1* Foodborne Toxin Detection and Prevention Research Unit, Western Regional Research Center, United States Department of Agriculture (USDA), 800 Buchanan Street, Albany, CA 94710, USA
2 Michigan State College of Human Medicine, 15 Michigan St. NE, Grand Rapids, MI, 49503, USA
3 Interpath Laboratory, Pendleton, OR 97801, USA
4 Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Ave, Los Angeles, CA 90033, USA
Keywords: toxin; sensitivity; resistance; survival; immunity; Drosophila melanogaster
Abbreviations: Jun related antigen (Jra); immune deficiency (Imd); antimicrobial peptides (AMP); mitogen-activated protein kinase (MAPK); Bloomington Drosophila stock center (BDSC); wild type (WT); lipopolysaccharide (LPS); adenosine diphosphate (ADP); adenosine triphosphate (ATP); adenosine monophosphate (cAMP); 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); soluble N-ethylmale-imide-sensitive factorattachment protein receptors (SNARE); basic leucine zipper (bZIP); deoxyribonucleic acid (DNA); eukaryotic elongation factor (eEF); programmed death-ligand (PD-L); cluster of differentiation (CD).
To defend against invading microbes, insects produce antimicrobial peptides (AMP) that target microbial cell walls and employ circulating hematocytes to eliminate microbes via reactive oxygen species damage and phagocytosis (24). Insects utilize two evolutionarily conserved signaling pathways, Toll and the immune deficiency (Imd), to express AMP in response to microbial pathogens. The Toll pathway is activated by lysine (lys)- type peptidoglycan found in cell walls of most Gram-positive bacteria (25). The Imd pathway is mainly activated by lys-derived diaminopimelate (dap)-type peptidoglycans found in cell walls of Gram-negative bacteria, Bacillus species, and most Clostridium species, and by fungal cell wall components (26-28). In addition to AMP and phagocytic responses, microbial pore-forming toxins induce fly intestinal epithelia to undergo a thinning (purging) followed by a rapid recovery of initial thickness, contributing to the maintenance of gut wall integrity during microbial infections (29).
While the innate immune response to microbes is wellknown in insects and mammals, the innate immune response to toxins is not well understood. Recent studies with Staphylococcus aureus pore-forming α-toxin showed that the bacteria induce the secretion of toxin receptor-bearing exosomes from mammalian cells, which act as toxin decoys by scavenging and preventing toxins from binding to target cells (30). Furthermore, conserved recovery mechanisms allow host cells to repair mechanical damage inflicted by toxins, such as plasma membrane repair by the lipogenic process and clogging and removing toxin pores (31). The intracellular responses to toxins include cytoskeleton remodeling and cell survival pathways such as Mitogen-Activated Protein Kinase (MAPK) pathways (31). Moreover, host cell autophagy of toxin-containing organelles is a central defense mechanism against toxins (31).
Several reports showed that microbial toxins increase the abundance of a stress response transcription factor, Jun, in human cells (32, 33) and the homolog Jun-related antigen, Jra, in Drosophila (34, 35). In the absence of bacteria, Jun is activated by S. aureus α-toxin and is necessary for the resistance of host cells to this toxin, shown by increased sensitivity to α-toxin in Jundeficient cells (32). In Drosophila, expression of Jra is activated in response to cholera toxins and E. coli lipopolysaccharide (LPS) treatments (34-36). These observations suggest that Jun may be essential for activating the response to both endo- and exotoxins of bacterial pathogens and microbial toxins. We hypothesized that Jra is necessary for innate immune response to microbial toxins in Drosophila. This study investigated insect sensitivity to several endo- and exotoxins. It began to uncover the mechanism of the response to many toxins in the D. melanogaster model of oral intoxication. Our experiments provide further insights into mammals’ innate immunity mechanisms against bacterial toxins.
Toxins used for Drosophila feeding assays (Table 1) were purchased from List Biological Laboratories (Campbell, California). Toxins (product numbers) used for screening in Drosophila included: Bordetella pertussis toxin (180) and adenylate cyclase toxin (188L), Clostridium difficile toxins A (152C) and B (155B), Clostridium perfringens ε-toxin (126A), Clostridium septicum α-toxin (116L), Corynebacterium diphtheriae toxin (150), Escherichia coli O111:B4 lipopolysaccharide (201), Pasteurella multocida toxin (156), Salmonella typhimurium lipopolysaccharide (225), Salmonella minnesota R595 lipopolysaccharide (304) and monophosphoryl lipid A (401), and Vibrio cholerae cholera toxin (100B).
Drosophila rearing
Oregon-R Drosophila melanogaster (Bloomington Drosophila Stock Center (BDSC) stock #2376) fly strain was used to conduct wild-type (WT) experiments. Mutant strains (stock # from BDSC) used in this study were: Jra (7218), Jra (7217), and JraIA109 (3273). 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. (37). 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 added in 50 mM sucrose solution at a concentration of 1 μg/mL, which falls within the range of the concentration of plasma-circulating toxin components in infected mammals during the late stages of infections, and as previously tested (37). This concentration is also within the range of toxins found in water used to rinse food contaminated with bacteria (38). 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 per condition. All experiments were performed with male flies to avoid a potential variability in survival and immunity due to confounding sex differences. Because the immunity of flies changes with age (24, 39-41), the tested flies were of mixed ages. Experimental vials were incubated at 30°C and checked at least 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 Gram-positive and -negative bacteria as well as fungi at 30°C. Thus, we designed our experimental intoxication model consistent with such studies (42-45). 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 30°C 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.
Data analysis
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 nonparametric 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 2N A shows the average and standard errors of median survival of flies exposed to each toxin. Predictions of delivery,
Table 1.Toxins used in this study. |
||
Toxin |
Toxin type |
Pathogen (Gram + or -) |
Alpha (a) toxin |
Pore-forming |
Clostridium septicum (+) |
Epsilon (e) toxin |
Pore-forming |
Clostridium perfringens (+) |
Adenylate cyclase |
Converting ATP to cAMP |
Bordetella pertussis (-) |
Pertussis toxin |
ADP-ribosylating |
Bordetella pertussis (-) |
Cholera toxin |
ADP-ribosylating |
Vibrio cholerae (-) |
Diphtheria toxin |
ADP-ribosylating |
Corynebacterium diphtheriae (+) |
Toxin A |
Glycosylating |
Clostridium difficile (+) |
Toxin B |
Glycosylating |
|
Pasteurella multocida toxin |
Deaminating |
Pasteurella multocida (-) |
Lipopolysaccharide (LPS) |
Outer membrane endotoxin |
Escherichia coli (-) |
LPS |
Outer membrane endotoxin |
Salmonella typhimurium (-) |
LPS |
Outer membrane endotoxin |
Salmonella minnesota (-) |
Lipid A |
Component of LPS |
Insects can be exposed to various toxin-producing human bacterial pathogens. The effect of ingested toxins on insects has not been extensively studied. Thus, we tested the response of D. melanogaster to twelve orally fed exotoxins and endotoxins produced by ten human bacterial pathogens. These toxins were chosen because they originate from various Gram-negative and Gram-positive bacteria with diversity in molecular structures and biochemical functions. Toxins and their characteristics are summarized in Table 1.
Wild-type (WT) fly survival varied when exposed to the various toxins of human bacterial pathogens (Fig. 1). Poreforming α- and ε-toxins from C. septicum and C. perfringens significantly increased the median survival of flies by 85.5 h and 84.5 h, respectively (Fig. 1A-B). Conversely, B. pertussis adenylate cyclase toxin and E. coli LPS decreased the median survival by 94.5 h and 66 h, respectively (Fig. 1C-D). All other tested toxins did not significantly affect the survival of WT flies (Fig. 1EM). Among those is Salmonella LPS, consisting of lipid A and a polysaccharide, as well as lipid A alone. These results show that only some toxins adversely affect Drosophila survival, and that insects are innately insensitive to many microbial toxins.
Bacterial toxins affect Drosophila survival through stress response regulator Jra
To determine if Jra is necessary for the response of Drosophila to microbial toxins, we measured the survival of a Jra mutant strain of D. melanogaster relative to that of WT flies following exposure to the same toxins as in Fig. 1. Jra is a stress-responsive transcription factor consisting of a bZIP DNA binding domain and a transcription activation domain. The mutant strain expresses a dominant-negative Jra allele consisting of its bZIP domain absent of the transcription activation domain (46-49).
The extended survival of WT flies in response to pore-forming toxins was negated in Jra mutant flies. After exposure to α- and ε-toxins, the extension of survival decreased from 85.5 h and 84.5 h, respectively, in WT flies (Fig. 1A-B) to 24 h and 36 h, respectively, in Jra flies (Fig. 2A-B). Of the toxins that shortened the survival of WT flies, B. pertussis adenylate cyclase and E. coli LPS further decreased the survival of Jra flies by 117.5 h and 93 h, respectively (Fig. 2C-D). The toxins that did not affect the survival of WT flies all shortened the survival of Jra flies by a range of 24h to 108.5 h, except the Diphtheria toxin, which did not affect either strain (Fig. 2E-M). Notably, Salmonella LPS and lipid A (Fig. 2E, F, M) shortened the survival of Jra flies by 86.5h to 98.5 h, in contrast to having no effect on WT flies (Fig. 1E, F, M), indicating that the toxicity of LPS in Jra flies is likely caused by the lipid A component. Collectively, these results suggest that Jra is necessary for the response of Drosophila to many toxins of various human pathogens (Fig. 2N).
Validation of Jra’s role in response to ε-toxin
In addition to expressing a partial Jra gene sequence that codes for a dominant negative form of a protein, the Jra 7218 strain also harbors loss of function point mutations in genes y and
To further investigate whether the loss of response to α-toxin in Jra strain 7218 is due to w1118 and y1 mutations, we tested the sensitivity of another Jra mutant strain, 3273, harboring wildtype y and w genes in a different genetic background cn1 bw1 speck1/CyO JraIA109 (50). This strain carries Jra with a loss of function point mutation, where a single base change at position 651 of the Jra sequence introduces a stop codon. Therefore, the mutation results in a truncated Jra protein lacking the DNA binding and dimerization domain (51). We observed that, similar to Jra strain 7218, the survival of Jra strain 3273 is unaffected by α-toxin (Fig. 3B-C). This further supports the finding that Jra is responsible for toxin response in flies.

Numerous human pathogens exert their harmful effects by expressing toxins. Unlike known effects of insecticidal microbial toxins, such as the pore-forming Cry toxin of Bacillus thuringiensis (52), very little is known about the effects of toxins of human microbial pathogens on insects. Since host targets of toxins and immune responses are evolutionarily conserved between mammals and insects, D. melanogaster has been utilized as a model organism to study the function of toxins of human pathogens and host sensitivity (53-64). In most of these studies, bacterial toxins were genetically expressed in flies and adversely affected the developmental and immunological processes in D. melanogaster. For example, just like in humans, it was shown that i) tetanus toxin and Botulinum neurotoxins disable Drosophila neuronal function by targeting fly SNARE homolog (53-55), ii) Diphtheria toxin and Pseudomonas exotoxin A inhibit protein synthesis in D. melanogaster by catalyzing the ADP-ribosylation of insect homolog of eEF-2 (54, 56), and iii) cholera and Pertussis toxins catalyze the ADP-ribosylation of a fly homolog of Gs protein, which activates cellular adenylate cyclase and results in a toxic level of cyclic adenosine monophosphate (cAMP) (57- 59). Other secreted toxins studied by genetic expression in flies include AvrA of S. typhimurium (60) and CagA of Helicobacter pylori (61). In other studies, the contribution of toxins to bacterial pathogenicity was demonstrated by infecting flies with toxinexpressing and toxin-deficient bacterial strains. This was done for cholera toxin (62) and pore-forming hemolysins from S. aureus and Serratia marcescens (29, 63, 64).
However, only a few studies tested the effect of toxins on flies by a physiologically relevant feeding assay. This was accomplished by exposing flies to purified recombinant cholera and anthrax toxins (37, 65). In contrast to toxins’ effect on mammals, orally ingested anthrax and cholera toxins did not affect the survival of flies, and the anthrax toxin component activated Drosophila resistance to bacteria through immune pathway mechanisms. Our study tested the effect of twelve toxins of human bacterial pathogens on the survival of flies by the feeding assay and investigated the mechanism of toxin response in flies. When ingested, most of these toxins do not affect the survival of flies at 1 μg/mL. Notably,the enhanced survival of Drosophila fed with pore-forming toxins (Fig. 1A-B) resembles the hormetic response, a phenomenon by which adaptive responses of organisms or cells to low dosages of stresses enhance their survival and stress resistance (66).
Several confounding factors limit the interpretations of our study. Only one concertation, 1 μg/mL, was tested for each toxin, and future tests should determine possible lethal and sub-lethal doses for each toxin. In addition, although each experiment was performed at least three times, they were performed with flies of various ages. This was done because the immunity of flies changes with age (24, 39-41). Within an experiment, the age distribution of flies in control and experimental conditions is similar. However, the age distribution may not be the same the next time the experiment is performed. Thus, overall survival may vary from experiment to experiment. Notwithstanding, the effect of microbial toxins on survival was reproducible, as seen by error bars for median survival in Figs 2N and 3C. Additionally, changing Whatman papers during the experiment was not practical; thus, one of the factors that limited fly survival in our experiments was the evaporation of the sucrose solution. Moreover, sucrose solution is not a well-balanced food, and flies’ survival is not expected to be as long as on the nutrient-rich fly food. Another factor that could affect flies’ survival in our experimental design is the deposition of feces and eggs on the surface of the Whatman paper, which could potentially limit access to food. Other confounding limitations are the possibility that the Jra mutation may affect the amount of food and microbial toxins consumed by flies.
In humans, Jun has been shown to have several functions. A robust Jun expression has been observed in all cell lines from patients with classical Hodgkin lymphoma and anaplastic largecell lymphoma (67). Additionally, Jun is essential for neuronal microtubule assembly and apoptosis (68), activation of Junmediated transcription cell cycle regulation (69), protective immunity through increased CD47 and PD-L1, and involvement in toxins signaling (60, 70, 71).
Our study supports the role of Jra in response to many microbial toxins. Drosophila Jra expression was induced in response to cholera toxin and LPS treatments (33, 34). Moreover, the JNK (Jun N-terminal Kinase) pathway, a MAPK cascade known to activate Jra, is induced by H. pylori CagA toxin (72). We propose that insects respond to toxins through Jra activity, which allows mounting the innate immunity to toxins and toxin-producing pathogens. To evade this Jra-mediated toxin immunity, other toxins may block and inactivate Jra-response: the Drosophila JNK pathway is inhibited by anthrax toxin Lethal Factor and by Salmonella enterica toxin AvrA, similarly to how they act in mammals (60, 70, 71). In human cells, anthrax toxin Lethal Factor acts as a proteinase that decreases Jun levels. Such levels were restored with a 26S proteasome inhibitor, indicating that anthrax toxin promoted the degradation of Jun protein through a proteosome-dependent pathway (70, 71). It was also shown that the secreted S. typhimurium effector protein AvrA possesses acetyltransferase activity toward specific MAPKKs and potently inhibits JNK-Jun and NF-κB signaling pathways in Drosophila and mice (60).
Previous studies demonstrated that Jra could promote longevity in Drosophila (73). Various stresses, such as pathogens and heat, activate Jra through JNK (bsk) protein (74). As a result, Jra regulates and promotes many physiological processes that influence insect homeostasis, including cytoprotection, wound healing, cell proliferation, and the extension of the lifespan (73, 75). Previous studies have shown that Jun activates the expression of a set of genes that mediate the synthesis of fatty acids and lipids, thus driving lipogenesis (3, 76). Other studies showed that the pore-forming α-toxin of S. aureus and aerolysin of Aeromonas hydrophila induce lipogenesis and restoration of the bilayer integrity (77, 78). We hypothesize that α-toxin, at the dosage examined in our study, affected the survival of flies by activating Jra, which induced lipogenesis and epithelial septate junction repair, allowing for cytoprotection and extension of survival. Meanwhile, studies revealed that prolonged longevity (hormetic response) involves multiple integrative signal transduction processes that are dependent on types of stressors and dosage levels (79, 80). Hence, elucidating the comprehensive mechanism of prolonged survival of flies warrants future indepth investigation with varying doses of toxins.
This study identifies the insect Jra as a new protein target for future insecticidal compounds capable of protecting mammals from pathogens-vectoring insect pests. A literature survey was performed to facilitate future optimization of promising compounds displaying Jra antagonism, resulting in several hits against the mammalian homolog, Jun. A short list exemplifying starting points for chemical space for optimization of Jra antagonism is proposed in Table 2 based on published selectivity and potency (81), coupled with in silico evaluation of permeability potential, biological distribution characteristics, and chemical/metabolic stability (i.e., ADME properties, using ADMET Predictor v. 10.3) as additional critical governing factors (82, 83). Additionally, since Jra is known to form complexes with other transcription factors, it is pertinent to consider compounds within the context of equilibrium interactions of these transcription response elements. To bind effectively to the target, an essential putative Jra antagonist must be selective and overcome potential off-target competitive interactions. From a stoichiometric perspective, small molecule antagonists possess physical, chemical, and biopharmaceutical properties that enable formulation and delivery at pharmacological quantities, which can outcompete known probabilities, such as hetero-dimerization and interactions with off-target components. Within this context, most prior work has focused on disrupting the interaction of Jun with DNA or with Jun-binding heterodimers. These functional regions on the surface of Jun represent defined target surfaces suited for small molecule inhibitors. While published drug candidates targeting mammalian Jun have historically been small molecules, earlier work on short peptide-based disruptors may offer additional options.
Declarations
a. Conflict of interest: NA
b. Ethical approval: NA
c. Clinical trial registration: NA
- 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.
- WHO. Anthrax in humans and animals. 4th ed. ed. Geneva, Switzerland: World Health Organization; 2008. 12 p.
- Turell MJ and Knudson GB. Mechanical transmission of Bacillus anthracis by stable flies (Stomoxys calcitrans) and mosquitoes (Aedes aegypti and Aedes taeniorhynchus). Infect Immun. 1987 Aug;55(8):1859-1861.doi: 10.1128/iai.55.8.1859-1861.1987
- Stiles CW. Isolation of the Bacillus anthracis from Spinose Ear Ticks Ornithodorus megnini. American Journal of Veterinary Research. 1944;5(17):318-319.
- Morris H. Blood-sucking insects as transmitters of anthrax or charbon: Agricultural Experiment Station of the Louisiana State University and A. & M. College; 1918.
- Hoffmann C, Zimmermann F, Biek R, Kuehl H, Nowak K, Mundry R, et al. Persistent anthrax as a major driver of wildlife mortality in a tropical rainforest. Nature. 2017 Aug 2;548(7665):82-86.doi.org/10.1038/nature23309
- Carlson CJ, Kracalik IT, Ross N, Alexander KA, Hugh-Jones ME, Fegan M, et al. The global distribution of Bacillus anthracis and associated anthrax risk to humans, livestock and wildlife. Nat Microbiol. 2019 May 13.doi.org/10.1038/s41564-019-0435-4
- Blackburn JK, Van Ert M, Mullins JC, Hadfield TL, Hugh-Jones ME. The necrophagous fly anthrax transmission pathway: empirical and genetic evidence from wildlife epizootics. Vector Borne Zoonotic Dis. 2014 Aug;14(8):576-583.doi: 10.1089/vbz.2013.1538
- Gogarten JF, Düx A, Mubemba B, Pléh K, Hoffmann C, Mielke A, et al. Tropical rainforest flies carrying pathogens form stable associations with social nonhuman primates. Mol Ecol. 2019 Sep;28(18):4242-4258. doi: 10.1111/mec.15145
- Zohdy S and Schwartz TS. Shoo fly don't bother me: Flies track social primates and carry viable anthrax. Mol Ecol. 2019 Sep;28(18):4135-4137.doi.org/10.1111/mec.15215
- 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
- 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.doi: 10.1128/aem.46.1.32-36.1983
- 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
- Werdmuller BF, Brakman M, Vreede RW. [A tropical ulcer; cutaneous diphtheria]. Ned Tijdschr Geneeskd. 1996 Nov 30;140(48):2414-2416.
- Berger A, Dangel A, Schober T, Schmidbauer B, Konrad R, Marosevic D, et al. Whole genome sequencing suggests transmission of Corynebacterium diphtheriae-caused cutaneous diphtheria in two siblings, Germany, 2018. Euro Surveill. 2019 Jan;24(2). doi: 10.2807/1560-7917.ES.2019.24.2.1800683
- de Benoist AC, White JM, Efstratiou A, Kelly C, Mann G, Nazareth B, et al. Imported cutaneous diphtheria, United Kingdom. Emerg Infect Dis. 2004 Mar;10(3):511-513. doi: 10.3201/eid1003.030524
- Neupane S, Nayduch D, Zurek L. House Flies (Musca domestica) Pose a Risk of Carriage and Transmission of Bacterial Pathogens Associated with Bovine Respiratory Disease (BRD). Insects. 2019 Oct 18;10(10).doi: 10.3390/insects10100358
- Thao ML, Gullan PJ, Baumann P. Secondary (gamma-Proteobacteria) endosymbionts infect the primary (beta-Proteobacteria) endosymbionts of mealybugs multiple times and coevolve with their hosts. Appl Environ Microbiol. 2002 Jul;68(7):3190-3197. doi: 10.1128/AEM.68.7.3190-3197.2002
- Davies MP, Anderson M, Hilton AC. The housefly Musca domestica as a mechanical vector of Clostridium difficile. J Hosp Infect. 2016 Nov;94(3):263-267. doi: 10.1016/j.jhin.2016.08.023
- Burt SA, Siemeling L, Kuijper EJ, Lipman LJ. Vermin on pig farms are vectors for Clostridium difficile PCR ribotypes 078 and 045. Vet Microbiol. 2012 Nov 9;160(1-2):256-258. doi: 10.1016/j.vetmic.2012.05.014
- Koransky JR, Stargel MD, Dowell VR, Jr. Clostridium septicum bacteremia. Its clinical significance. Am J Med. 1979 Jan;66(1):63-66. doi:10.1016/0002-9343(79)90483-2
- 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.
- Olsen AR and 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
- 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
- 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
- Schleifer KH and 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
- Takumi K and awata T. Quantitative chemical analyses and antigenic properties of peptidoglycans from Clostridium botulinum and other clostridia. Jpn J Microbiol. 1976 Aug;20(4):287-92.doi: 10.1111/j.1348-0421.1976.tb00990.x
- 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
- Lee KZ, Lestradet M, Socha C, Schirmeier S, Schmitz A, Spenle C, et al. Enterocyte Purge and Rapid Recovery Is a Resilience Reaction of the Gut Epithelium to Pore-Forming Toxin Attack. Cell Host Microbe. 2016 Dec 14;20(6):716-730. doi: 10.1016/j.chom.2016.10.010
- 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.org/10.1038/s41586-020-2066-6
- 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
- Moyano AJ, Racca AC, Soria G, Saka HA, Andreoli V, Smania AM, et al. c-Jun Proto-Oncoprotein Plays a Protective Role in Lung Epithelial Cells Exposed to Staphylococcal α-Toxin. Front Cell Infect Microbiol. 2018;8:170.doi.org/10.3389/fcimb.2018.00170
- Briata P, D'Anna F, Franzi AT, Gherzi R. AP-1 activity during normal human keratinocyte differentiation: evidence for a cytosolic modulator of AP-1/DNA binding. Exp Cell Res. 1993 Jan;204(1):136-146. doi: 10.1006/excr.1993.1018
- Kim T, Yoon J, Cho H, Lee WB, Kim J, Song YH, et al. Downregulation of lipopolysaccharide response in Drosophila by negative crosstalk between the AP1 and NF-kappaB signaling modules. Nat Immunol. 2005 Feb;6(2):211-218. doi: 10.1038/ni1159
- Kim LK, Choi UY, Cho HS, Lee JS, Lee WB, Kim J, et al. Down-regulation of NF-kappaB target genes by the AP-1 and STAT complex during the innate immune response in Drosophila. PLoS Biol. 2007 Sep;5(9):e238.doi: 10.1371/journal.pbio.0050238
- Sluss HK, Han Z, Barrett T, Goberdhan DC, Wilson C, Davis RJ, et al. A JNK signal transduction pathway that mediates morphogenesis and an immune response in Drosophila. Genes Dev. 1996 Nov 1;10(21):2745-2758.doi: 10.1371/journal.pbio.0050238
- 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
- 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.doi: 10.4172/1948-5948.1000046
- 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.org/10.3389/fimmu.2019.03075
- Garschall K, Flatt T. The interplay between immunity and aging in Drosophila. F1000Res. 2018;7:160doi: 10.12688/f1000research.13117.1. eCollection 2018
- Sciambra N, Chtarbanova S. The Impact of Age on Response to Infection in Drosophila. Microorganisms. 2021 Apr 29;9(5)doi: 10.3390/microorganisms9050958
- 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):5622-8doi: 10.4049/jimmunol.172.9.5622
- 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):4169-81. doi: 10.1128/IAI.02318-14
- 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):218-23.doi: 10.1111/j.1472-765X.2006.02040.x
- 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):2347-55.doi: 10.1099/mic.0.27116-0
- Perkins KK, Admon A, Patel N, Tjian R. The Drosophila Fos-related AP-1 protein is a developmentally regulated transcription factor. Genes Dev. 1990 May;4(5):822-34 doi: 10.1101/gad.4.5.822
- Bohmann D, Ellis MC, Staszewski LM, Mlodzik M. Drosophila Jun mediates Ras-dependent photoreceptor determination. Cell. 1994 Sep 23;78(6):973-86doi: 10.1016/0092-8674(94)90273-9
- Eresh S, Riese J, Jackson DB, Bohmann D, Bienz M. A CREB-binding site as a target for decapentaplegic signalling during Drosophila endoderm induction. Embo j. 1997 Apr 15;16(8):2014-22doi: 10.1093/emboj/16.8.2014
- Haussmann IU, White K, Soller M. Erect wing regulates synaptic growth in Drosophila by integration of multiple signaling pathways. Genome Biol. 2008 Apr 17;9(4):R73doi: 10.1186/gb-2008-9-4-r73
- Nüsslein-Volhard C, Wieschaus E, Kluding H. Mutations affecting the pattern of the larval cuticle inDrosophila melanogaster : I. Zygotic loci on the second chromosome. Wilehm Roux Arch Dev Biol. 1984 Sep;193(5):267-82doi: 10.1007/BF00848156
- Riesgo-Escovar JR, Hafen E. Drosophila Jun kinase regulates expression of decapentaplegic via the ETS-domain protein Aop and the AP-1 transcription factor DJun during dorsal closure. Genes Dev. 1997 Jul 1;11(13):1717-27doi: 10.1101/gad.11.13.1717
- Soberón M, Pardo L, Muñóz-Garay C, Sánchez J, Gómez I, Porta H, et al. Pore formation by Cry toxins. Adv Exp Med Biol. 2010;677:127-42doi: 10.1007/978-1-4419-6327-7_11
- Backhaus P, Langenhan T, Neuser K. Effects of transgenic expression of botulinum toxins in Drosophila. J Neurogenet. 2016 Mar;30(1):22-31 doi: 10.3109/01677063.2016.1166223.
- Thum AS, Knapek S, Rister J, Dierichs-Schmitt E, Heisenberg M, Tanimoto H. Differential potencies of effector genes in adult Drosophila. J Comp Neurol. 2006 Sep 10;498(2):194-203doi: 10.1002/cne.21022
- Umezaki Y, Yasuyama K, Nakagoshi H, Tomioka K. Blocking synaptic transmission with tetanus toxin light chain reveals modes of neurotransmission in the PDF-positive circadian clock neurons of Drosophila melanogaster. J Insect Physiol. 2011 Sep;57(9):1290-9doi: 10.1016/j.jinsphys.2011.06.004
- Avet-Rochex A, Bergeret E, Attree I, Meister M, Fauvarque MO. Suppression of Drosophila cellular immunity by directed expression of the ExoS toxin GAP domain of Pseudomonas aeruginosa. Cell Microbiol. 2005 Jun;7(6):799-810doi: 10.1111/j.1462-5822.2005.00512.x
- Guichard A, Cruz-Moreno B, Aguilar B, van Sorge NM, Kuang J, Kurkciyan AA, et al. Cholera toxin disrupts barrier function by inhibiting exocyst-mediated trafficking of host proteins to intestinal cell junctions. Cell Host Microbe. 2013 Sep 11;14(3):294-305doi: 10.1016/j.chom.2013.08.001
- Fitch CL, de Sousa SM, O'Day PM, Neubert TA, Plantilla CM, Spencer M, et al. Pertussis toxin expression in Drosophila alters the visual response and blocks eating behaviour. Cell Signal. 1993 Mar;5(2):187-207doi: 10.1016/0898-6568(93)90070-3
- Vecsey CG, Pirez N, Griffith LC. The Drosophila neuropeptides PDF and sNPF have opposing electrophysiological and molecular effects on central neurons. J Neurophysiol. 2014 Mar;111(5):1033-45doi: 10.1152/jn.00712.2013
- Jones RM, Wu H, Wentworth C, Luo L, Collier-Hyams L, Neish AS. Salmonella AvrA Coordinates Suppression of Host Immune and Apoptotic Defenses via JNK Pathway Blockade. Cell Host Microbe. 2008 Apr 17;3(4):233-44doi: 10.1016/j.chom.2008.02.016
- Botham CM, Wandler AM, Guillemin K. A transgenic Drosophila model demonstrates that the Helicobacter pylori CagA protein functions as a eukaryotic Gab adaptor. PLoS Pathog. 2008 May 16;4(5):e1000064doi: 10.1371/journal.ppat.1000064
- Hang S, Purdy AE, Robins WP, Wang Z, Mandal M, Chang S, et al. The acetate switch of an intestinal pathogen disrupts host insulin signaling and lipid metabolism. Cell Host Microbe. 2014 Nov 12;16(5):592-604doi: 10.1016/j.chom.2014.10.006
- Nehme NT, Liégeois S, Kele B, Giammarinaro P, Pradel E, Hoffmann JA, et al. A model of bacterial intestinal infections in Drosophila melanogaster. PLoS Pathog. 2007 Nov;3(11):e173doi: 10.1371/journal.ppat.0030173
- Needham AJ, Kibart M, Crossley H, Ingham PW, Foster SJ. Drosophila melanogaster as a model host for Staphylococcus aureus infection. Microbiology. 2004 Jul;150(Pt 7):2347-2355doi: 10.1099/mic.0.27116-0
- 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):e8doi: 10.1371/journal.ppat.0010008
- Calabrese EJ, Mattson MP. How does hormesis impact biology, toxicology, and medicine? NPJ Aging Mech Dis. 2017;3:13doi: 10.1038/s41514-017-0013-z
- Mathas S, Hinz M, Anagnostopoulos I, Krappmann D, Lietz A, Jundt F, et al. Aberrantly expressed c-Jun and JunB are a hallmark of Hodgkin lymphoma cells, stimulate proliferation and synergize with NF-kappa B. Embo j. 2002 Aug 1;21(15):4104-4113doi: 10.1093/emboj/cdf389
- Nateri AS, Riera-Sans L, Da Costa C, Behrens A. The ubiquitin ligase SCFFbw7 antagonizes apoptotic JNK signaling. Science. 2004 Feb 27;303(5662):1374-1378doi: 10.1126/science.1092880
- Koyama-Nasu R, David G, Tanese N. The F-box protein Fbl10 is a novel transcriptional repressor of c-Jun. Nat Cell Biol. 2007 Sep;9(9):1074-1080doi: 10.1038/ncb1628. Epub 2007 Aug 19
- Guichard A, Park JM, Cruz-Moreno B, Karin M, Bier E. Anthrax lethal factor and edema factor act on conserved targets in Drosophila. Proc Natl Acad Sci U S A. 2006 Feb 28;103(9):3244-3249doi: 10.1073/pnas.0510748103
- Ouyang W, Guo P, Fang H, Frucht DM. Anthrax lethal toxin rapidly reduces c-Jun levels by inhibiting c-Jun gene transcription and promoting c-Jun protein degradation. J Biol Chem. 2017 Oct 27;292(43):17919-17927doi: 10.1074/jbc.M117.805648
- Wandler AM, Guillemin K. Transgenic expression of the Helicobacter pylori virulence factor CagA promotes apoptosis or tumorigenesis through JNK activation in Drosophila. PLoS Pathog. 2012;8(10):e1002939doi: 10.1371/journal.ppat.1002939
- Biteau B, Jasper H. EGF signaling regulates the proliferation of intestinal stem cells in Drosophila. Development. 2011 Mar;138(6):1045-1055doi: 10.1242/dev.056671
- Tafesh-Edwards G, Eleftherianos I. JNK signaling in Drosophila immunity and homeostasis. Immunol Lett. 2020 Oct;226:7-11doi: 10.1016/j.imlet.2020.06.017
- Wang MC, Bohmann D, Jasper H. JNK Signaling Confers Tolerance to Oxidative Stress and Extends Lifespan in Drosophila. Developmental Cell. 2003 2003/11/01/;5(5):811-816doi: 10.1016/s1534-5807(03)00323-x
- Desert C, Baéza E, Aite M, Boutin M, Le Cam A, Montfort J, et al. Multi-tissue transcriptomic study reveals the main role of liver in the chicken adaptive response to a switch in dietary energy source through the transcriptional regulation of lipogenesis. BMC Genomics. 2018 2018/03/07;19(1):187 doi: 10.1186/s12864-018-4520-5
- Bhakdi S, Tranum-Jensen J. Alpha-toxin of Staphylococcus aureus. Microbiol Rev. 1991 Dec;55(4):733-751doi: 10.1128/mr.55.4.733-751.1991
- Gurcel L, Abrami L, Girardin S, Tschopp J, van der Goot FG. Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival. Cell. 2006 Sep 22;126(6):1135-1145doi:https://doi.org/10.1016/j.cell.2006.07.033
- Le Bourg E. Hormesis, aging and longevity. Biochim Biophys Acta. 2009 Oct;1790(10):1030-1039doi.org/10.1016/j.bbagen.2009.01.004
- Mantha M, Jumarie C. Cadmium-induced hormetic effect in differentiated Caco-2 cells: ERK and p38 activation without cell proliferation stimulation. J Cell Physiol. 2010 Jul;224(1):250-261 doi: 10.1002/jcp.22128
- Brennan A, Leech JT, Kad NM, Mason JM. Selective antagonism of cJun for cancer therapy. J Exp Clin Cancer Res. 2020 Sep 11;39(1):184doi: 10.1186/s13046-020-01686-9
- Wunberg T, Hendrix M, Hillisch A, Lobell M, Meier H, Schmeck C, et al. Improving the hit-to-lead process: data-driven assessment of drug-like and lead-like screening hits. Drug Discov Today. 2006 Feb;11(3-4):175-180 doi: 10.1016/S1359-6446(05)03700-1
- Fraczkiewicz R, Lobell M, Göller AH, Krenz U, Schoenneis R, Clark RD, et al. Best of both worlds: combining pharma data and state of the art modeling technology to improve in Silico pKa prediction. J Chem Inf Model. 2015 Feb 23;55(2):389-397doi.org/10.1021/ci500585w