies by 54 hours (Fig. 1D). Collectively, these results suggest that Myc is necessary for the response of female flies to exotoxins of various human pathogens, and that in the absence of Myc, male flies remain insensitive or become insensitive to the tested exotoxins.

The effect of endotoxins on the survival of wild-type and Myc mutant Drosophila melanogaster
Bacterial endotoxins are members of a class of toxins inside bacterial cells, which are released when the cell disintegrates. Such toxins include LPS, found in the outer membrane of gramnegative bacteria and consisting of lipid A and a polysaccharide. We tested the effect of LPS from S. typhimurium and S. minnesota on the survival of males and females of WT and MYC mutant strains. Both toxins did not affect the survival of male WT and female WT flies, as the median survival of flies was not significantly changed by either LPS (Fig. 2A-D). Male MYC flies responded similarly to male WT flies to both LPS treatments (Fig. 2A, C), showing no reduction in survival relative to untreated flies. In contrast, the median survival of female MYC flies treated with S. minnesota LPS (Fig. 2B) or S. typhimurium LPS (Fig. 2D) decreased by 72.5 and 56 hours, respectively.

Additionally, the S. minnesota LPS component lipid A did not affect the survival of WT and male MYC flies relative to untreated flies (Fig. 2E), but lipid A reduced the median survival of female MYC flies by 104 hours relative to untreated MYC females (Fig. 2F). As with LPS, female WT flies were insensitive to lipid A. These results indicate that the toxicity of LPS endotoxins in female MYC flies is at least partly due to the lipid A component. As summarized in Fig. 3, this study suggests that Myc is necessary for the response of female Drosophila to many exo- and endotoxins produced by various human pathogens.

This study investigates whether D. melanogaster Myc gene affects the sensitivity of fruit flies to ingested microbial toxins. We observed that both sexes of wild-type flies were insensitive to the tested microbial toxins. In contrast, Myc mutant females, but not males, were significantly more sensitive to all tested toxins than wild-type females. This study identifies the toxin-response function of the Myc gene in D. melanogaster. Only a few studies tested the effect of microbial toxins on flies by a physiologically relevant feeding assay. This was accomplished by exposing flies to purified recombinant cholera and anthrax toxins (39, 49). In contrast to toxins’ effect on mammals, orally ingested anthrax and cholera toxins did not affect the survival of flies, and anthrax toxin component activated Drosophila resistance to bacteria through immune pathway mechanisms. Additionally,

orally fed LPS from E. coli was tested in D. melanogaster, although the effect of LPS on flies’ survival was not investigated (50). This study tested the sensitivity of flies to four toxins of human bacterial pathogens by the feeding assay and investigated the mechanism of toxin resistance. We observed that while α-toxin prolonged the survival of male and female flies, other tested toxins did not affect the flies’ survival at 1 μg/mL. This study shows that D. melanogaster is insensitive to many bacterial toxins, and that the toxin tolerance in females is mediated by the Myc protein. The role of Myc in male flies is toxin-specific: while the mutation in Myc negated α-toxin-induced survival in males, the response of Myc mutant males to other toxins was similar to that of WT males.

There are several confounding factors that limit the interpretations of our study. Only one toxin concertation was tested for each toxin, and future tests should study whether other concentrations of toxins affect flies’ survival. In addition, the experiments were performed with flies of various ages, because the immunity of flies changes with age (8, 41-43). The age distribution of flies in control and experimental groups within an experiment should be similar. However, the age distribution may not be the same the next time the experiment is performed. Thus, although overall survival may have varied from experiment to experiment, the effect of microbial toxins on survival was reproducible, as seen by error bars for median survival in Figure 3. Additionally, since changing Whatman papers during the experiment was not practical, the evaporation of the sucrose solution limited the survival. Moreover, since the sucrose solution is not a well-balanced food, flies’ survival is not expected to be as long as on the nutrient-rich fly food. Another factor that could potentially limit access to food is the deposition of feces and eggs on the surface of the Whatman paper, which could limit access to food. Moreover, the sex and the Myc mutation may affect the amount of food and microbial toxins consumed by flies.

Previously, the anthrax toxin component, Protective Antigen, was shown to protect insects against pathogenic microbes by activating the Toll pathway (39). However, in contrast to the non-pathogenic and non-toxic anthrax toxin component, other toxins, such as pore-forming α-toxin, ADP ribosylating cholera toxin, and pro-apoptotic LPS, can have harmful activity. Based on our results, we hypothesize that once ingested, microbial toxins trigger stress and immune responses in the gut. The stress to cells of the intestinal lining can cause damage or death of enterocytes in the midgut. In response to the loss of enterocytes, intestinal stem cells proliferate and differentiate to regenerate new enterocytes in the midgut. This step is mainly dependent on Myc in females. The expression of Myc itself is induced in mammalian systems by microbial toxins (24-31). The protein that transcriptionally regulates this process in male flies has not been discovered yet, and future work should focus on identifying such proteins in males. The new enterocytes can induce immunity pathways to inhibit toxin-producing microbes. Based on our previous study (39), bacterial toxins may activate the Toll pathway to reduce the sensitivity of flies to pathogens (Fig. 4). Previous studies demonstrated that cholera toxin does not activate the expression of human c-Myc (32), and c-Myc does not contribute to the pathogenicity of the C. perfringens α-toxin (33). Nevertheless, our study showed that in Drosophila, the response to both toxins requires Myc (Fig. 1). Future studies should validate this model by gut-specific Myc knockdown by RNAi or mutagenesis by CRISPR/Cas9, as well as determine the gene that mediates toxin response in male D. melanogaster. Moreover, future studies should investigate specific tissues, in which Myc mediates microbial toxin response in females by tissue-specific gene inactivation. Additionally, as our results show that Myc mutation shortens flies’ survival, more tests should determine the relationship between survival and toxin response.

While our study helps demonstrate how insects tolerate toxins of human microbial pathogens, resulting in their greater opportunity to transmit the disease-causing microbes, it further suggests that LPS in combination with Myc inhibitors could be used beneficially in agriculture as a new insecticidal combination. More generally, this study identifies insect Myc protein as a new target for future insecticidal compounds for controlling insect vectors of human pathogens. Although human c-Myc is identified as a critical player in tumor development, no specific c-Myc-targeting drugs are available clinically. Existing literature identifying potential compounds targeting mammalian c-Myc may be useful as a basis for optimizing their use as inhibitors of insect Myc. A short list of such possible Myc antagonists is proposed in Table 1. The list of Myc inhibitors in Table 1 was assembled based on the published potency information (51-54), coupled with in silico evaluation of permeability potential, distribution characteristics in biorelevant systems, and chemical or metabolic stability (i.e., ADME properties, using ADMET Predictor v. 10.3).

Myc is known to heterodimerize with another transcription factor, Max (23, 55). All four small molecules presented in Table 1 were discovered in drug screens to inhibit the cMyc- Max interaction. Most prior work has focused on developing cMyc-Max heterodimer inhibitors (55). While published drug candidates targeting mammalian c-Myc have historically tended to be small molecules, there is an effort to develop larger-sized inhibitors of c-Myc. For example, omomyc (56), a 90 amino acid-long peptide that comprises the DNA binding domain of c-Myc, acts as a dominant-negative c-Myc, by competing with the fulllength c-Myc protein for DNA binding. However, while offering additional options for inhibiting insect Myc, there is a greater complexity for its delivery to target insect cells than small molecules (55). In that regard, small molecules targeting Mycmediated insect response to microbial toxins, particularly those produced by vectored human pathogens, may prove useful in agricultural applications.

a. Conflict of interest: NA
b. Ethical approval: NA
c. Clinical trial registration: NA

1. De Vos V, Turnbull PC. Anthrax. In: Coetzer JA, Thomson GR, Tustin RC, eds. Infectious diseases of livestock, with special reference to Southern Africa. 2 ed. Cape Town: Oxford University Press Southern Africa; 2004.
2. Echeverria P, Harrison BA, Tirapat C, McFarland A. Flies as a source of enteric pathogens in a rural village in Thailand. Appl Environ Microbiol. 1983 Jul;46(1):32-36.doi: 10.1128/aem.46.1.32-36.1983
3. Yap KL, Kalpana M, Lee HL. Wings of the common house fly (Musca domestica L.): importance in mechanical transmission of Vibrio cholerae. Trop Biomed. 2008 Apr;25(1):1-8.
4. Monyama MC, Onyiche ET, Taioe MO, Nkhebenyane JS, Thekisoe OMM. Bacterial pathogens identified from houseflies in different human and animal settings: A systematic review and meta-analysis. Vet Med Sci. 2021 May 6.doi: 10.1002/vms3.496
5. Looveren NV, Vandeweyer D, Schelt Jv, Campenhout LV. Occurrence of Clostridium perfringens vegetative cells and spores throughout an industrial production process of black soldier fly larvae (Hermetia illucens). Journal of Insects as Food and Feed. 2022;8(4):399-407.doi.org/10.3920/JIFF2021.0073
6. Mian LS, Maag H, Tacal JV. Isolation of Salmonella from muscoid flies at commercial animal establishments in San Bernardino County, California. J Vector Ecol. 2002 Jun;27(1):82-85.
7. Olsen AR, Hammack TS. Isolation of Salmonella spp. from the housefly, Musca domestica L., and the dump fly, Hydrotaea aenescens (Wiedemann) (Diptera: Muscidae), at caged-layer houses. J Food Prot. 2000 Jul;63(7):958-960.doi: 10.4315/0362-028x-63.7.958
8. Buchon N, Silverman N, Cherry S. Immunity in Drosophila melanogaster--from microbial recognition to whole-organism physiology. Nat Rev Immunol. 2014 Dec;14(12):796-810.doi: 10.1038/nri3763
9. Wang L, Weber AN, Atilano ML, Filipe SR, Gay NJ, Ligoxygakis P. Sensing of Gram-positive bacteria in Drosophila: GNBP1 is needed to process and present peptidoglycan to PGRP-SA. EMBO J. 2006 Oct 18;25(20):5005-5014.doi: 10.1038/sj.emboj.7601363
10. Schleifer KH, Kandler O. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev. 1972 Dec;36(4):407-477.doi: 10.1128/br.36.4.407-477.1972
11. Takumi K, Kawata T. Quantitative chemical analyses and antigenic properties of peptidoglycans from Clostridium botulinum and other clostridia. Jpn J Microbiol. 1976 Aug;20(4):287-292.doi:10.1111/j.1348-0421.1976.tb00990.x
12. Iatsenko I, Kondo S, Mengin-Lecreulx D, Lemaitre B. PGRP-SD, an Extracellular Pattern-Recognition Receptor, Enhances Peptidoglycan-Mediated Activation of the Drosophila Imd Pathway. Immunity. 2016 Nov 15;45(5):1013-1023. doi: 10.1016/j.immuni.2016.10.029
13. Keller MD, Ching KL, Liang FX, Dhabaria A, Tam K, Ueberheide BM, et al. Decoy exosomes provide protection against bacterial toxins. Nature. 2020 Mar;579(7798):260-264.doi: 10.1038/s41586-020-2066-6
14. Brito C, Cabanes D, Sarmento Mesquita F, Sousa S. Mechanisms protecting host cells against bacterial pore-forming toxins. Cell Mol Life Sci. 2019 Apr;76(7):1319-1339. doi: 10.1007/s00018-018-2992-8
15. Micchelli CA, Perrimon N. Evidence that stem cells reside in the adult Drosophila midgut epithelium. Nature. 2006 Jan 26;439(7075):475-479.doi.org/10.1038/nature04371
16. Ohlstein B, Spradling A. The adult Drosophila posterior midgut is maintained by pluripotent stem cells. Nature. 2006 Jan 26;439(7075):470-474.doi: 10.1038/nature04333
17. Jiang H, Patel PH, Kohlmaier A, Grenley MO, McEwen DG, Edgar BA. Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila midgut. Cell. 2009 Jun 26;137(7):1343-1355.doi: 10.1038/nature04333
18. Amcheslavsky A, Jiang J, Ip YT. Tissue damage-induced intestinal stem cell division in Drosophila. Cell Stem Cell. 2009 Jan 9;4(1):49-61.doi: 10.1016/j.stem.2008.10.016
19. Buchon N, Broderick NA, Poidevin M, Pradervand S, Lemaitre B. Drosophila intestinal response to bacterial infection: activation of host defense and stem cell proliferation. Cell Host Microbe. 2009 Feb 19;5(2):200-211. doi: 10.1016/j.chom.2009.01.003
20. Gallant P. Drosophila Myc. Adv Cancer Res. 2009;103:111-144.
21. Ren F, Shi Q, Chen Y, Jiang A, Ip YT, Jiang H, et al. Drosophila Myc integrates multiple signaling pathways to regulate intestinal stem cell proliferation during midgut regeneration. Cell Res. 2013 Sep;23(9):1133-1146.doi:10.1038/cr.2013.101
22. Cordero JB, Stefanatos RK, Scopelliti A, Vidal M, Sansom OJ. Inducible progenitor-derived Wingless regulates adult midgut regeneration in Drosophila. Embo j. 2012 Oct 3;31(19):3901-3917.doi: 10.1038/emboj.2012.248
23. Meyer N, Penn LZ. Reflecting on 25 years with MYC. Nat Rev Cancer. 2008 Dec;8(12):976-990.doi: 10.1038/nrc2231
24. Introna M, Hamilton TA, Kaufman RE, Adams DO, Bast RC, Jr. Treatment of murine peritoneal macrophages with bacterial lipopolysaccharide alters expression of c-fos and c-myc oncogenes. J Immunol. 1986 Oct 15;137(8):2711-2715.
25. Huang C, Zhang Y, Qi H, Xu X, Yang L, Wang J. Myc is involved in Genistein protecting against LPS-induced myocarditis in vitro through mediating MAPK/JNK signaling pathway. Biosci Rep. 2020 Jun 26;40(6).doi: 10.1042/BSR20194472
26. Yao D, Dong Q, Tian Y, Dai C, Wu S. Lipopolysaccharide stimulates endogenous β-glucuronidase via PKC/NF-κB/c-myc signaling cascade: a possible factor in hepatolithiasis formation. Mol Cell Biochem. 2018 Jul;444(1-2):93-102.doi: 10.1007/s11010-017-3234-3
27. Ganguly N, Giang PH, Basu SK, Mir FA, Siddiqui I, Sharma P. Mycobacterium tuberculosis 6-kDa early secreted antigenic target (ESAT-6) protein downregulates lipopolysaccharide induced c-myc expression by modulating the extracellular signal regulated kinases 1/2. BMC Immunol. 2007 Oct 3;8:24.doi: 10.1186/1471-2172-8-24
28. Li H, Xie P, Li G, Hao L, Xiong Q. In vivo study on the effects of microcystin extracts on the expression profiles of proto-oncogenes (c-fos, c-jun and c-myc) in liver, kidney and testis of male Wistar rats injected i.v. with toxins. Toxicon. 2009 Jan;53(1):169-117.doi: 10.1016/j.toxicon.2008.10.027
29. Wu S, Morin PJ, Maouyo D, Sears CL. Bacteroides fragilis enterotoxin induces c-Myc expression and cellular proliferation. Gastroenterology. 2003 Feb;124(2):392-400.doi: 10.1053/gast.2003.50047
30. Soriani M, Williams NA, Hirst TR. Escherichia coli enterotoxin B subunit triggers apoptosis of CD8(+) T cells by activating transcription factor c-myc. Infect Immun. 2001 Aug;69(8):4923-4930.doi: 10.1128/IAI.69.8.4923-4930.2001
31. Marcatili A, D'Isanto M, Vitiello M, Galdiero R, Galdiero M. p53 and c-myc activation by Pasteurella haemolytica leukotoxin is correlated with bovine mononuclear cells apoptosis. New Microbiol. 2002 Apr;25(2):195-204.
32. Muñoz E, Zubiaga AM, Merrow M, Sauter NP, Huber BT. Cholera toxin discriminates between T helper 1 and 2 cells in T cell receptor-mediated activation: role of cAMP in T cell proliferation. J Exp Med. 1990 Jul 1;172(1):95-103.doi: 10.1084/jem.172.1.95
33. Ivie SE, Fennessey CM, Sheng J, Rubin DH, McClain MS. Gene-trap mutagenesis identifies mammalian genes contributing to intoxication by Clostridium perfringens ε-toxin. PLoS One. 2011 Mar 11;6(3):e17787.doi.org/10.1371/journal.pone.0017787
34. Gallant P. Myc/Max/Mad in invertebrates: the evolution of the Max network. Curr Top Microbiol Immunol. 2006;302:235-253. doi: 10.1007/3-540-32952-8_9
35. Chang PL, Dunham JP, Nuzhdin SV, Arbeitman MN. Somatic sex-specific transcriptome differences in Drosophila revealed by whole transcriptome sequencing. BMC genomics. 2011;12(1):1-20.doi.org/10.1186/1471-2164-12-364
36. Lott SE, Villalta JE, Schroth GP, Luo S, Tonkin LA, Eisen MB. Noncanonical compensation of zygotic X transcription in early Drosophila melanogaster development revealed through single-embryo RNA-seq. PLoS biology. 2011;9(2):e1000590.doi.org/10.1371/journal.pbio.1000590
37. Roberts DB, Evans-Roberts S. The X-linked α-chain gene of Drosophila LSP-1 does not show dosage compensation. Nature. 1979;280(5724):691-692.
38. Mathews KW, Cavegn M, Zwicky M. Sexual Dimorphism of Body Size Is Controlled by Dosage of the X-Chromosomal Gene Myc and by the Sex-Determining Gene tra in Drosophila. Genetics. 2017;205(3): 1215-1228.doi: 10.1534/genetics.116.192260
38. Alameh S, Bartolo G, O'Brien S, Henderson EA, Gonzalez LO, Hartmann S, et al. Anthrax toxin component, Protective Antigen, protects insects from bacterial infections. PLoS Pathog. 2020 Aug;16(8):e1008836.doi: 10.1371/journal.ppat.1008836
39. Wang A, Molina G, Prima V, Wang K. Anti-LPS test strip for the detection of food contaminated with Salmonella and E. coli. J Microb Biochem Technol. 2011;3:026-029.
40. Belmonte RL, Corbally MK, Duneau DF, Regan JC. Sexual Dimorphisms in Innate Immunity and Responses to Infection in Drosophila melanogaster. Front Immunol. 2019;10:3075.doi: 10.3389/fimmu.2019.03075
41. Garschall K, Flatt T. The interplay between immunity and aging in Drosophila. F1000Res. 2018;7:160.doi: 10.12688/f1000research.13117.1            
42. Sciambra N, Chtarbanova S. The Impact of Age on Response to Infection in Drosophila. Microorganisms. 2021 Apr 29;9(5).doi: 10.3390/microorganisms9050958
43. Alarco AM, Marcil A, Chen J, Suter B, Thomas D, Whiteway M. Immune-deficient Drosophila melanogaster: a model for the innate immune response to human fungal pathogens. J Immunol. 2004 May 1;172(9): 5622-5628.doi: 10.4049/jimmunol.172.9.5622
44. Eleftherianos I, More K, Spivack S, Paulin E, Khojandi A, Shukla S. Nitric oxide levels regulate the immune response of Drosophila melanogaster reference laboratory strains to bacterial infections. Infect Immun. 2014 Oct;82(10):4169-4181.doi: 10.1128/IAI.02318-14
45. Jensen RL, Pedersen KS, Loeschcke V, Ingmer H, Leisner JJ. Limitations in the use of Drosophila melanogaster as a model host for gram-positive bacterial infection. Lett Appl Microbiol. 2007 Feb;44(2):218-223.doi: 10.1111/j.1472-765X.2006.02040.x
46. Needham AJ, Kibart M, Crossley H, Ingham PW, Foster SJ. Drosophila melanogaster as a model host for Staphylococcus aureus infection. Microbiology (Reading). 2004 Jul;150(Pt 7):2347-2355.doi: 10.1099/mic.0.27116-0
47. Pierce SB, Yost C, Britton JS, Loo LW, Flynn EM, Edgar BA, et al. dMyc is required for larval growth and endoreplication in Drosophila. Development. 2004 May;131(10):2317-2327.doi: 10.1242/dev.01108
48. Blow NS, Salomon RN, Garrity K, Reveillaud I, Kopin A, Jackson FR, et al. Vibrio cholerae infection of Drosophila melanogaster mimics the human disease cholera. PLoS Pathog. 2005 Sep;1(1):e8.doi: 10.1371/journal.ppat.0010008
49. Soldano A, Alpizar YA, Boonen B, Franco L, López-Requena A, Liu G, et al. Gustatory-mediated avoidance of bacterial lipopolysaccharides via TRPA1 activation in Drosophila. Elife. 2016 Jun 14;5. doi: 10.7554/eLife.13133
50. Castell A, Yan Q, Fawkner K, Hydbring P, Zhang F, Verschut V, et al. A selective high affinity MYC-binding compound inhibits MYC:MAX interaction and MYC-dependent tumor cell proliferation. Sci Rep. 2018 07 03;8(1):10064.doi:10.1038/s41598-018-28107-4
51. Choi SH, Mahankali M, Lee SJ, Hull M, Petrassi HM, Chatterjee AK, et al. Targeted Disruption of Myc-Max Oncoprotein Complex by a Small Molecule. ACS Chem Biol. 2017 11 17;12(11):2715-2719. doi:10.1021/acschembio.7b00799
52. Hart JR, Garner AL, Yu J, Ito Y, Sun M, Ueno L, et al. Inhibitor of MYC identified in a Kröhnke pyridine library. Proc Natl Acad Sci U S A. 2014 Aug 26;111(34):12556-12561. doi: 10.1073/pnas.1319488111
53. Jeong KC, Ahn KO, Yang CH. Small-molecule inhibitors of c-Myc transcriptional factor suppress proliferation and induce apoptosis of promyelocytic leukemia cell via cell cycle arrest. Mol Biosyst. 2010 Aug;6(8):1503-1509.doi:10.1039/c002534h
54. Llombart V, Mansour MR. Therapeutic targeting of "undruggable" MYC. EBioMedicine. 2022 Jan;75:103756.doi:10.1016/j.ebiom.2021.103756
55. Beaulieu ME, Jauset T, Massó-Vallés D, Martínez-Martín S, Rahl P, Maltais L, et al. Intrinsic cell-penetrating activity propels Omomyc from proof of concept to viable anti-MYC therapy. Sci Transl Med. 2019 Mar 20;11(484). doi:10.1126/scitranslmed.aar5012