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Immunity in Drosophila melanogaster-from microbial recognition to whole-organism physiology
Abstract
Since the discovery of antimicrobial peptide responses 40 years ago, the fruit fly Drosophila melanogaster has proven to be a powerful model for the study of innate immunity. Early work focused on innate immune mechanisms of microbial recognition and subsequent nuclear factor-κB signal transduction. More recently, D. melanogaster has been used to understand how the immune response is regulated and coordinated at the level of the whole organism. For example, researchers have used this model in studies investigating interactions between the microbiota and the immune system at barrier epithelial surfaces that ensure proper nutritional and immune homeostasis both locally and systemically. In addition, studies in D. melanogaster have been pivotal in uncovering how the immune response is regulated by both endocrine and metabolic signalling systems, and how the immune response modifies these systems as part of a homeostatic circuit. In this Review, we briefly summarize microbial recognition and antiviral immunity in D. melanogaster, and we highlight recent studies that have explored the effects of organism-wide regulation of the immune response and, conversely, the effects of the immune response on organism physiology.
... Given the evolutionary conservation of basic innate immune mechanisms in Drosophila and mammals [26], including humans, it is highly probable that insights gained from the Drosophila infection system are applicable to mammalian infections. The innate immune system of Drosophila encompasses a humoral response, marked by the production of antimicrobial substances, and a cellular response, represented by phagocytosis and bactericidal action of macrophages [26,27]. ...
... Given the evolutionary conservation of basic innate immune mechanisms in Drosophila and mammals [26], including humans, it is highly probable that insights gained from the Drosophila infection system are applicable to mammalian infections. The innate immune system of Drosophila encompasses a humoral response, marked by the production of antimicrobial substances, and a cellular response, represented by phagocytosis and bactericidal action of macrophages [26,27]. In the humoral response, the intracellular information pathway is activated by the recognition of cell wall components from Gram-positive or Gram-negative bacteria by specific receptors. ...
... In the humoral response, the intracellular information pathway is activated by the recognition of cell wall components from Gram-positive or Gram-negative bacteria by specific receptors. The receptors, information molecules, and transcription factors comprising this pathway are shared between Drosophila and humans [26]. ...
It is widely acknowledged that smoking exacerbates the severity of infectious diseases. A presumed mechanism involves the damage inflicted by tobacco smoke on the organs of host organisms. In this study, an alternative hypothesis was explored: smoking enhances the virulence of bacteria. This possibility was investigated using Escherichia coli as the bacterial agent and Drosophila as the host organism. Our inquiry focused on the potential gene expression changes in E. coli subsequent to exposure to tobacco smoke. Analysis of the transcription promoter activity of genes encoding proteins within the E. coli two-component system, a regulatory machinery governing gene expression, revealed the activation of twelve out of 50 promoters in response to tobacco smoke. Subsequently, Drosophila was infected with E. coli exposed to tobacco smoke or left untreated. Interestingly, there were no significant differences observed in the survival periods of Drosophila following infection with E. coli, whether treated or untreated with tobacco smoke. Contrary to the initial hypothesis, the findings suggest that while tobacco smoke alters gene expression in E. coli, these changes do not appear to impact bacterial virulence. Although this study has illuminated the influence of tobacco smoke on the gene expression of E. coli, further analyses are necessary to elucidate the implications of these changes. Nevertheless, the results imply that smoking affects not only host organisms but may also exert influence on invading bacteria.
... Seminal proof of concept studies in the 1990s and early 2000s, utilising wild-type and mutant Drosophila strains and fungi, provided a comprehensive framework for employing Drosophila in fungal research (20)(21)(22)(23)(24)(25). Over the last decade, more extensive Drosophila-fungi work has taken place, leading to a better understanding of virulence, pathogenicity, and host immune responses (26)(27)(28)(29)(30). ...
... Drosophila, affectionately dubbed the biology "work horse", has been used in fundamental biology, inbreeding and heredity studies since the early 1900s (27,31) and has led to substantial contributions to our understanding of genetics, cellular biology, neurobiology and immunology (31, 32). Of note, the discovery of Drosophila Toll receptor nearly 3 decades ago elucidated the function of the analogous mammalian Toll-like receptor (TLR) pathway, which is indispensable to innate immunity (20; Lemaitre,21,26). Drosophila genome can be genetically manipulated, and genome-wide studies performed to determine genes crucial for survival and infection (27,33). ...
... AMPs are small, positively charged peptides that interact with hydrophobic regions of microbial cells walls and cause cell wall degradation and microbial death and are secreted into the haemolymph by the fat body (45,46). In addition to AMPs, reactive oxygen species (ROS) are produced by Dual Oxidase (DUOX) and NADPH (Nox) at the epithelial cells (26,32). The humoral response also provides protection against viral attack through RNAi and autophagy processes (36,47). ...
Invasive fungal diseases have profound effects upon human health and are on increase globally. The World Health Organization (WHO) in 2022 published the fungal priority list calling for improved public health interventions and advance research. Drosophila melanogaster presents an excellent model system to dissect host-pathogen interactions and has been proved valuable to study immunopathogenesis of fungal diseases. In this review we highlight the recent advances in fungal-Drosophila interplay with an emphasis on the recently published WHO’s fungal priority list and we focus on available tools and technologies.
... The Drosophila immune system is composed of the immune deficiency (IMD) pathway and the Toll pathway as well as hemocyte activity (Buchon et al., 2014;Koranteng et al., 2022). First, we investigated the effects of IMD immune activity on larval development stimulated by Ap mono-association. ...
... The mutant Ap [Ap(P3G5)], which lacks PQQ-ADH activity, did not significantly stimulate larval development, serving as a negative control for our experimental condition (see below). In flies with either mutated imd or rel, which are the genes encoding components of the IMD immune pathway (Buchon et al., 2014), mono-association of GF larva with Ap(DM001) stimulated larval development by 30% or 40%, respectively, compared with GF larva; these were significantly greater than the 23% stimulation observed in wild-type flies (Figs. 1B-1D, Supplementary Figs. S1B and S1C). ...
... The Toll immune pathway is the other branch of fly innate immunity known to be activated mainly by infection of gram-positive bacteria or fungi. Upon infection, the activation of a protease cascade converts the Toll receptor ligand encoded by spaetzle (spz) into the active form, which stimulates the Toll immune pathway (Buchon et al., 2014). Thus, using a spz-mutant host, we tested whether the Toll immune pathway functions in the same manner as the IMD immune pathway in suppressing development-promoting effects of commensal bacteria. ...
The physiology of most organisms, including Drosophila, is heavily influenced by their interactions with certain types of commensal bacteria. Acetobacter and Lactobacillus, two of the most representative Drosophila commensal bacteria, have stimulatory effects on host larval development and growth. However, how these effects are related to host immune activity remains largely unknown. Here, we show that the Drosophila development-promoting effects of commensal bacteria are suppressed by host immune activity. Mono-association of germ-free Drosophila larvae with Acetobacter pomorum stimulated larval development, which was accelerated when host immune deficiency (IMD) pathway genes were mutated. This phenomenon was not observed in the case of mono-association with Lactobacillus plantarum. Moreover, the mutation of Toll pathway, which constitutes the other branch of the Drosophila immune pathway, did not accelerate A. pomorum-stimulated larval development. The mechanism of action of the IMD pathway-dependent effects of A. pomorum did not appear to involve previously known host mechanisms and bacterial metabolites such as gut peptidase expression, acetic acid, and thiamine, but appeared to involve larval serum proteins. These findings may shed light on the interaction between the beneficial effects of commensal bacteria and host immune activity.
... Overall, the study of AMPs has offered invaluable insights into the ancient origins of defense mechanisms and their evolution, inspiring potential applications as alternatives to conventional antibiotics and fueling biomedical research for novel therapeutic purposes. Some reviews have summarized the AMPs from the model insect Drosophila (Insecta: Diptera), which have mainly focused on the classification, activity, and regulation of AMPs [21,22]. In this review, we summarize the current knowledge and recent advances on AMPs from various model insects, highlight the regulatory pathways and evolution of insect AMPs, and present a perspective on the potential applications of insect AMPs. ...
... As antimicrobial effectors, insect AMPs are produced in hemocytes, fat bodies, and epithelial cells via two major nuclear factor-κB (NF-κB) pathways during infection: the Toll and the IMD (immune deficiency) pathways [21,[111][112][113]. Some AMPs are produced only upon immune stimulation, for example, cecropins. ...
Antimicrobial peptides (AMPs), as immune effectors synthesized by a variety of organisms, not only constitute a robust defense mechanism against a broad spectrum of pathogens in the host but also show promising applications as effective antimicrobial agents. Notably, insects are significant reservoirs of natural AMPs. However, the complex array of variations in types, quantities, antimicrobial activities, and production pathways of AMPs, as well as evolution of AMPs across insect species, presents a significant challenge for immunity system understanding and AMP applications. This review covers insect AMP discoveries, classification, common properties, and mechanisms of action. Additionally, the types, quantities, and activities of immune-related AMPs in each model insect are also summarized. We conducted the first comprehensive investigation into the diversity, distribution, and evolution of 20 types of AMPs in model insects, employing phylogenetic analysis to describe their evolutionary relationships and shed light on conserved and distinctive AMP families. Furthermore, we summarize the regulatory pathways of AMP production through classical signaling pathways and additional pathways associated with Nitric Oxide, insulin-like signaling, and hormones. This review advances our understanding of AMPs as guardians in insect immunity systems and unlocks a gateway to insect AMP resources, facilitating the use of AMPs to address food safety concerns.
... These cascades can be regulated at multiple levels by processes such as receptor localization, ubiquitination or phosphorylation, all ways that cells can inhibit or enhance the activity of immune signalling [5][6][7]. Ultimately, the cascade converges to activate transcription factors (TFs) [8,9], master regulators of gene expression. Most immune pathways result in the activation of just one or a select few TFs. ...
... Meanwhile, Acetobacter bacteria are common mutualist microbes of the Drosophila gut, and typically remain in the gut compartments [14]. The reason Pectobacterium can cause a systemic immune response upon oral infection is partly the damage it does to the host gut, but more importantly, it invades compartments where immune surveillance and potential for systemic response is high, such as the body cavity [8]. Mutualists like Acetobacter do not puncture the gut epithelium and enter the body cavity in the same way, preventing local or systemic immune hyperactivation. ...
The microbiome includes both ‘mutualist’ and ‘pathogen’ microbes, regulated by the same innate immune architecture. A major question has therefore been: how do hosts prevent pathogenic infections while maintaining beneficial microbes? One idea suggests hosts can selectively activate innate immunity upon pathogenic infection, but not mutualist colonization. Another idea posits that hosts can selectively attack pathogens, but not mutualists. Here I review evolutionary principles of microbe recognition and immune activation, and reflect on newly observed immune effector–microbe specificity perhaps supporting the latter idea. Recent work in Drosophila has found a surprising importance for single antimicrobial peptides in combatting specific ecologically relevant microbes. The developing picture suggests these effectors have evolved for this purpose. Other defence responses like reactive oxygen species bursts can also be uniquely effective against specific microbes. Signals in other model systems including nematodes, Hydra, oysters, and mammals, suggest that effector–microbe specificity may be a fundamental principle of host–pathogen interactions. I propose this effector–microbe specificity stems from weaknesses of the microbes themselves: if microbes have intrinsic weaknesses, hosts can evolve effectors that exploit those weaknesses. I define this host–microbe relationship as ‘the Achilles principle of immune evolution’. Incorporating this view helps interpret why some host–microbe interactions develop in a coevolutionary framework (e.g. Red Queen dynamics), or as a one-sided evolutionary response. This clarification should be valuable to better understand the principles behind host susceptibilities to infectious diseases.
This article is part of the theme issue ‘Sculpting the microbiome: how host factors determine and respond to microbial colonization’.
... As antimicrobial effectors, insect AMPs are produced in hemocytes, fat body, and epithelial cells via two major nuclear factor-κB (NF-κB) pathways during infection: the Toll and the IMD (immune deficiency) pathways [21,[105][106][107]. Some AMPs are produced only upon immune stimulation, for example cecropins. ...
... (www.preprints.org) | NOT PEER-REVIEWED | Posted: 5 March 2024 doi:10.20944/preprints202403.0123.v121 ...
Abstract: Antimicrobial Peptides (AMPs), as immune effectors synthesized by a variety of or-ganisms, not only constitute a robust defense mechanism against a broad spectrum of pathogens in the host, but also show promising applications as effective antimicrobial agents. Notably, in-sects are significant reservoirs of natural AMPs. However, the complex array of variations in types, quantities, antimicrobial activities, production pathways of AMPs, and evolution of AMPs across insect species presents a significant challenge for immunity system understanding and AMP applications. This review covers insect AMP discoveries, classification, common properties, and mechanisms of action. Additionally, the types, quantities, and activities of immune-related AMPs in each model insect are also summarized. We conducted the first comprehensive inves-tigation into the diversity, distribution, and evolution of 20 types of AMPs in model insects, em-ploying phylogenetic analysis to describe their evolutionary relationships and shed light on conserved and distinctive AMP families. Furthermore, we summarize the regulatory pathways of AMP production through classical signaling pathways and additional pathways associated with Nitric Oxide, insulin-like signaling, and hormones. This review advances our understanding of AMPs as guardians in insect immunity system and unlocks a gateway to insect AMP resources, facilitating the use of AMPs to address food safety concerns.
... Further Gene Set Enrichment Analysis (GSEA) displayed positive correlations between BubR1 depletion and glycerolipid metabolism ( Figure 3C). Furthermore, we found that IMD signaling pathway, the main immunity pathway responsible for Gram-bacterial infection and lipolysis [16,56,57], showed the substantially decreased level in BubR1-insufficient fat bodies, analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis ( Figure 3D). The antibacterial peptide (AMP) DptA decreased, whereas pirk increased in BubR1-deficient flies upon fasting, which were both implicated in the IMD signaling pathway ( Figure 3E). ...
... Beyond its role on the lipolysis, the IMD signaling pathway is also involved in anti-bacterial infection [56,70,71], somatic sex determination [72], homeostatic synaptic plasticity [73], intestinal epithelial turnover [70], elimination of unfit tissue cells [74,75], sleep [76,77], neurodegeneration [78][79][80], female oviposition [81], lifespan regulation [82][83][84][85] and tumorigenesis [86]. Since BubR1 attenuates the fasting-induced AGING lipolysis through the IMD signaling pathway, it will be interesting to uncover whether BubR1 play roles on forementioned biological processes via the IMD signaling pathway, and whether BubR1 exerts its effects on other IMD-mediated physiological activities through the key downstream target Relish. ...
Lipolysis, the key process releasing fat acids to generate energy in adipose tissues, correlates with starvation resistance. Nevertheless, its detail mechanisms remain elusive. BubR1, an essential mitotic regulator, ensures proper chromosome alignment and segregation during mitosis, but its physiological functions are largely unknown. Here, we use Drosophila adult fat body, the major lipid storage organ, to study the functions of BubR1 in lipolysis. We show that both whole body- and fat body-specific BubR1 depletions increase lipid degradation and shorten the lifespan under fasting but not feeding. Relish, the conserved regulator of IMD signaling pathway, acts as the downstream target of BubR1 to control the expression level of Bmm and modulate the lipolysis upon fasting. Thus, our study reveals new functions of BubR1 in starvation-induced lipolysis and provides new insights into the molecular mechanisms of lipolysis mediated by IMD signaling pathway.
... Furthermore, Drosophila shares many similarities with mammals in various aspects such as functional organs and physiology (Hyun 2013;Buchon et al. 2014;Lee SH and Kim 2021). For example, insulin/ insulin-like (IIS) and target of rapamycin (TOR) signaling pathway are a highly conserved signaling pathway that plays a major role in metazoan growth and development in response to the nutritional status of the host. ...
... In flies, two different pathways respond to microbial infections: the Toll and IMD pathways. In general, the Toll pathway recognizes Gram-positive bacteria with lysine-type peptidoglycan in the cell wall, while the IMD pathway recognizes Gram-negative and Gram-positive bacteria with diaminopimelic acid (DAP)type peptidoglycan (Leulier et al. 2003;Buchon et al. 2014). Over-activation of the IMD pathway during the juvenile growth period results in smaller final size, developmental delay, and disruption of metabolic homeostasis via decreased IIS/TOR signaling, but the mechanism underlying the influence of gut commensal bacteria on Figure 2. The molecular pathways mediating the effects of Lactiplantibacillus plantarum (Lp) and Acetobacter pomorum (Ap) on Drosophila juvenile development and growth. ...
The gut microbiome plays a crucial role in maintaining health in a variety of organisms, from insects to humans. Further, beneficial symbiotic microbes are believed to contribute to improving the quality of life of the host. Drosophila is an optimal model for studying host–commensal microbe interactions because it allows for convenient manipulation of intestinal microbial composition. Fly microbiota has a simple taxonomic composition and can be cultivated and genetically tracked. This permits functional studies and analyses of the molecular mechanisms underlying their effects on host physiological processes. In this context, we briefly introduce the principle of juvenile developmental growth in Drosophila. Then, we discuss the current understanding of the molecular mechanisms underlying the effects of gut commensal bacteria, such as Lactiplantibacillus plantarum and Acetobacter pomorum, in the fly gut microbiome on Drosophila juvenile growth, including specific actions of gut hormones and metabolites in conserved cellular signaling systems, such as the insulin/insulin-like (IIS) and the target of rapamycin (TOR) pathways. Given the similarities in tissue function/structure, as well as the high conservation of physiological systems between Drosophila and mammals, findings from the Drosophila model system will have significant implications for understanding the mechanisms underlying the interaction between the host and the gut microbiome in metazoans.
... While the humoral immunity is mainly regulated by Toll and immunodeficiency (IMD) pathways (33)(34)(35). The D. melanogaster Toll and IMD pathways share significant similarities with the mammalian TNF-a pathway and the Toll-like receptor pathway (TLR), respectively (36)(37)(38). The IMD pathway activates in immune tissues throughout the body, and recognizes diaminopimelic acid (DAP) type peptidoglycans present on cell walls of most Gramnegative bacteria and some Gram-positive bacteria (39). ...
... In contrast, the Toll pathway activates primarily in the fat body or hemolymph or immune cells, and respond to MAMPs from Gram-positive and fungi containing lysine-type peptidoglycans and b-glucans, respectively (40). In response to pathogen infection, the Toll and IMD signaling pathways of D. melanogaster activate the p65-like transcription factor Dif and p105-like transcription factor Relish, respectively; The transcription factor then enters the nucleus and induces the expression of antimicrobial peptide (AMP) by binding to the kB binding site in the AMP gene promoter region (33,36,41) (Figure 1). ...
Multicellular organisms live in environments containing diverse nutrients and a wide variety of microbial communities. On the one hand, the immune response of organisms can protect from the intrusion of exogenous microorganisms. On the other hand, the dynamic coordination of anabolism and catabolism of organisms is a necessary factor for growth and reproduction. Since the production of an immune response is an energy-intensive process, the activation of immune cells is accompanied by metabolic transformations that enable the rapid production of ATP and new biomolecules. In insects, the coordination of immunity and metabolism is the basis for insects to cope with environmental challenges and ensure normal growth, development and reproduction. During the activation of insect immune tissues by pathogenic microorganisms, not only the utilization of organic resources can be enhanced, but also the activated immune cells can usurp the nutrients of non-immune tissues by generating signals. At the same time, insects also have symbiotic bacteria in their body, which can affect insect physiology through immune-metabolic regulation. This paper reviews the research progress of insect immune-metabolism regulation from the perspective of insect tissues, such as fat body, gut and hemocytes. The effects of microorganisms (pathogenic bacteria/non-pathogenic bacteria) and parasitoids on immune-metabolism were elaborated here, which provide guidance to uncover immunometabolism mechanisms in insects and mammals. This work also provides insights to utilize immune-metabolism for the formulation of pest control strategies.
... These cascades can be regulated at multiple levels by processes such as receptor localization, ubiquitination, or phosphorylation, all ways that cells can inhibit or enhance the activity of immune signalling [4][5][6]. Ultimately the cascade converges to activate transcription factors (TFs) [7,8], master regulators of gene expression. Most immune pathways result in the activation of just one or a select few TFs. ...
... Meanwhile, Acetobacter bacteria are common mutualist microbes of the Drosophila gut, and typically remain in the gut compartments [12]. The reason Pseudomonas causes a systemic immune response upon oral infection is partly the damage it does to the host gut, but more importantly, it invades compartments where immune surveillance and potential for systemic response is high, such as the body cavity [7]. Mutualists do not puncture the gut epithelium and enter the body cavity in the same way, preventing local or systemic immune hyperactivation. ...
The microbiome includes both "mutualist" and "pathogen" microbes, regulated by the same innate immune architecture. A major question has therefore been: how do hosts prevent pathogenic infections while maintaining beneficial microbes? One idea suggests hosts can selectively activate innate immunity upon pathogenic infection, but not mutualist colonisation. Another idea posits that hosts can selectively attack pathogens, but not mutualists. Here I review evolutionary principles of microbe recognition and immune activation, and reflect on newly-observed immune effector-microbe specificity perhaps supporting the latter idea. Recent work in Drosophila has found a surprising importance for single antimicrobial peptides in combatting specific ecologically-relevant microbes. The developing picture suggests these effectors have evolved for this purpose. Other defence responses like ROS bursts can also be uniquely effective against specific microbes. Signals in other model systems including nematodes, Hydra, oysters, and mammals, suggest that effector-microbe specificity may be a fundamental principle of host-pathogen interactions. I propose this effector-microbe specificity stems from weaknesses of the microbes themselves: if microbes have intrinsic weaknesses, hosts can evolve effectors that exploit those weaknesses. I define this host-microbe relationship as "the Achilles principle of immune evolution." Incorporating this view helps interpret why some host-microbe interactions develop in a coevolutionary framework (e.g. Red Queen dynamics), or as a one-sided evolutionary response. This clarification should be valuable to better understand the principles behind host susceptibilities to infectious diseases.
... Sexual dimorphism as well as effects in mating and reproduction have been extensively reported in D. melanogaster studies [8][9][10]. This, in addition to its well-characterized immune system [11] and to the great benefits in terms of cost, time-consumption and genetic tools availability [12], make D. melanogaster a good model to study a possible trade-off between the two physiological aspects. Immunity in D. melanogaster relies solely on innate immune response. ...
... Immunity in D. melanogaster relies solely on innate immune response. Nevertheless, as in mammals, this immune response is also divided into humoral and cellular arms, and have Nuclear factor kappa B(NF-κB) family transcription factors and signal transduction pathways [11]. The humoral response is mainly based on the synthesis of antimicrobial peptides (AMPs) in the fat body of the fly regulated by two signalling pathways: the Toll and the immune deficiency (Imd) pathways, which induce the production of Drosomycin and Diptericin (as the main AMP of each pathway), respectively [13,14]. ...
Sex and reproductive status of the host have a major impact on the immune response against infection. Our aim was to understand their impact on host tolerance or resistance in the systemic Mycobacterium marinum infection of Drosophila melanogaster. We measured host survival and bacillary load at time of death, as well as expression by quantitative real-time polymerase chain reaction of immune genes (diptericin and drosomycin). We also assessed the impact of metabolic and hormonal regulation in the protection against infection by measuring expression of upd3, impl2 and ecR. Our data showed increased resistance in actively mating flies and in mated females, while reducing their tolerance to infection. Data suggests that Toll and immune deficiency (Imd) pathways determine tolerance and resistance, respectively, while higher basal levels of ecR favours the stimulation of the Imd pathway. A dual role has been found for upd3 expression, linked to increased/decreased mycobacterial load at the beginning and later in infection, respectively. Finally, impl2 expression has been related to increased resistance in non-actively mating males. These results allow further assessment on the differences between sexes and highlights the role of the reproductive status in D. melanogaster to face infections, demonstrating their importance to determine resistance and tolerance against M. marinum infection.
... They provide a low-cost, in vivo model for evaluating bacteria-phage interactions in the presence of an immune system, prior to use of mammalian models and thus, offer an important step-up from in vitro experiments. G. mellonella has a complex innate immune system, with similarity to mammalian systems [34] and can be kept at 37 °C during experiments to approximate conditions in human infections. G. mellonella have been used to assess changes in virulence of phage-resistant A. baumannii to further understand mechanisms of resistance [18,32]. ...
Introduction . Acinetobacter baumannii is a critical priority pathogen for novel antimicrobials (World Health Organization) because of the rise in nosocomial infections and its ability to evolve resistance to last resort antibiotics. A. baumannii is thus a priority target for phage therapeutics. Two strains of a novel, virulent bacteriophage (LemonAid and Tonic) able to infect carbapenem-resistant A. baumannii (strain NCTC 13420), were isolated from environmental water samples collected through a citizen science programme.
Gap statement. Phage-host coevolution can lead to emergence of host resistance, with a concomitant reduction in the virulence of host bacteria; a potential benefit to phage therapy applications.
Methodology. In vitro and in vivo assays, genomics and microscopy techniques were used to characterize the phages; determine mechanisms and impact of phage resistance on host virulence, and the efficacy of the phages against A. baumannii .
Results. A. baumannii developed resistance to both viruses, LemonAid and Tonic. Resistance came at a cost to virulence, with the resistant variants causing significantly reduced mortality in a Galleria mellonella larval in vivo model. A replicated 8 bp insertion increased in frequency (~40 % higher frequency than in the wild-type) within phage-resistant A. baumannii mutants, putatively resulting in early truncation of a protein of unknown function. Evidence from comparative genomics and an adsorption assay suggests this protein acts as a novel phage receptor site in A. baumannii . We find no evidence linking resistance to changes in capsule structure, a known virulence factor. LemonAid efficiently suppressed growth of A. baumanni in vitro across a wide range of titres. However, in vivo , while survival of A. baumannii infected larvae significantly increased with both remedial and prophylactic treatment with LemonAid (10 ⁷ p.f.u. ml –1 ), the effect was weak and not sufficient to save larvae from morbidity and mortality.
Conclusion. While LemonAid and Tonic did not prove effective as a treatment in a Galleria larvae model, there is potential to harness their ability to attenuate virulence in drug-resistant A. baumannii .
... While the IMD pathway is present in several insect tissues and recognizes mostly peptidoglycan from Gram-negative bacteria (Kaneko & Silverman, 2005), the Toll pathway essentially operates in the fat body and hemocytes, responding to Grampositive bacteria or fungi PAMP (El Chamy et al., 2008). Beyond these mechanisms, insulin-like peptides and target of rapamycin (TOR) as well as Akt, FOXO, and AMP, complement the array of innate immune response mechanisms in insects (Buchon et al., 2014;Galenza & Foley, 2019;Myllymäki et al., 2014;Valanne et al., 2011). ...
Aedes aegypti females are natural vectors of important arboviruses such as dengue, zika, and yellow fever. Mosquitoes activate innate immune response signaling pathways upon infection, as a resistance mechanism to fight pathogens and limit their propagation. Despite the beneficial effects of immune activation for insect vectors, phenotypic costs ultimately affect their fitness. However, the underlying mechanisms that mediate these fitness costs remain poorly understood. Given the high energy required to mount a proper immune response, we hypothesized that systemic activation of innate immunity would impair flight muscle mitochondrial function, compromising tissue energy demand and flight activity. Here, we investigated the dynamic effects of activation of innate immunity by intra‐thoracic zymosan injection on A. aegypti flight muscle mitochondrial metabolism. Zymosan injection significantly increased defensin A expression in fat bodies in a time‐dependent manner that compromised flight activity. Although oxidant levels in flight muscle were hardly altered, ATP‐linked respiratory rates driven by mitochondrial pyruvate+proline oxidation were significantly reduced at 24 h upon zymosan injection. Oxidative phosphorylation coupling was preserved regardless of innate immune response activation along 24 h. Importantly, rotenone‐sensitive respiration and complex I‐III activity were specifically reduced 24 h upon zymosan injection. Also, loss of complex I activity compromised ATP‐linked and maximal respiratory rates mediated by mitochondrial proline oxidation. Finally, the magnitude of innate immune response activation negatively correlated with respiratory rates, regardless of the metabolic states. Collectively, we demonstrate that activation of innate immunity is strongly associated with reduced flight muscle complex I activity with direct consequences to mitochondrial proline oxidation and flight activity. Remarkably, our results indicate a trade‐off between dispersal and immunity exists in an insect vector, underscoring the potential consequences of disrupted flight muscle mitochondrial energy metabolism to arbovirus transmission.
... Insects' systemic immune response involves the production of antimicrobial peptides (Agyemang-Badu et al., 2023) by the fat body, which is then released into the hemolymph. AMP gene expression is primarily regulated by two signaling pathways: the Imd pathway, activated in response to Gram-negative bacteria, and the Toll pathway, triggered by Gram-positive bacteria, fungi, and yeast (Buchon et al., 2014). ...
The escalating challenge of malaria control necessitates innovative approaches that extend beyond traditional control strategies. This review explores the incorporation of traditional vector control techniques with emerging Wolbachia-based interventions. Wolbachia, a naturally occurring bacteria, offers a novel approach for combatting vector-borne diseases, including malaria, by reducing the mosquitoes’ ability to transmit these diseases. The study explores the rationale for this integration, presenting various case studies and pilot projects that have exhibited significant success. Employing a multi-dimensional approach that includes community mobilization, environmental modifications, and new biological methods, the paper posits that integrated efforts could mark a turning point in the struggle against malaria. Our findings indicate that incorporating Wolbachia-based strategies into existing vector management programs not only is feasible but also heightens the efficacy of malaria control initiatives in different countries especially in Pakistan. The paper concludes that continued research and international collaboration are imperative for translating these promising methods from the laboratory to the field, thereby offering a more sustainable and effective malaria control strategy.
... Indeed, the midgut gene expression seems to indicate an active response to a perceived pathogen during November, which suggests bacterial opportunism. In Drosophila melanogaster, immune response to pathogens is highly regionalized in the gut with the Toll pathway active in the foregut and hindgut, and IMD regulating the midgut 37 . This may explain the robust immune response seen in queen midguts through the Toll and IMD pathways, as most gene expression of the ileum was not significantly different. ...
The health of honey bee queens is crucial for colony success, particularly during stressful periods like overwintering. To accompany a previous longitudinal study of colony and worker health, we explored niche-specific gut microbiota, host gene expression, and pathogen prevalence in honey bee queens overwintering in a warm southern climate. We found differential gene expression and bacterial abundance with respect to various pathogens throughout the season. Biologically older queens had larger microbiotas, particularly enriched in Bombella and Bifidobacterium. Both Deformed Wing Virus A and B subtypes were highest in the fat body tissue in January, correlating with colony Varroa levels, and Deformed Wing Virus titers in workers. High viral titers in queens were associated with decreased vitellogenin expression, suggesting a potential trade-off between immune function and reproductive capacity. Additionally, we found a complex and dynamic relationship between these viral loads and immune gene expression, indicating a possible breakdown in the coordinated immune response as the season progressed. Our study also revealed a potential link between Nosema and Melissococcus plutonius infections in queens, demonstrating that seasonal opportunism is not confined to just workers. Overall, our findings highlight the intricate interplay between pathogens, metabolic state, and immune response in honey bee queens. Combined with worker and colony-level metrics from the same colonies, our findings illustrate the social aspect of queen health and resilience over the winter dearth.
... For efficient infection, pathogens have to adapt to the microenvironments within the host organism. Host-elicited oxidative stress is a common stress that microbes have to cope with when they infect plants, insects and mammals, including humans [6,7,[47][48][49]. Studying oxidative stress responses of pathogens can help us to understand how they can survive in the hostile environment within the host. ...
Pathogens have to cope with oxidative, iron- and carbon(glucose)-limitation stresses in the human body. To understand how combined iron–carbon limitation alters oxidative stress responses, Aspergillus fumigatus was cultured in glucose–peptone or peptone containing media supplemented or not with deferiprone as an iron chelator. Changes in the transcriptome in these cultures were recorded after H2O2 treatment. Responses to oxidative stress were highly dependent on the availability of glucose and iron. Out of the 16 stress responsive antioxidative enzyme genes, only the cat2 catalase–peroxidase gene was upregulated in more than two culturing conditions. The transcriptional responses observed in iron metabolism also varied substantially in these cultures. Only extracellular siderophore production appeared important regardless of culturing conditions in oxidative stress protection, while the enhanced synthesis of Fe-S cluster proteins seemed to be crucial for oxidative stress treated iron-limited and fast growing (glucose rich) cultures. Although pathogens and host cells live together in the same place, their culturing conditions (e.g., iron availability or occurrence of oxidative stress) can be different. Therefore, inhibition of a universally important biochemical process, like Fe-S cluster assembly, may selectively inhibit the pathogen growth in vivo and represent a potential target for antifungal therapy.
... Accumulating evidence suggests that microbial symbionts participate in numerous host biological processes such as growth, feeding, digestion, immunity, adaptation, and detoxification [3][4][5][6]. Simultaneously, hosts can affect the community assembly process of symbiotic microbiota via various mechanisms, for example, variations in gene expression and differential adhesion to mucosal surfaces [7][8][9]. ...
Host-associated microbiomes can play key roles in the metamorphosis of animals. Most scyphozoan jellyfish undergo strobilation in their life cycles, similar to metamorphosis in classic bilaterians. The exploration of jellyfish microbiomes may elucidate the ancestral mechanisms and evolutionary trajectories of metazoan–microbe associations and interactions during metamorphosis. However, current knowledge of the functional features of jellyfish microbiomes remains limited. Here, we performed a genome-centric analysis of associated microbiota across four successive life stages (polyp, early strobila, advanced strobila, and ephyra) during strobilation in the common jellyfish Aurelia coerulea. We observed shifts in taxonomic and functional diversity of microbiomes across distinct stages and proposed that the low microbial diversity in ephyra stage may be correlated with the high expression of the host-derived antimicrobial peptide aurelin. Furthermore, we recovered 43 high-quality metagenome-assembled genomes and determined the nutritional potential of the dominant Vibrio members. Interestingly, we observed increased abundances of genes related to the biosynthesis of amino acids, vitamins, and cofactors, as well as carbon fixation during the loss of host feeding ability, indicating the functional potential of Aurelia-associated microbiota to support the synthesis of essential nutrients. We also identified several potential mechanisms by which jellyfish-associated microbes establish stage-specific community structures and maintain stable colonisation in dynamic host environments, including eukaryotic-like protein production, bacterial secretion systems, restriction-modification systems, and clustered regularly interspaced short palindromic repeats-Cas systems. Our study characterises unique taxonomic and functional changes in jellyfish microbiomes during strobilation and provides foundations for uncovering the ancestral mechanism of host–microbe interactions during metamorphosis.
... Importantly, immune responses have not been studied extensively in connection with mitochondrial dysfunction, and the impact of tissue-specific mitochondrial metabolism on the immune response is currently not well understood. D. melanogaster has long been used as a model for studying innate immunity, due to the conservation of signaling cascades and effector molecules between the fly and humans (reviewed in [16]). Two tissue types crucial for a fly immune response both at larval and adult stages are the fat body, functionally akin to mammalian liver and adipose tissue [17], and the hemocytes, the fly immune cells [18]. ...
Background
Mitochondria participate in various cellular processes including energy metabolism, apoptosis, autophagy, production of reactive oxygen species, stress responses, inflammation and immunity. However, the role of mitochondrial metabolism in immune cells and tissues shaping the innate immune responses are not yet fully understood. We investigated the effects of tissue-specific mitochondrial perturbation on the immune responses at the organismal level. Genes for oxidative phosphorylation (OXPHOS) complexes cI-cV were knocked down in the fruit fly Drosophila melanogaster, targeting the two main immune tissues, the fat body and the immune cells (hemocytes).
Results
While OXPHOS perturbation in the fat body was detrimental, hemocyte-specific perturbation led to an enhanced immunocompetence. This was accompanied by the formation of melanized hemocyte aggregates (melanotic nodules), a sign of activation of cell-mediated innate immunity. Furthermore, the hemocyte-specific OXPHOS perturbation induced immune activation of hemocytes, resulting in an infection-like hemocyte profile and an enhanced immune response against parasitoid wasp infection. In addition, OXPHOS perturbation in hemocytes resulted in mitochondrial membrane depolarization and upregulation of genes associated with the mitochondrial unfolded protein response.
Conclusions
Overall, we show that while the effects of mitochondrial perturbation on immune responses are highly tissue-specific, mild mitochondrial dysfunction can be beneficial in immune-challenged individuals and contributes to variation in infection outcomes among individuals.
... The D. melanogaster genome is 60% homologous to that of humans and since about 75% of the genes responsible for human diseases have homologues in flies [9], these insects have recently become useful tools for studying human diseases, including rare Mendelian diseases [10], neurodegenerative diseases [11] and cancer [12]. The molecular mechanisms of pathogenic proteins encoded by viral and bacterial genomes have also been studied in Drosophila [13]. ...
The study of pathogenicity and virulence of fungal strains, in vivo in the preclinical phase, is carried out through the use of animal models belonging to various classes of mammals (rodents, leproids, etc.). Although animals are functionally more similar to humans, these studies have some limitations in terms of ethics (animal suffering), user-friendliness, cost-effectiveness, timing (physiological response time) and logistics (need for adequately equipped laboratories). A good in vivo model must possess some optimal characteristics to be used, such as rapid growth, small size and short life cycle. For this reason, insects, such as Galleria mellonella (Lepidoptera), Drosophila melanogaster (Diptera) and Bombyx mori (Lepidoptera), have been widely used as alternative non-mammalian models. Due to their simplicity of use and low cost, the larvae of G. mellonella represent an optimal model above all to evaluate the virulence of fungal pathogens and the use of antifungal treatments (either single or in combination with biologically active compounds). A further advantage is also represented by their simple neuronal system limiting the suffering of the animal itself, their ability to survive at near-body ambient temperatures as well as the expression of proteins able to recognise combined pathogens following the three R principles (replacement, refinement and reduction). This review aims to assess the validity as well as the advantages and disadvantages of replacing mammalian classes with G. mellonella as an in vivo study model for preclinical experimentation.
... To protect themselves from invasion by environmental pathogens, insects are armed with a well-developed innate immunity (1)(2)(3)(4). Unlike vertebrates, insects lack acquired immunity and do not possess immune memory against pathogens. However, recent studies have reported that insects exhibit high defensive capabilities against pathogen infection through a phenomenon called immune priming (5)(6)(7)(8). ...
Insects lack acquired immunity and were thought to have no immune memory, but recent studies reported a phenomenon called immune priming, wherein sublethal dose of pathogens or nonpathogenic microbes stimulates immunity and prevents subsequential pathogen infection. Although the evidence for insect immune priming is accumulating, the underlying mechanisms are still unclear. The bean bug Riptortus pedestris acquires its gut microbiota from ambient soil and spatially structures them into a multispecies and variable community in the anterior midgut and a specific, monospecies Caballeronia symbiont population in the posterior region. We demonstrate that a particular Burkholderia strain colonizing the anterior midgut stimulates systemic immunity by penetrating gut epithelia and migrating into the hemolymph. The activated immunity, consisting of a humoral and a cellular response, had no negative effect on the host fitness, but on the contrary protected the insect from subsequent infection by pathogenic bacteria. Interruption of contact between the Burkholderia strain and epithelia of the gut weakened the host immunity back to preinfection levels and made the insects more vulnerable to microbial infection, demonstrating that persistent acquisition of environmental bacteria is important to maintain an efficient immunity.
... Interestingly, these receptors are expressed on immune-competent cells as well as on neuronal cells 18,29,30 . Work from several laboratories has shown that bacterial peptidoglycan, an essential component of the bacterial cell wall, mediates many interactions between bacteria and flies [31][32][33][34][35] . Its recognition by PGRP family members activates NF-κB pathways on immunocompetent cells, leading to the production of immune effectors and regulators. ...
The survival of animals depends, among other things, on their ability to identify threats in their surrounding environment. Senses such as olfaction, vision and taste play an essential role in sampling their living environment, including microorganisms, some of which are potentially pathogenic. This study focuses on the mechanisms of detection of bacteria by the Drosophila gustatory system. We demonstrate that the peptidoglycan (PGN) that forms the cell wall of bacteria triggers an immediate feeding aversive response when detected by the gustatory system of adult flies. Although we identify ppk23+ and Gr66a+ gustatory neurons as necessary to transduce fly response to PGN, we demonstrate that they play very different roles in the process. Time-controlled functional inactivation and in vivo calcium imaging demonstrate that while ppk23+ neurons are required in the adult flies to directly transduce PGN signal, Gr66a+ neurons must be functional in larvae to allow future adults to become PGN sensitive. Furthermore, the ability of adult flies to respond to bacterial PGN is lost when they hatch from larvae reared under axenic conditions. Recolonization of germ-free larvae, but not adults, with a single bacterial species, Lactobacillus brevis, is sufficient to restore the ability of adults to respond to PGN. Our data demonstrate that the genetic and environmental characteristics of the larvae are essential to make the future adults competent to respond to certain sensory stimuli such as PGN.
... However, progress has been hindered by the inherent difficulty of studying inflammaging in mammalian models with long lifespans. Fruit flies may be a promising alternative model since they share important innate immune pathways with mammals but have a lifespan of just 50-70 days [6][7][8]. Most importantly, there is evidence that the fundamental mechanisms of inflammaging are well-conserved between fruit flies and mammals. ...
Background: A prominent hallmark of aging is inflammaging-the increased expression of innate immune genes without identifiable infection. Model organisms with shorter lifespans, such as the fruit fly, provide an essential platform for probing the mechanisms of inflammaging. Multiple groups have reported that, like mammalian models, old flies have significantly higher levels of expression of anti-microbial peptide genes. However, whether some of these genes-or any others-can serve as reliable markers for assessing and comparing inflammaging in different strains remains unclear.
Methods and Results: We compared RNA-Seq datasets generated by different groups. Although the fly strains used in these studies differ significantly, we found that they share a core group of genes with strong aging-associated expression. In addition to anti-microbial peptide genes, we identified other genes that have prominently increased expression in old flies, especially SPH93. We further showed that machine learning models can be used to predict the "inflammatory age" of the fruit fly.
Conclusion: A core group of genes may serve as markers for studying inflammaging in Drosophila. RNA-Seq profiles, in combination with machine-learning models, can be applied to measure the acceleration or deceleration of inflammaging.
... When a parasitoid wasp lays its egg inside D. melanogaster, the fly larva launches an antiparasitoid immune response following infection, which can be divided into cellular and humoral responses [4,13,14]. Cellular responses include the proliferation of hemocytes and the differentiation of specialist lamellocytes that function to encapsulate the parasitoid egg in a multilayered cellular capsule [15][16][17]. The capsule becomes melanized through phenoloxidases activity mediated by crystal cells and lamellocytes, eventually killing the parasite [18]. ...
Both constitutive and inducible immune mechanisms are employed by hosts for defense against infection. Constitutive immunity allows for a faster response, but it comes with an associated cost that is always present. This trade-off between speed and fitness costs leads to the theoretical prediction that constitutive immunity will be favored where parasite exposure is frequent. We selected populations of Drosophila melanogaster under high parasite pressure from the parasitoid wasp Leptopilina boulardi . With RNA sequencing, we found the evolution of resistance in these populations was associated with them developing constitutively active humoral immunity, mediated by the larval fat body. Furthermore, these evolved populations were also able to induce gene expression in response to infection to a greater level, which indicates an overall more activated humoral immune response to parasitization. The anti-parasitoid immune response also relies on the JAK/STAT signaling pathway being activated in muscles following infection, and this induced response was only seen in populations that had evolved under high parasite pressure. We found that the cytokine Upd3, which induces this JAK/STAT response, is being expressed by immature lamellocytes. Furthermore, these immune cells became constitutively present when populations evolved resistance, potentially explaining why they gained the ability to activate JAK/STAT signaling. Thus, under intense parasitism, populations evolved resistance by increasing both constitutive and induced immune defenses, and there is likely an interplay between these two forms of immunity.
... Successful transformation of our basic understanding of fruit fly biology to the discovery of new genetic disorders in human disease has promoted the recent expansion of employing Drosophila to model human diseases, which will accelerate the scientific discovery of new signaling and metabolic pathways related to human disease and facilitate the translation of the basic science to clinical applications. The flies are permissive to infection by diverse species of microorganisms that often cause diseases in humans, and as such, is an attractive model for studying bacterial virulence and pathogenesis (Edwards and Kjellerup, 2012;Buchon et al., 2014). Several features of D. melanogaster make it an attractive model to study bacterial interactions, including simple endogenous microbial community, high-throughput screening potential, genetic tractability, cost-effectiveness, and ability to recapitulate key virulence events such as in vitro biofilm formation in microbial colonization of the fly's crop. ...
The fruit fly Drosophila melanogaster has emerged as a valuable model for investigating human biology, including the role of the microbiome in health and disease. Historically, studies involving the infection of D. melanogaster with single microbial species have yielded critical insights into bacterial colonization and host innate immunity. However, recent evidence has underscored that multiple microbial species can interact in complex ways through physical connections, metabolic cross-feeding, or signaling exchanges, with significant implications for healthy homeostasis and the initiation, progression, and outcomes of disease. As a result, researchers have shifted their focus toward developing more robust and representative in vivo models of co-infection to probe the intricacies of polymicrobial synergy and dysbiosis. This review provides a comprehensive overview of the pioneering work and recent advances in the field, highlighting the utility of Drosophila as an alternative model for studying the multifaceted microbial interactions that occur within the oral cavity and other body sites. We will discuss the factors and mechanisms that drive microbial community dynamics, as well as their impacts on host physiology and immune responses. Furthermore, this review will delve into the emerging evidence that connects oral microbes to systemic conditions in both health and disease. As our understanding of the microbiome continues to evolve, Drosophila offers a powerful and tractable model for unraveling the complex interplay between host and microbes including oral microbes, which has far-reaching implications for human health and the development of targeted therapeutic interventions.
... The production of antimicrobial peptides (AMPs) is governed by the Toll and IMD pathways, which are two NF-κB signal transduction pathways [1,2]. AMPs are mainly synthesized in the fat body and then secreted into the hemolymph to control systemic infection [3][4][5]. Melanization, which is catalyzed by active phenoloxidase (PO), is a universal defense response to invading pathogens in arthropods [1,[6][7][8]. ...
The prophenoloxidase (PPO) activation and Toll antimicrobial peptide synthesis pathways are two critical immune responses in the insect immune system. The activation of these pathways is mediated by the cascade of serine proteases, which is negatively regulated by serpins. In this study, we identified a typical serpin, BmSerpin-4, in silkworms, whose expression was dramatically up-regulated in the fat body and hemocytes after bacterial infections. The pre-injection of recombinant BmSerpin-4 remarkably decreased the antibacterial activity of the hemolymph and the expression of the antimicrobial peptides (AMPs) gloverin-3, cecropin-D, cecropin-E, and moricin in the fat body under Micrococcus luteus and Yersinia pseudotuberculosis serotype O: 3 (YP III) infection. Meanwhile, the inhibition of systemic melanization, PO activity, and PPO activation by BmSerpin-4 was also observed. Hemolymph proteinase 1 (HP1), serine protease 2 (SP2), HP6, and SP21 were predicted as the candidate target serine proteases for BmSerpin-4 through the analysis of residues adjacent to the scissile bond and comparisons of orthologous genes in Manduca sexta. This suggests that HP1, SP2, HP6, and SP21 might be essential in the activation of the serine protease cascade in both the Toll and PPO pathways in silkworms. Our study provided a comprehensive characterization of BmSerpin-4 and clues for the further dissection of silkworm PPO and Toll activation signaling.
... In Drosophila, the innate immune response is initiated by pattern recognition receptors (PRRs), which recognise pathogen associated molecular patterns (PAMPs) on the surface of microbes to induce the conserved immune signal transduction pathways: Immune deficiency (IMD), Toll and JAK/STAT [15,16]. Activation of innate immune pathways results in the up regulation of a cadre of structurally diverse antimicrobial peptides (AMPs) with bactericidal properties. ...
Mutations in the GBA1 gene cause the lysosomal storage disorder Gaucher disease (GD) and are the greatest known genetic risk factors for Parkinson’s disease (PD). Communication between the gut and brain and immune dysregulation are increasingly being implicated in neurodegenerative disorders such as PD. Here, we show that flies lacking the Gba1b gene, the main fly orthologue of GBA1 , display widespread NF -k B signalling activation, including gut inflammation, and brain glial activation. We also demonstrate intestinal autophagic defects, gut dysfunction, and microbiome dysbiosis. Remarkably, modulating the microbiome of Gba1b knockout flies, by raising them under germ-free conditions, partially ameliorates lifespan, locomotor and immune phenotypes. Moreover, we show that modulation of the immune deficiency (IMD) pathway is detrimental to the survival of Gba1 deficient flies. We also reveal that direct stimulation of autophagy by rapamycin treatment achieves similar benefits to germ-free conditions independent of gut bacterial load. Consistent with this, we show that pharmacologically blocking autophagosomal-lysosomal fusion, mimicking the autophagy defects of Gba1 depleted cells, is sufficient to stimulate intestinal immune activation. Overall, our data elucidate a mechanism whereby an altered microbiome, coupled with defects in autophagy, drive chronic activation of NF -k B signaling in a Gba1 loss-of-function model. It also highlights that elimination of the microbiota or stimulation of autophagy to remove immune mediators, rather than prolonged immunosuppression, may represent effective therapeutic avenues for GBA1- associated disorders.
... In vertebrates, the conservation of immune cells between species is well-known [28], and recent studies have shown conserved marker genes and regulatory programs between Drosophila and vertebrates at the whole organism level [29,30]. Although the homology between Drosophila hemocytes and vertebrate immune cells has been previously discussed [31], comparative transcriptomic analyses across immune cell types have not been performed. To address this issue, we compared the expression and conservation of cluster of differentiation (CD) genes and validated functions in Drosophila hemocytes (Fig 4). ...
Drosophila hemocytes serve as the primary defense system against harmful threats, allowing the animals to thrive. Hemocytes are often compared to vertebrate innate immune system cells due to the observed functional similarities between the two. However, the similarities have primarily been established based on a limited number of genes and their functional homologies. Thus, a systematic analysis using transcriptomic data could offer novel insights into Drosophila hemocyte function and provide new perspectives on the evolution of the immune system. Here, we performed cross-species comparative analyses using single-cell RNA-sequencing data from Drosophila and vertebrate immune cells. We found several conserved markers for the cluster of differentiation (CD) genes in Drosophila hemocytes and validated the role of CG8501 ( CD59 ) in phagocytosis by plasmatocytes, which function much like macrophages in vertebrates. By comparing whole transcriptome profiles in both supervised and unsupervised analyses, we showed that Drosophila hemocytes are largely homologous to vertebrate myeloid cells, especially plasmatocytes to monocytes/macrophages and prohemocyte 1 (PH1) to hematopoietic stem cells. Furthermore, a small subset of prohemocytes with hematopoietic potential displayed homology with hematopoietic progenitor populations in vertebrates. Overall, our results provide a deeper understanding of molecular conservation in the Drosophila immune system.
... 23 As a result, there were increased expression of inflammatory factors and AMPs following the immune responses. 24 These resulted in stronger antimicrobial responses of the infected larvae compared to the non-infected ones as portrayed in our study. Hemolymph extracted from BSF larvae immunized by E. coli showed promising bactericidal properties at low concentration compared to MRSA-infected larvae. ...
The larvae of the black soldier fly (BSFL), Hermetia illucens (Linnaeus, 1758) (Diptera: Stratiomyidae), can survive in environments contaminated with various bacteria by producing antimicrobial compounds. This study, for the very first time, investigated the potential antibacterial activity of hemolymph extracted from BSFL in Malaysia using diffusion and dilution methods. Prior to extraction, the larvae were infected with either Methicillin-resistant Staphylococcus aureus (MRSA) or Escherichia coli. Then, the hemolymph was collected. Serial dilutions from 200 to 12.5 mg/ml of the hemolymph extracts were screened against ten different bacteria. The results showed inhibition of eight out of ten tested bacteria (i.e., MRSA, Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis, Micrococcus luteus, Escherichia coli, Klebsiella pneumoniae and Acinetobacter sp.). We found that immunological-challenge larvae have stronger antimicrobial activity than the control groups. The Minimum Inhibitory Concentrations (MIC) for bacteria against for infected larvae were 12.5 mg/ml for MRSA, S. pyogenes, B. subtilis, M. luteus, E. coli, and K. pneumoniae. As for bactericidal activity, the MBC of E. coli infected larvae was 25mg/ml against S. pyogenes and B. subtilis. In conclusion, BSFL hemolymph has antibacterial activity against a range of bacteria and could be a candidate for novel antimicrobial development.
... Although Drosophila is considered a powerful model to study the host immune, hormonal, and metabolic responses to pathogenic bacteria [107][108][109][110][111][112][113][114][115][116], the specific effects these infections cause on pheromone production and, consequently, on chemical communication are still largely unexplored. ...
Pathogens can influence the physiology and behavior of both animal and plant hosts in a manner that promotes their own transmission and dispersal. Recent research focusing on insects has revealed that these manipulations can extend to the production of pheromones, which are pivotal in chemical communication. This review provides an overview of the current state of research and available data concerning the impacts of bacterial, viral, fungal, and eukaryotic pathogens on chemical communication across different insect orders. While our understanding of the influence of pathogenic bacteria on host chemical profiles is still limited, viral infections have been shown to induce behavioral changes in the host, such as altered pheromone production, olfaction, and locomotion. Entomopathogenic fungi affect host chemical communication by manipulating cuticular hydrocarbons and pheromone production, while various eukaryotic parasites have been observed to influence insect behavior by affecting the production of pheromones and other chemical cues. The effects induced by these infections are explored in the context of the evolutionary advantages they confer to the pathogen. The molecular mechanisms governing the observed pathogen-mediated behavioral changes, as well as the dynamic and mutually influential relationships between the pathogen and its host, are still poorly understood. A deeper comprehension of these mechanisms will prove invaluable in identifying novel targets in the perspective of practical applications aimed at controlling detrimental insect species.
... As a Gram-negative bacterium, Apo has cell walls that contain DAP-type peptidoglycan, which can be recognized by the insect immune deficiency (IMD) pathway and trigger immune responses . Pathogen infections induce the expression of immune genes in many insect hosts (Bai et al., 2021;Buchon et al., 2014). In D. melanogaster, oral infection of Erwinia carotovora, a bacterial phytopathogen, increases the expression of antimicrobial peptides under the control of the IMD pathway (Buchon et al., 2009) and significantly delays larval development (Houtz et al., 2019). ...
Insects are rich in various microorganisms, which play diverse roles in affecting host biology. Although most Drosophila species prefer rotten fruits, the agricultural pest Drosophila suzukii attacks ripening fruits before they are harvested. We have reported that the microbiota has positive and negative impacts on the agricultural pest D. suzukii on nutrient‐poor and ‐rich diets, respectively. On nutrient‐poor diets, microbes provide protein to facilitate larval development. But how they impede D. suzukii development on nutrient‐rich diets is unknown. Here we report that Acetobacter pomorum (Apo), a commensal bacterium in many Drosophila species and rotting fruit, has several detrimental effects in D. suzukii . Feeding D. suzukii larvae nutrient‐rich diets containing live Apo significantly delayed larval development and reduced the body weight of emerged adults. Apo induced larval immune responses and downregulated genes of digestion and juvenile hormone metabolism. Knockdown of these genes in germ‐free larvae reproduced Apo‐like weakened phenotypes. Apo was confirmed to secrete substantial amounts of gluconic acid. Adding gluconic acid to the D. suzukii larval diet hindered larval growth and decreased adult body weight. Moreover, the dose of gluconic acid that adversely affected D. suzukii did not negatively affect Drosophila melanogaster , suggesting that D. suzukii is less tolerant to acid than D. melanogaster . Taken together, these findings indicate that D. suzukii is negatively affected by gluconic acid, which may explain why it prefers ripening fruit over Apo‐rich rotting fruit. These results show an insect's tolerance to microbes can influence its ecological niche.
... Excessive ROS production produces cell damage via inflammation by DAMPs activation. When DAMPs are released, excessive ROS production can cause inflammation and cell damage [39]. ROS molecules produced during infection are associated with antibacterial responses [38], e.g., the hydrogen peroxide produced by damaged tissues in Drosophila embryos mediates the recruitment of hemocytes [15]. ...
(1) Background: The global population is projected to reach a staggering 9.8 billion people by the year 2050, leading to major concerns about food security. The necessity to increase livestock production is inevitable. The black soldier fly (BSF) is known for its ability to consume a wide range of organic waste, and BSF larvae have already been used as a partial substitute for fishmeal. In contrast, the use of antibiotics in livestock feed for growth promotion and prophylaxis poses a severe threat to global health owing to antimicrobial resistance. Insect antimicrobial peptides (AMPs) have shown the potential to rapidly disrupt target bacterial membranes, making bacterial resistance to AMPs a less likely concern. (2) Methods: In this study, we explored various methods for stimulating AMP synthesis in BSF larvae and found that thermal injury effectively induced the production of various AMP types. Additionally, we investigated the activation of innate immune response pathways that lead to AMP production following thermal injury. (3) Results: Interestingly, thermal injury treatment, although not involving bacteria, exhibited a similar response to that observed following Gram-positive bacterial infection in eliciting the expression of AMP genes. (4) Conclusions: Our findings offer support for the industrial use of BSF to enhance livestock production and promote environmental health.
... In insects, these cascades consist of so-called clip-containing serine proteases (cSPs), a large protein family (consisting of 5 subfamilies, A-E), which is characterized by one or more clip prodomains and one or more protease domains with trypsin-like specificity, and their homologs (cSPHs), which have lost one or more residues of the catalytic triad required for proteolytic activity. Our knowledge of the molecular makeup of these cascades is mainly derived from insect model species that are either large in size, enabling biochemical investigations of their hemolymph (e.g., Manduca sexta [11] and Tenebrio molitor [12]), or hold an excellent genetic and molecular toolbox (in case of Drosophila melanogaster [13,14]). ...
Insect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist of clip-domain serine proteases (cSPs) and/or their non-catalytic homologs, which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquito Anopheles gambiae as a central node in this protease network. CLIPB4 is tightly co-expressed with SRPN2 and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network in An . gambiae , by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.
Pathogen host shifts – where a pathogen jumps from one host species to another – are important sources of emerging infectious diseases. Although viral pathogens are a major source of emerging infectious diseases, there is a growing focus on the emergence of novel bacterial infections through host shifts. However, compared to viruses we know relatively little about the factors that determine whether a bacteria can infect a novel host, such as how host phylogenetics constrains variation in pathogen host range. Here, we experimentally examined susceptibility to bacterial infections using a panel of 36 Drosophilidae species and four bacterial pathogens (Providencia rettgeri, Pseudomonas entomophila, Enterococcus faecalis, Staphylococcus aureus). The outcomes of infection differed greatly among pathogens and across host species. The host phylogeny explains a considerable amount of variation in susceptibility, with the greatest phylogenetic signal for P. rettgeri infection, with 94% of variation in mortality being explained by the host phylogeny. Positive correlations were observed between mortality and bacterial load for three out of the four pathogens. Correlations in susceptibility between the four pathogens were positive but largely non-significant, suggesting susceptibility is mostly pathogen-specific. These results suggest that susceptibility to bacterial pathogens may be predicted by the host phylogeny, but the effect may vary in magnitude between different bacteria.
This study delved into the consequences of prolonged administration of vitamin D3 on innate immune systems, particularly NF-κB and JAK/STAT, in Drosophila melanogaster. The outcomes indicated that vitamin D3 treatment exhibited a notable capacity to improve the survival of adult flies with compromised immune functions, a condition induced by the loss of PGRP-LB, particularly when the flies were exposed to heat-killed Escherichia coli. The PGRP-LBΔ mutant line that was treated with heat-killed E. coli experienced reduced survival. Treatment of heat-killed E. coli-treated PGRP-LBΔ with vitamin D3 resulted in improved survival, and this phenotypic feature might be due to the downregulation of gene expression in the NF-κB and JAK/STAT pathways. However, a higher concentration of vitamin D3 was associated with decreased survival, potentially linked to intricate immunological responses. The research also underscored the influence of vitamin D3 on the expression of antioxidant genes, sod1 and sod2, indicating an augmented resistance to oxidative stress. Further, this study revealed the effect of vitamin D3 on the reproductive status of the autoinflammatory model, showing an increase in pupae and adult flies with a treatment of 10 mM vitamin D3, suggesting the potential benefits of vitamin D3 on the reproductive profile. Overall, this study provides preliminary insights into the complex interactions between vitamin D3, immune pathways, oxidative responses in the cell, and reproduction in Drosophila.
Nutrition and resilience are linked, though it is not yet clear how diet confers stress resistance or the breadth of stressors that it can protect against. We have previously shown that transiently restricting an essential amino acid can extend lifespan and also protect against nicotine exposure in Drosophila melanogaster, raising the possibility that amino acid restriction is geroprotective because of elevated detoxification capacity. Here, we sought to characterise the nature of this dietary mediated protection, and determine whether it was sex, amino acid, and/or nicotine specific. When we compared between sexes, we found that isoleucine deprivation increases female, but not male, nicotine resistance. Surprisingly, we found that this protection afforded to females was not replicated by dietary protein restriction and was instead specific to individual amino acid restriction. Other studies have documented methionine or leucine restriction conferring stress resistance, though we previously found that individually depriving them did not increase nicotine resistance. We therefore wondered whether reducing the severity of restriction of these amino acids could confer nicotine resistance. This was true for methionine restriction, and we found that flies fed a diet containing 25% methionine for 7 days protected against subsequent nicotine poisoning (~30% longer lived than controls with all amino acids). However, when dietary leucine was altered, nicotine resistance changed, but no single diet was protective. To understand whether these beneficial effects of diet were specific to nicotine or were generalisable across stressors, we pre-treated with amino acid restriction diets and exposed them to other types of stress. We did not find any diets that protected against heat stress or infection with the bacterium Enterococcus faecalis. However, we found that some of the diets that protected against nicotine also protected against oxidative and starvation stress, and improved survival following cold shock. Interestingly, we found that a diet lacking isoleucine was the only diet to protect against all these stressors. These data point to isoleucine as a critical determinant of robustness in the face of environmental challenges.
The silkworm, a crucial model organism of the Lepidoptera, offers an excellent platform for investigating the molecular mechanisms underlying the innate immune response of insects toward pathogens. Over the years, researchers worldwide have identified numerous immune‐related genes in silkworms. However, these identified silkworm immune genes are not well classified and not well known to the scientific community. With the availability of the latest genome data of silkworms and the extensive research on silkworm immunity, it has become imperative to systematically categorize the immune genes of silkworms with different database IDs. In this study, we present a meticulous organization of prevalent immune‐related genes in the domestic silkworm, using the SilkDB 3.0 database as a reliable source for updated gene information. Furthermore, utilizing the available data, we classify the collected immune genes into distinct categories: pattern recognition receptors, classical immune pathways, effector genes and others. In‐depth data analysis has enabled us to predict some potential antiviral genes. Subsequently, we performed antiviral experiments on selected genes, exploring their impact on Bombyx mori nucleopolyhedrovirus replication. The outcomes of this research furnish novel insights into the immune genes of the silkworm, consequently fostering advancements in the field of silkworm immunity research by establishing a comprehensive classification and functional understanding of immune‐related genes in the silkworm. This study contributes to the broader understanding of insect immune responses and opens up new avenues for future investigations in the domain of host–pathogen interactions.
Background: A prominent hallmark of aging is inflammaging—the increased expression of innate immune genes without identifiable infection. Model organisms with shorter lifespans, such as the fruit fly, provide an essential platform for probing the mechanisms of inflammaging. Multiple groups have reported that, like mammalian models, old flies have significantly higher levels of expression of anti-microbial peptide genes. However, whether some of these genes—or any others—can serve as reliable markers for assessing and comparing inflammaging in different strains remains unclear.
Methods and Results: We compared RNA-Seq datasets generated by different groups. Although the fly strains used in these studies differ significantly, we found that they share a core group of genes with strong aging-associated expression. In addition to anti-microbial peptide genes, we identified other genes that have prominently increased expression in old flies, especially SPH93. We further showed that machine learning models can be used to predict the “inflammatory age” of the fruit fly.
Conclusion: A core group of genes may serve as markers for studying inflammaging in Drosophila. RNA-Seq profiles, in combination with machine-learning models, can be applied to measure the acceleration or deceleration of inflammaging.
In insects, the production of antimicrobial peptides is considered to be the solo arm of the innate immune response. The Toll and immune deficiency pathways are two major signaling pathways that lead to the production of antimicrobial peptides as final effectors. The dynamic functions of Toll/Toll‐like receptors have been thoroughly reviewed in mammals and Drosophila . During the last decade, we have attempted to clarify the immunological roles of different Toll receptor variants and their ligands in Tenebrio molitor . Accordingly, we showed that TmToll‐8 , −9 , and −10 mRNA transcripts are induced following systemic injections of Gram‐negative, Gram‐positive, and fungal infections. Our data revealed harmonic expression patterns of TmToll‐8 , −9 , and −10 throughout the developmental stages of healthy individuals and in response to infections in the examined tissues (Malpighian tubules, gut, fat bodies, and hemolymph), illustrating similar evolutionary conversions of different Toll receptor variants in T. molitor . Taken together, our findings highlight the immunological actions of TmToll‐8 , −9 , and −10 in response to pathogenic insults in T. molitor .
Insects and fungi, both megadiverse groups of organisms, have a long history of associations with each other. Among the fungi, Hypocreales (Ascomycota) and Entomophthoromycotina (Zoopagomycota), contain the most common pathogens of insects. Ascomycete entomopathogenic fungi (EPF) are mostly well-studied and have been found to be either heterothallically or homothallically sexual with a divergent number of genes at the mating-type loci of different species. So far more than 100 strains of >60 EPF species have been genome sequenced which demonstrated substantial variations in genome size and gene content among each other. However, convergent evolution of fungal entomopathogenicity has been coined, especially in terms of the similar expansion of protease and chitinase gene families in EPF for targeting the protein- and chitin-rich insect cuticles. Functional genetic studies have unveiled an array of EPF genes involved in mediating host recognition and interactions with insect hosts, especially mechanisms of invading or evading host immunity to facilitate fungal colonization of insect body cavities. The discovery of small molecules and their essential roles in mediating EPF associations with insect hosts also have been advanced. In addition to advancing the mechanisms of fungus–insect interactions, these works highly benefit the development of cost-effective mycoinsecticides and the protection of ecologically and economically important beneficial insects.
The tactics used by animal pathogens to combat host immunity are largely unclear. Here, we report the depiction of the virulence-required effector Tge1 deployed by the entomopathogen Metarhizium robertsii to suppress Drosophila antifungal immunity. Tge1 can target both GNBP3 and GNBP-like 3 (GL3), and the latter can
bind to b-glucans like GNBP3, whereas the glucan binding by both receptors can be attenuated by Tge1. As opposed to the surveillance GNBP3, GL3 is inducible in Drosophila depending on the Toll pathway via a positive feedback loop mechanism. Losses of GNBP3 and GL3 genes result in the deregulations of protease
cascade, Spa¨ tzle maturation, and antimicrobial gene expressions in Drosophila upon fungal challenges. Fly survival assays confirm that GL3 plays a more essential role than GNBP3 in combating fungal infections. In addition to evidencing the gene-for-gene interactions between fungi and insects, our data advance insights into Drosophila antifungal immunity.
Overnutrition with dietary sugar can worsen infection outcomes in diverse organisms including insects and humans, generally through unknown mechanisms. In the present study, we show that adult Drosophila melanogaster fed high-sugar diets became more susceptible to infection by the Gram-negative bacteria Providencia rettgeri and Serratia marcescens, although diet had no significant effect on infection by Gram-positive bacteria Enterococcus faecalis or Lactococcus lactis. We found that P. rettgeri and S. marcescens proliferate more rapidly in D. melanogaster fed a high-sugar diet, resulting in increased probability of host death. D. melanogaster become hyperglycemic on the high-sugar diet, and we find evidence that the extra carbon availability may promote S. marcescens growth within the host. However, we found no evidence that increased carbon availability directly supports greater P. rettgeri growth. D. melanogaster on both diets fully induce transcription of antimicrobial peptide (AMP) genes in response to infection, but D. melanogaster provided with high-sugar diets show reduced production of AMP protein. Thus, overnutrition with dietary sugar may impair host immunity at the level of AMP translation. Our results demonstrate that dietary sugar can shape infection dynamics by impacting both host and pathogen, depending on the nutritional requirements of the pathogen and by altering the physiological capacity of the host to sustain an immune response.
Author Summary
Diet has critical impact on the quality of immune defense, and high-sugar diets increase susceptibility to bacterial infection in many animals. Yet it is unknown which aspects of host and pathogen physiology are impacted by diet to influence infection dynamics. Here we show that high-sugar diets increase susceptibility to some, but not all, bacterial infections in Drosophila . We find that feeding on high sugar diet impairs the host immune response by reducing the level of antimicrobial peptides produced. The expression of genes encoding these peptides is not affected, so we infer that protein translation is impaired. We further show that flies on high-sugar diets are hyperglycemic, and that some pathogens may use the excess sugar in the host to promote growth during the infection. Thus, our study demonstrates that dietary impacts on infection outcome arise through physiological effects on both the host and pathogen.
Leishmaniasis transmission cycles are maintained and sustained in nature by the complex crosstalk of the Leishmania parasite, sandfly vector, and the mammalian hosts (human, as well as zoonotic reservoirs). Regardless of the vast research on human host-parasite interaction, there persists a substantial knowledge gap on the parasite’s development and modulation in the vector component. This review focuses on some of the intriguing aspects of the Leishmania-sandfly interface, beginning with the uptake of the intracellular amastigotes from an infected host to the development of the parasite within the sandfly’s alimentary canal, followed by the transmission of infective metacyclic stages to another potential host. Upon ingestion of the parasite, the sandfly hosts an intricate repertoire of immune barriers, either to evade the parasite or to ensure its homeostatic coexistence with the vector gut microbiome. Sandfly salivary polypeptides and Leishmania exosomes are co-egested with the parasite inoculum during the infected vector bite. This has been attributed to the modulation of the parasite infection and subsequent clinical manifestation in the host. While human host–based studies strive to develop effective therapeutics, a greater understanding of the vector-parasite-microbiome and human host interactions could help us to identify the targets and to develop strategies for effectively preventing the transmission of leishmaniasis.
Host-microbe interactions influence intestinal stem cell (ISC) activity to modulate epithelial turnover and composition. Here we investigated the functional impacts of viral infection on intestinal homeostasis and the mechanisms by which viral infection alters ISC activity. We report that Drosophila A virus (DAV) infection disrupts intestinal homeostasis in Drosophila by inducing sustained ISC proliferation, resulting in intestinal dysplasia, loss of gut barrier function, and reduced lifespan. We found that additional viruses common in laboratory-reared Drosophila also promote ISC proliferation. The mechanism of DAV-induced ISC proliferation involves progenitor-autonomous EGFR signaling, JNK activity in enterocytes, and requires Sting-dependent NF-kB (Relish) activity. We further demonstrate that activating Sting-Relish signaling is sufficient to induce ISC proliferation, promote intestinal dysplasia, and reduce lifespan in the absence of infection. Our results reveal that viral infection can significantly disrupt intestinal physiology, highlight a novel role for Sting-Relish signaling, and support a role for viral infection in aging.
Graphical Abstract
The innate immune system provides hosts with a crucial first line of defense against pathogens. While immune genes are often among the fastest evolving genes in the genome, in Drosophila, antimicrobial peptides (AMPs) are notable exceptions. Instead, AMPs may be under balancing selection, such that over evolutionary timescales multiple alleles are maintained in populations. In this study, we focus on the Drosophila antimicrobial peptide Diptericin A, which has a segregating amino acid polymorphism associated with differential survival after infection with the Gram-negative bacteria Providencia rettgeri. Diptericin A also helps control opportunistic gut infections by common Drosophila gut microbes, especially those of Lactobacillus plantarum. In addition to genotypic effects on gut immunity, we also see strong sex-specific effects that are most prominent in flies without functional diptericin A. To further characterize differences in microbiomes between different diptericin genotypes, we used 16S metagenomics to look at the microbiome composition. We used both lab reared and wild caught flies for our sequencing and looked at overall composition as well as the differential abundance of individual bacterial families. Overall, we find flies that are homozygous serine for diptericin A are better equipped to survive a systemic infection from P. rettgeri, but in general homozygous arginine flies have a longer lifespan after being fed common gut commensals. Our results suggest a possible mechanism for the maintenance of genetic variation of diptericin A through the complex interactions of sex, systemic immunity, and the maintenance of the gut microbiome.
Aedes aegypti females are natural vectors of important arboviruses such as Dengue, Zika, and yellow fever. Mosquitoes activate innate immune response signaling pathways upon infection, as a resistance mechanism to fight pathogens and limit their propagation. Despite the beneficial effects of immune activation for insect vectors, phenotypic costs ultimately affect their fitness. However, the underlying mechanisms that mediate these fitness costs remain poorly understood. Given the high energy required to mount a proper immune response, we hypothesized that systemic activation of innate immunity would impair flight muscle mitochondrial function, compromising tissue energy demand and flight activity. Here, we investigated the dynamic effects of activation of innate immunity by intra-thoracic zymosan injection on A. aegypti flight muscle mitochondrial metabolism. Zymosan injection significantly increased defensin expression in fat bodies in a time-dependent manner that compromised flight activity. Although oxidant levels in flight muscle were hardly altered, ATP-linked respiratory rates driven by mitochondrial pyruvate+proline oxidation were significantly reduced at 24h upon zymosan injection. Oxidative phosphorylation coupling was preserved regardless of innate immune response activation along 24h. Importantly, rotenone-sensitive respiration and complex I-III activity were specifically reduced 24h upon zymosan injection. Also, loss of complex I activity compromised ATP-linked and maximal respiratory rates mediated by mitochondrial proline oxidation. Finally, the magnitude of innate immune response activation negatively correlated with respiratory rates, regardless of the metabolic states. Collectively, we demonstrate that activation of innate immunity is strongly associated with reduced flight muscle complex I activity with direct consequences to mitochondrial proline oxidation and flight activity. Remarkably, our results indicate a trade-off between dispersal and immunity exists in an insect vector, underscoring the potential consequences of disrupted flight muscle mitochondrial energy metabolism to arbovirus transmission.
An active immune response is energetically demanding and requires reallocation of nutrients to support resistance to and tolerance of infection. Insulin signaling is a critical global regulator of metabolism and whole-body homeostasis in response to nutrient availability and energetic needs, including those required for mobilization of energy in support of the immune system. In this review, we share findings that demonstrate interactions between innate immune activity and insulin signaling primarily in the insect model Drosophila melanogaster as well as other insects like Bombyx mori and Anopheles mosquitos. These studies indicate that insulin signaling and innate immune activation have reciprocal effects on each other, but that those effects vary depending on the type of pathogen, route of infection, and nutritional status of the host. Future research will be required to further understand the detailed mechanisms by which innate immunity and insulin signaling activity impact each other.
Obesity is associated with various metabolic disorders, such as insulin resistance and adipose tissue inflammation (ATM), characterized by macrophage infiltration into adipose cells. This study presents a novel Drosophila model (OBL) to investigate the mechanisms underlying these obesity-related pathologies. We employed genetic manipulation to reduce ecdysone levels to prolong the larval stage. These animals are hyperphagic and exhibit features resembling obesity in mammals, including increased lipid storage, adipocyte hypertrophy, and high circulating glucose levels. Moreover, we observed a significant infiltration of immune cells (hemocytes) in the fat bodies accompanied by insulin resistance. We found that attenuation of Eiger/TNFα signaling reduced ATM and improved insulin sensitivity. Furthermore, using metformin and the antioxidant anthocyanins, we ameliorated both phenotypes. Our data highlights evolutionarily conserved mechanisms allowing the development of Drosophila models for discovering therapeutic pathways in adipose tissue immune cell infiltration and insulin resistance. Our model can also provide a platform to perform genetic screens or test the efficacy of novel therapeutic interventions for diseases such as obesity, type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD).
Insects respond to microbial infection by the rapid and transient expression of several genes encoding potent antimicrobial peptides. Herein we demonstrate that this antimicrobial response of Drosophila is not aspecific but can discriminate between various classes of microorganisms. We first observe that the genes encoding antibacterial and antifungal peptides are differentially expressed after injection of distinct microorganisms. More strikingly, Drosophila that are naturally infected by entomopathogenic fungi exhibit an adapted response by producing only peptides with antifungal activities. This response is mediated through the selective activation of the Toll pathway.
Significance
The airways of the lungs are lined by ciliated and secretory epithelial cells important for mucociliary clearance. When these cells are damaged or lost, they are replaced by the differentiation of basal stem cells. Little is known about how this repair is orchestrated by signaling pathways in the epithelium and underlying stroma. We present evidence using cultured airway cells and genetic manipulation of a mouse model of airway repair that the cytokine IL-6 promotes the differentiation of ciliated vs. secretory cells. This process involves direct Stat3 regulation of genes controlling both cell fate (Notch1) and the differentiation of multiciliated cells (Multicilin and forkhead box protein J1). Moreover, the major producer of IL-6 appears to be mesenchymal cells in the stroma rather than immune cells.
Unlabelled:
To elucidate mechanisms underlying the complex relationships between a host and its microbiota, we used the genetically tractable model Drosophila melanogaster. Consistent with previous studies, the microbiota was simple in composition and diversity. However, analysis of single flies revealed high interfly variability that correlated with differences in feeding. To understand the effects of this simple and variable consortium, we compared the transcriptome of guts from conventionally reared flies to that for their axenically reared counterparts. Our analysis of two wild-type fly lines identified 121 up- and 31 downregulated genes. The majority of these genes were associated with immune responses, tissue homeostasis, gut physiology, and metabolism. By comparing the transcriptomes of young and old flies, we identified temporally responsive genes and showed that the overall impact of microbiota was greater in older flies. In addition, comparison of wild-type gene expression with that of an immune-deficient line revealed that 53% of upregulated genes exerted their effects through the immune deficiency (Imd) pathway. The genes included not only classic immune response genes but also those involved in signaling, gene expression, and metabolism, unveiling new and unexpected connections between immunity and other systems. Given these findings, we further characterized the effects of gut-associated microbes on gut morphology and epithelial architecture. The results showed that the microbiota affected gut morphology through their impacts on epithelial renewal rate, cellular spacing, and the composition of different cell types in the epithelium. Thus, while bacteria in the gut are highly variable, the influence of the microbiota at large has far-reaching effects on host physiology.
Importance:
The guts of animals are in constant association with microbes, and these interactions are understood to have important roles in animal development and physiology. Yet we know little about the mechanisms underlying the establishment and function of these associations. Here, we used the fruit fly to understand how the microbiota affects host function. Importantly, we found that the microbiota has far-reaching effects on host physiology, ranging from immunity to gut structure. Our results validate the notion that important insights on complex host-microbe relationships can be obtained from the use of a well-established and genetically tractable invertebrate model.
The triggering of innate immune mechanisms is emerging as a crucial component of major neurodegenerative diseases. Microglia and other cell types in the brain can be activated in response to misfolded proteins or aberrantly localized nucleic acids. This diverts microglia from their physiological and beneficial functions, and leads to their sustained release of pro-inflammatory mediators. In this Review, we discuss how the activation of innate immune signalling pathways - in particular, the NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome - by aberrant host proteins may be a common step in the development of diverse neurodegenerative disorders. During chronic activation of microglia, the sustained exposure of neurons to pro-inflammatory mediators can cause neuronal dysfunction and contribute to cell death. As chronic neuroinflammation is observed at relatively early stages of neurodegenerative disease, targeting the mechanisms that drive this process may be useful for diagnostic and therapeutic purposes.
Beyond their role in cell metabolism, development, and reproduction, hormones are also important modulators of the immune system. In the context of inflammatory disorders, systemic administration of pharmacological doses of synthetic glucocorticoids (GCs) is widely used as an anti-inflammatory treatment [1, 2]. However, not all actions of GCs are immunosuppressive, and many studies have suggested that physiological concentrations of GCs can have immunoenhancing effects [3-7]. For a more comprehensive understanding of how steroid hormones regulate immunity and inflammation, a simple in vivo system is required. The Drosophila embryo has recently emerged as a powerful model system to study the recruitment of immune cells to sterile wounds [8] and host-pathogen dynamics [9]. Here we investigate the immune response of the fly embryo to bacterial infections and find that the steroid hormone 20-hydroxyecdysone (20-HE) can regulate the quality of the immune response and influence the resolution of infection in Drosophila embryos.
Sterile inflammation triggered by endogenous factors is thought to contribute to the pathogenesis of acute and chronic inflammatory diseases. Here, we demonstrate that apoptosis-deficient mutants spontaneously develop a necrosis-driven systemic immune response in Drosophila and provide an in vivo model for studying the organismal response to sterile inflammation. Metabolomic analysis of hemolymph from apoptosis-deficient mutants revealed increased sarcosine and reduced S-adenosyl-methionine (SAM) levels due to glycine N-methyltransferase (Gnmt) upregulation. We showed that Gnmt was elevated in response to Toll activation induced by the local necrosis of wing epidermal cells. Necrosis-driven inflammatory conditions induced dFoxO hyperactivation, leading to an energy-wasting phenotype. Gnmt was cell-autonomously upregulated by dFoxO in the fat body as a possible rheostat for controlling energy loss, which functioned during fasting as well as inflammatory conditions. We propose that the dFoxO-Gnmt axis is essential for the maintenance of organismal SAM metabolism and energy homeostasis.
Host defense against pathogenic infection is composed of resistance and tolerance. Resistance is the ability of the host to limit a pathogen burden, whereas tolerance is the ability to limit the deleterious effects of a given pathogen burden. This distinction recognizes that the fittest host does not necessarily have the most aggressive immune system, suggesting that host-pathogen co-evolution involves more than an escalating arms race between pathogen virulence factors and host antimicrobial activity. How a host balances resistance and tolerance and how this balance influences the evolution of host defense remains unanswered. In order to determine how genotype-by-diet interactions and evolutionary costs of each strategy may constrain the evolution of host defense, we measured survival, fecundity, and pathogen burden over five days in ten genotypes of Drosophila melanogaster reared on two diets and infected with the Gram-negative bacterial pathogen Providencia rettgeri.
We demonstrated two distinct phases of infection: an acute phase that consists of high mortality, low fecundity, and high pathogen loads, and a chronic phase where there was a substantial but stable pathogen load and mortality and fecundity returned to uninfected levels. We demonstrated genetic variation for resistance in both phases of infection, but find genetic variation for tolerance only in the acute phase. We found genotype-by-diet interactions for tolerance, especially in the acute phase, but genotype-by-diet interaction did not significantly shape resistance. We found a diet-dependent positive relationship between resistance and tolerance and a weak evolutionary cost of resistance, but did not detect any costs of tolerance.
Existing models of tolerance and resistance are overly simplistic. Multi-phase infections such as that studied here are rarely considered, but we show important differences in determination and evolutionary constraints on tolerance and resistance over the two phases of infection. Our observation of genetic variation for tolerance is inconsistent with simple models that predict evolutionary fixation of tolerance alleles, and instead indicate that genetic variation for resistance and tolerance is likely to be maintained by non-independence between resistance and tolerance, condition-dependent evolutionary costs, and environmental heterogeneity.
Drosophila melanogaster has become a system-of-choice for functional genomic studies. Many resources, including online databases and software tools, are now available to support design or identification of relevant fly stocks and reagents, or analysis and mining of existing functional genomic, transcriptomic, proteomic, etc. datasets. These include large community collections of fly stocks and plasmid clones, 'meta' information sites like FlyBase and FlyMine, and an increasing number of more specialized reagents, databases and online tools. Here, we introduce key resources useful to plan large-scale functional genomics studies in Drosophila and to analyze, integrate and mine the results of those studies in ways that facilitate identification of highest-confidence results and generation of new hypotheses. We also discuss ways in which existing resources can be used and might be improved, and suggest a few areas of future development that would further support large- and small-scale studies in Drosophila and facilitate use of Drosophila information by the research community more generally.
In recent years, the synergistic relationship between NADPH Oxidases (NOX)/Dual Oxidases (DUOX) enzymes and peroxidases has received increased attention. Peroxidases utilize NOX/DUOX-generated H2O2 for a myriad of functions including, but not limited to thyroid hormone biosynthesis, cross-linking extracellular matrices (ECM) and immune defense. We postulated that one or more peroxidases produced by Caenorhabditis elegans would act in host defense, possibly in conjunction with BLI-3, the only NOX/DUOX enzyme encoded by the genome that is expressed. Animals exposed to RNAi of the putative peroxidase genes were screened for susceptibility to the human pathogen Enterococcus faecalis. One of three genes identified, skpo-1 (ShkT-containing peroxidase), was studied in-depth. Animals mutant for this gene were significantly more susceptible to E. faecalis, but not Pseudomonas aeruginosa. A slight decrease in longevity was also observed. The skpo-1 mutant animals had a dumpy phenotype of incomplete penetrance; half the animals displayed a dumpy phenotype ranging from slight to severe, and half were morphologically wild type. The SKPO-1 protein contains the critical catalytic residues necessary for peroxidase activity, and in a whole animal assay more H2O2 was detected from the mutant compared to the wild type, consistent with the loss of an H2O2 sink. By using tissue-specific skpo-1 RNAi and immunohistochemical localization with an anti-SKPO-1 antibody, it was determined that the peroxidase is functionally and physically present in the hypodermis. In conclusion, these results characterize a peroxidase that functions protectively in the hypodermis during exposure to E. faecalis.
Dengue is one of the most widespread mosquito-borne diseases in the world. The causative agent, dengue virus (DENV), is primarily transmitted by the mosquito Aedes aegypti, a species that has proved difficult to control using conventional methods. The discovery that A. aegypti transinfected with the wMel strain of Wolbachia showed limited DENV replication led to trial field releases of these mosquitoes in Cairns, Australia as a biocontrol strategy for the virus.
Field collected wMel mosquitoes that were challenged with three DENV serotypes displayed limited rates of body infection, viral replication and dissemination to the head compared to uninfected controls. Rates of dengue infection, replication and dissemination in field wMel mosquitoes were similar to those observed in the original transinfected wMel line that had been maintained in the laboratory. We found that wMel was distributed in similar body tissues in field mosquitoes as in laboratory ones, but, at seven days following blood-feeding, wMel densities increased to a greater extent in field mosquitoes.
Our results indicate that virus-blocking is likely to persist in Wolbachia-infected mosquitoes after their release and establishment in wild populations, suggesting that Wolbachia biocontrol may be a successful strategy for reducing dengue transmission in the field.
Apoptosis is an evolutionarily conserved mechanism that removes damaged or unwanted cells, effectively maintaining cellular
homeostasis. It has long been suggested that a deficiency in this type of naturally occurring cell death could potentially
lead to necrosis, resulting in the release of endogenous immunogenic molecules such as damage-associated molecular patterns
(DAMPs) and a noninfectious inflammatory response. However, the details about how danger signals from apoptosis-deficient
cells are detected and translated to an immune response are largely unknown. In this study, we found that Drosophila mutants deficient for Dronc, the key initiator caspase required for apoptosis, produced the active form of the endogenous
Toll ligand Spätzle (Spz). We speculated that, as a system for sensing potential DAMPs in the hemolymph, the dronc mutants constitutively activate a proteolytic cascade that leads to Spz proteolytic processing. We demonstrated that Toll
signaling activation required the action of Persephone, a CLIP domain serine protease that usually reacts to microbial proteolytic
activities. Our findings show that the Persephone proteolytic cascade plays a crucial role in mediating DAMP-induced systemic
responses in apoptosis-deficient Drosophila mutants.
Wolbachia are intracellular bacterial symbionts that are able to protect various insect hosts from viral infections. This tripartite interaction was initially described in Drosophila melanogaster carrying wMel, its natural Wolbachia strain. wMel has been shown to be genetically polymorphic and there has been a recent change in variant frequencies in natural populations. We have compared the antiviral protection conferred by different wMel variants, their titres and influence on host longevity, in a genetically identical D. melanogaster host. The phenotypes cluster the variants into two groups - wMelCS-like and wMel-like. wMelCS-like variants give stronger protection against Drosophila C virus and Flock House virus, reach higher titres and often shorten the host lifespan. We have sequenced and assembled the genomes of these Wolbachia, and shown that the two phenotypic groups are two monophyletic groups. We have also analysed a virulent and over-replicating variant, wMelPop, which protects D. melanogaster even better than the closely related wMelCS. We have found that a ∼21 kb region of the genome, encoding eight genes, is amplified seven times in wMelPop and may be the cause of its phenotypes. Our results indicate that the more protective wMelCS-like variants, which sometimes have a cost, were replaced by the less protective but more benign wMel-like variants. This has resulted in a recent reduction in virus resistance in D. melanogaster in natural populations worldwide. Our work helps to understand the natural variation in wMel and its evolutionary dynamics, and inform the use of Wolbachia in arthropod-borne disease control.
Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. Here, we reveal that regulation of macrophage-like cells (hemocytes) by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils) is still not well understood. Here in a simpler model system, we used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. We show that in response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of immunity in vivo in Drosophila, and paves the way for genetic dissection of the mechanisms at work behind steroid regulation of innate immune cells.
The resident prokaryotic microbiota of the metazoan gut elicits profound effects on the growth and development of the intestine. However, the molecular mechanisms of symbiotic prokaryotic-eukaryotic cross-talk in the gut are largely unknown. It is increasingly recognized that physiologically generated reactive oxygen species (ROS) function as signalling secondary messengers that influence cellular proliferation and differentiation in a variety of biological systems. Here, we report that commensal bacteria, particularly members of the genus Lactobacillus, can stimulate NADPH oxidase 1 (Nox1)-dependent ROS generation and consequent cellular proliferation in intestinal stem cells upon initial ingestion into the murine or Drosophila intestine. Our data identify and highlight a highly conserved mechanism that symbiotic microorganisms utilize in eukaryotic growth and development. Additionally, the work suggests that specific redox-mediated functions may be assigned to specific bacterial taxa and may contribute to the identification of microbes with probiotic potential.
The neuroendocrine architecture and insulin/insulin-like signaling (IIS) events in Drosophila are remarkably conserved. As IIS pathway governs growth and development, metabolism, reproduction, stress response, and longevity; temporal, spatial, and nutrient regulation of dilps encoding Drosophila insulin-like peptides (DILPs) provides potential mechanisms in modulating IIS. Of eight DILPs (DILP1-8) identified, recent studies have furthered our understanding of physiological roles of DILP2, DILP3, DILP5, and DILP6 in metabolism, aging, and responses to dietary restriction (DR), which will be the focus of this review. While the DILP producing IPCs of the brain secrete DILP2, 3, and 5, fat body produces DILP6. Identification of factors that influence dilp expression and DILP secretion has provided insight into the intricate regulatory mechanisms underlying transcriptional regulation of those genes and the activity of each peptide. Studies involving loss-of-function dilp mutations have defined the roles of DILP2 and DILP6 in carbohydrate and lipid metabolism, respectively. While DILP3 has been implicated to modulate lipid metabolism, a metabolic role for DILP5 is yet to be determined. Loss of dilp2 or adult fat body specific expression of dilp6 has been shown to extend lifespan, establishing their roles in longevity regulation. The exact role of DILP3 in aging awaits further clarification. While DILP5 has been shown associated with DR-mediated lifespan extension, contradictory evidence that precludes a direct involvement of DILP5 in DR exists. This review highlights recent findings on the importance of conserved DILPs in metabolic homeostasis, DR, and aging, providing strong evidence for the use of DILPs in modeling metabolic disorders such as diabetes and hyperinsulinemia in the fly that could further our understanding of the underlying processes and identify therapeutic strategies to treat them.
In antiviral RNA interference (RNAi), the DICER enzyme processes virus-derived double-stranded RNA (dsRNA) into small interfering
RNAs (siRNAs) that guide ARGONAUTE proteins to silence complementary viral RNA. As a counterdefense, viruses deploy viral
suppressors of RNAi (VSRs). Well-established in plants and invertebrates, the existence of antiviral RNAi remains unknown
in mammals. Here, we show that undifferentiated mouse cells infected with encephalomyocarditis virus (EMCV) or Nodamura virus
(NoV) accumulate ~22-nucleotide RNAs with all the signature features of siRNAs. These derive from viral dsRNA replication
intermediates, incorporate into AGO2, are eliminated in Dicer knockout cells, and decrease in abundance upon cell differentiation. Furthermore, genetically ablating a NoV-encoded VSR
that antagonizes DICER during authentic infections reduces NoV accumulation, which is rescued in RNAi-deficient mouse cells.
We conclude that antiviral RNAi operates in mammalian cells.
Viral Defenses
In plants and invertebrates, RNA interference (RNAi) functions as an innate antiviral defense mechanism. Viruses that infect plants and invertebrates have evolved viral suppressors of RNAi (VSRs) that disable the RNAi pathway. Whether mammals use RNAi as a defense against viruses has been less clear (see the Perspective by Sagan and Sarnow ). Li et al. (p. 231 ) and Maillard et al. (p. 235 ) studied mammalian cell lines and baby mice productively infected with RNA viruses and observed the production of virus-derived small RNAs (vsRNAs). When the putative VSR proteins of the infecting viruses were disabled, host RNAi-derived vsRNAs were much increased and the viruses were rapidly cleared and unable to mount a full-blown infection. Thus, RNAi also has an innate antiviral function in mammals.
Animals deploy various molecular sensors to detect pathogen infections. RIG-like receptor (RLR) proteins identify viral RNAs and initiate innate immune responses. The three human RLRs recognize different types of RNA molecules and protect against different viral pathogens. The RLR protein family is widely thought to have originated shortly before the emergence of vertebrates and rapidly diversified through a complex process of domain grafting. Contrary to these findings, here we show that full-length RLRs and their downstream signaling molecules were present in the earliest animals, suggesting that the RLR-based immune system arose with the emergence of multicellularity. Functional differentiation of RLRs occurred early in animal evolution via simple gene duplication followed by modifications of the RNA binding pocket, many of which may have been adaptively driven. Functional analysis of human and ancestral RLRs revealed that the ancestral RLR displayed RIG-1-like RNA-binding. MDA5-like binding arose through changes in the RNA binding pocket following the duplication of the ancestral RLR, which may have occurred either early in Bilateria or later, after deuterostomes split from protostomes. The sensitivity and specificity with which RLRs bind different RNA structures has repeatedly adapted throughout mammalian evolution, suggesting a long-term evolutionary arms race with viral RNA or other molecules.
Infections disturb metabolic homeostasis in many contexts, but the underlying connections are not completely understood. To address this, we use paired genetic and computational screens in Drosophila to identify transcriptional regulators of immunity and pathology and their associated target genes and physiologies. We show that Mef2 is required in the fat body for anabolic function and the immune response. Using genetic and biochemical approaches, we find that MEF2 is phosphorylated at a conserved site in healthy flies and promotes expression of lipogenic and glycogenic enzymes. Upon infection, this phosphorylation is lost, and the activity of MEF2 changes-MEF2 now associates with the TATA binding protein to bind a distinct TATA box sequence and promote antimicrobial peptide expression. The loss of phosphorylated MEF2 contributes to loss of anabolic enzyme expression in Gram-negative bacterial infection. MEF2 is thus a critical transcriptional switch in the adult fat body between metabolism and immunity.
The role of specific gut microbes in shaping body composition remains unclear. We transplanted fecal microbiota from adult
female twin pairs discordant for obesity into germ-free mice fed low-fat mouse chow, as well as diets representing different
levels of saturated fat and fruit and vegetable consumption typical of the U.S. diet. Increased total body and fat mass, as
well as obesity-associated metabolic phenotypes, were transmissible with uncultured fecal communities and with their corresponding
fecal bacterial culture collections. Cohousing mice harboring an obese twin’s microbiota (Ob) with mice containing the lean
co-twin’s microbiota (Ln) prevented the development of increased body mass and obesity-associated metabolic phenotypes in
Ob cage mates. Rescue correlated with invasion of specific members of Bacteroidetes from the Ln microbiota into Ob microbiota
and was diet-dependent. These findings reveal transmissible, rapid, and modifiable effects of diet-by-microbiota interactions.
Ubiquitin-mediated targeting of intracellular bacteria to the autophagy pathway is a key innate defence mechanism against invading microbes, including the important human pathogen Mycobacterium tuberculosis. However, the ubiquitin ligases responsible for catalysing ubiquitin chains that surround intracellular bacteria are poorly understood. The parkin protein is a ubiquitin ligase with a well-established role in mitophagy, and mutations in the parkin gene (PARK2) lead to increased susceptibility to Parkinson's disease. Surprisingly, genetic polymorphisms in the PARK2 regulatory region are also associated with increased susceptibility to intracellular bacterial pathogens in humans, including Mycobacterium leprae and Salmonella enterica serovar Typhi, but the function of parkin in immunity has remained unexplored. Here we show that parkin has a role in ubiquitin-mediated autophagy of M. tuberculosis. Both parkin-deficient mice and flies are sensitive to various intracellular bacterial infections, indicating parkin has a conserved role in metazoan innate defence. Moreover, our work reveals an unexpected functional link between mitophagy and infectious disease.
Intestinal homeostasis is achieved, in part, by the integration of a complex set of mechanisms that eliminate pathogens and tolerate the indigenous microbiota. Drosophila melanogaster feeds on microorganism-enriched matter and therefore has developed efficient mechanisms to control ingested microorganisms. Regulatory mechanisms ensure an appropriate level of immune reactivity in the gut to accommodate the presence of beneficial and dietary microorganisms, while allowing effective immune responses to clear pathogens. Maintenance of D. melanogaster gut homeostasis also involves regeneration of the intestine to repair damage associated with infection. Entomopathogenic bacteria have developed common strategies to subvert these defence mechanisms and kill their host.
Bunyaviruses are an emerging group of medically important viruses, many of which are transmitted from insects to mammals. To identify host factors that impact infection, we performed a genome-wide RNAi screen in Drosophila and identified 131 genes that impacted infection of the mosquito-transmitted bunyavirus Rift Valley fever virus (RVFV). Dcp2, the catalytic component of the mRNA decapping machinery, and two decapping activators, DDX6 and LSM7, were antiviral against disparate bunyaviruses in both insect cells and adult flies. Bunyaviruses 5' cap their mRNAs by "cap-snatching" the 5' ends of poorly defined host mRNAs. We found that RVFV cap-snatches the 5' ends of Dcp2 targeted mRNAs, including cell cycle-related genes. Loss of Dcp2 allows increased viral transcription without impacting viral mRNA stability, while ectopic expression of Dcp2 impedes viral transcription. Furthermore, arresting cells in late S/early G2 led to increased Dcp2 mRNA targets and increased RVFV replication. Therefore, RVFV competes for the Dcp2-accessible mRNA pool, which is dynamically regulated and can present a bottleneck for viral replication.
Throughout the animal kingdom, steroid hormones have been implicated in the defense against microbial infection, but how these systemic signals control immunity is unclear. Here, we show that the steroid hormone ecdysone controls the expression of the pattern recognition receptor PGRP-LC in Drosophila, thereby tightly regulating innate immune recognition and defense against bacterial infection. We identify a group of steroid-regulated transcription factors as well as two GATA transcription factors that act as repressors and activators of the immune response and are required for the proper hormonal control of PGRP-LC expression. Together, our results demonstrate that Drosophila use complex mechanisms to modulate innate immune responses, and identify a transcriptional hierarchy that integrates steroid signalling and immunity in animals.
Although the gut is a central organ of Eumetazoans and is essential for organismal health, our understanding of its morphological and molecular determinants remains rudimentary. Here, we provide a comprehensive atlas of Drosophila adult midgut. Specifically, we uncover a fine-grained regional organization consisting of 14 subregions with distinct morphological, histological, and genetic properties. We also show that Drosophila intestinal regionalization is defined after adult emergence, remains stable throughout life, and reestablishes following acute tissue damage. Additionally, we show that this midgut compartmentalization is achieved through the interplay between pan-midgut and regionalized transcription factors, in concert with spatial activities of morphogens. Interestingly, disruption of the midgut compartmentalization leads to a loss of intestinal homeostasis characterized by an increase in stem cell proliferation and aberrant immune responses. Our integrative analysis of Drosophila midgut compartmentalization provides insights into the conserved mechanisms underlying intestinal regionalization in metazoans.
Significance
Infection triggers the innate immune response in all metazoans, activating regulatory pathways that result in expression of effector proteins, including potent antimicrobial peptides. These pathways can also be activated in the brain by aging, stress, and injury. Although nominally protective, excessive neuroinflammatory responses may themselves contribute to neurodegenerative disease by mechanisms that remain unclear. We found that hyperactivation of innate immunity in the Drosophila brain as a result of mutation or bacterial injection causes neurodegeneration because of neurotoxic effects of antimicrobial peptides. These findings have important implications for the role of neuroinflammation in human neurodegenerative disease.
The fruit fly Drosophila has contributed significantly to our general understanding of the basic principles of signaling, cell and developmental biology, and neurobiology. However, answers to questions pertaining to energy metabolism have been so far mostly addressed in more complex model organisms such as mice. We review in this article recent studies that show how the genetic tractability and simplicity of Drosophila are being used to identify novel regulatory mechanisms at the organismal level, and to query the co-ordination between energy metabolism and other processes such as neurodegeneration, circadian rhythms, immunity, and tumor biology.
How persistent viral infections are established and maintained is widely debated and remains poorly understood. We found here that the persistence of RNA viruses in Drosophila melanogaster was achieved through the combined action of cellular reverse-transcriptase activity and the RNA-mediated interference (RNAi) pathway. Fragments of diverse RNA viruses were reverse-transcribed early during infection, which resulted in DNA forms embedded in retrotransposon sequences. Those virus-retrotransposon DNA chimeras produced transcripts processed by the RNAi machinery, which in turn inhibited viral replication. Conversely, inhibition of reverse transcription hindered the appearance of chimeric DNA and prevented persistence. Our results identify a cooperative function for retrotransposons and antiviral RNAi in the control of lethal acute infection for the establishment of viral persistence.
RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs), which are liberated from double-stranded (ds)RNA precursors by Dicer and guide the RNA-induced silencing complex (RISC) to targets. Although principles governing small RNA sorting into RISC have been uncovered, the spectrum of RNA species that can be targeted by Dicer proteins, particularly the viral RNAs present during an infection, are poorly understood. Dicer-2 potently restricts viral infection in insects by generating virus-derived siRNAs from viral RNA. To better characterize the substrates of Dicer-2, we examined the virus-derived siRNAs produced during the Drosophila antiviral RNAi response to four different viruses using high-throughput sequencing. We found that each virus was uniquely targeted by the RNAi pathway; dicing substrates included dsRNA replication intermediates and intramolecular RNA stem loops. For instance, a putative intergenic RNA hairpin encoded by Rift Valley Fever virus generates abundant small RNAs in both Drosophila and mosquito cells, while repetitive sequences within the genomic termini of Vaccinia virus, which give rise to abundant small RNAs in Drosophila, were found to be transcribed in both insect and mammalian cells. Moreover, we provide evidence that the RNA species targeted by Dicer-2 can be modulated by the presence of a viral suppressor of RNAi. This study uncovered several novel, heavily targeted features within viral genomes, offering insight into viral replication, viral immune evasion strategies, and the mechanism of antiviral RNAi.
A crucial early wound response is the recruitment of inflammatory cells drawn by danger cues released by the damaged tissue. Hydrogen peroxide (H(2)O(2)) has recently been identified as the earliest wound attractant in Drosophila embryos and zebrafish larvae [1, 2]. The H(2)O(2) signal is generated by activation of an NADPH oxidase, DUOX, and as a consequence, the first inflammatory cells are recruited to the wound within minutes. To date, nothing is known about how wounding activates DUOX. Here, we show that laser wounding of the Drosophila embryo epidermis triggers an instantaneous calcium flash, which travels as a wave via gap junctions several cell rows back from the wound edge. Blocking this calcium flash inhibits H(2)O(2) release at the wound site and leads to a reduction in the number of immune cells migrating to the wound. We suggest that the wound-induced calcium flash activates DUOX via an EF hand calcium-binding motif and thus triggers the production of the attractant damage cue H(2)O(2). Therefore, calcium represents the earliest signal in the wound inflammatory response.
Inflammation is an important component of normal responses to infection and injury. However, chronic activation of the immune system, due to aberrant responses to normal stimuli, can lead to the establishment of a persistent inflammatory state. Such inflammatory conditions are often debilitating, and are associated with a number of important co-morbidities including cardiovascular disease. Resting non-proliferative tissues have distinctive metabolic activities and requirements, which differ considerably from those in infiltrating immune cells, which are undergoing proliferation and differentiation. Immune responses in tissues may therefore be modulated by the relative abundance of substrates in the inflamed site. In turn immune cell activity can feed back and affect metabolic behaviour of the tissues, as most clearly demonstrated in cachexia – the loss of cellular mass driven by tumour necrosis factor-alpha (TNF-α) a key mediator of the inflammatory response. Here we discuss the potential for metabolomic analysis to clarify the interactions between inflammation and metabolic changes underlying many diseases. We suggest that an increased understanding of the interaction between inflammation and cellular metabolism, energy substrate use, tissue breakdown markers, the microbiome and drug metabolites, may provide novel insight into the regulation of inflammatory diseases.
Immunity and metabolism are intimately linked; manipulating metabolism, either through diet or genetics, has the power to alter survival during infection. However, despite metabolism's powerful ability to alter the course of infections, little is known about what being "sick" means metabolically. Here we describe the metabolic changes occurring in a model system when Listeria monocytogenes causes a lethal infection in Drosophila melanogaster. L. monocytogenes infection alters energy metabolism; the flies gradually lose both of their energy stores, triglycerides and glycogen, and show decreases in both intermediate metabolites and enzyme message for the two main energy pathways, beta-oxidation and glycolysis. L. monocytogenes infection also causes enzymatic reduction in the anti-oxidant uric acid, and knocking out the enzyme uric oxidase has a complicated effect on immunity. Free amino acid levels also change during infection, including a drop in tyrosine levels which may be due to robust L. monocytogenes induced melanization.
The fruit fly Drosophila melanogaster is a good model to unravel the molecular mechanisms of innate immunity and has led to some important discoveries about the sensing and signaling of microbial infections. The response of Drosophila to virus infections remains poorly characterized and appears to involve two facets. On the one hand, RNA interference involves the recognition and processing of dsRNA into small interfering RNAs by the host RNase Dicer-2 (Dcr-2), whereas, on the other hand, an inducible response controlled by the evolutionarily conserved JAK-STAT pathway contributes to the antiviral host defense. To clarify the contribution of the small interfering RNA and JAK-STAT pathways to the control of viral infections, we have compared the resistance of flies wild-type and mutant for Dcr-2 or the JAK kinase Hopscotch to infections by seven RNA or DNA viruses belonging to different families. Our results reveal a unique susceptibility of hop mutant flies to infection by Drosophila C virus and cricket paralysis virus, two members of the Dicistroviridae family, which contrasts with the susceptibility of Dcr-2 mutant flies to many viruses, including the DNA virus invertebrate iridescent virus 6. Genome-wide microarray analysis confirmed that different sets of genes were induced following infection by Drosophila C virus or by two unrelated RNA viruses, Flock House virus and Sindbis virus. Overall, our data reveal that RNA interference is an efficient antiviral mechanism, operating against a large range of viruses, including a DNA virus. By contrast, the antiviral contribution of the JAK-STAT pathway appears to be virus specific.
DEAD-box helicases play essential roles in RNA metabolism across species, but emerging data suggest that they have additional functions in immunity. Through RNAi screening, we identify an evolutionarily conserved and interferon-independent role for the DEAD-box helicase DDX17 in restricting Rift Valley fever virus (RVFV), a mosquito-transmitted virus in the bunyavirus family that causes severe morbidity and mortality in humans and livestock. Loss of Drosophila DDX17 (Rm62) in cells and flies enhanced RVFV infection. Similarly, depletion of DDX17 but not the related helicase DDX5 increased RVFV replication in human cells. Using crosslinking immunoprecipitation high-throughput sequencing (CLIP-seq), we show that DDX17 binds the stem loops of host pri-miRNA to facilitate their processing and also an essential stem loop in bunyaviral RNA to restrict infection. Thus, DDX17 has dual roles in the recognition of stem loops: in the nucleus for endogenous microRNA (miRNA) biogenesis and in the cytoplasm for surveillance against structured non-self-elements.
The use of fruit flies has recently emerged as a powerful experimental paradigm to study the core aspects of energy metabolism. The fundamental need for lipid and carbohydrate processing and storage across species dictates that the central regulators that control metabolism are highly conserved through evolution. Accordingly, the Drosophila system is being used to identify human disease genes and has the potential to model successfully human disorders that center on excessive caloric intake and metabolic dysfunction, including diet-induced lipotoxicity and type 2 diabetes. We review here recent progress on this front and contend that increasing such efforts will yield unexpectedly high rates of experimental return, thereby leading to novel approaches in the treatment of obesity and its comorbidities.
Colorectal cancer (CRC) presents a considerable disease burden worldwide. The human colon is also an anatomical location with the largest number of microbes. It is natural, therefore, to anticipate a role for microbes, particularly bacteria, in colorectal carcinogenesis. The increasing accessibility of microbial meta'omics is fueling a surge in our understanding of the role that microbes and the microbiota play in CRC. In this review, we will discuss recent insights into contributions of the microbiota to CRC and explore conceptual frameworks for evaluating the role of microbes in cancer causation. We also highlight new findings on candidate CRC-potentiating species and current knowledge gaps. Finally, we explore the roles of microbial metabolism as it relates to bile acids, xenobiotics, and diet in the etiology and therapeutics of CRC.
Antibiotic-associated infection with the bacterial pathogen Clostridium difficile is a major cause of morbidity and increased healthcare costs. C difficile infection (CDI) follows disruption of the indigenous gut microbiota by antibiotics. Antibiotics create an environment within the intestine that promotes C difficile spore germination, vegetative growth, and toxin production, leading to epithelial damage and colitis. Studies of patients with CDI and animal models have shown that the indigenous microbiota can inhibit expansion and persistence of C difficile. Although the specific mechanisms of these processes are not known, they are likely to interfere with key aspects of the pathogen’s physiology, including spore germination and competitive growth. Increasing our understanding of how the intestinal microbiota manage C difficile could lead to better means of controlling this important nosocomial pathogen.
Interactions between commensals and the host impact the metabolic and immune status of metazoans. Their deregulation is associated with age-related pathologies like chronic inflammation and cancer, especially in barrier epithelia. Maintaining a healthy commensal population by preserving innate immune homeostasis in such epithelia thus promises to promote health and longevity. Here, we show that, in the aging intestine of Drosophila, chronic activation of the transcription factor Foxo reduces expression of peptidoglycan recognition protein SC2 (PGRP-SC2), a negative regulator of IMD/Relish innate immune signaling, and homolog of the anti-inflammatory molecules PGLYRP1-4. This repression causes deregulation of Rel/NFkB activity, resulting in commensal dysbiosis, stem cell hyperproliferation, and epithelial dysplasia. Restoring PGRP-SC2 expression in enterocytes of the intestinal epithelium, in turn, prevents dysbiosis, promotes tissue homeostasis, and extends lifespan. Our results highlight the importance of commensal control for lifespan of metazoans and identify SC-class PGRPs as longevity-promoting factors.
Autophagy has been implicated as a component of host defense, but the significance of antimicrobial autophagy in vivo and the mechanism by which it is regulated during infection are poorly defined. Here we found that antiviral autophagy was conserved in flies and mammals during infection with Rift Valley fever virus (RVFV), a mosquito-borne virus that causes disease in humans and livestock. In Drosophila, Toll-7 limited RVFV replication and mortality through activation of autophagy. RVFV infection also elicited autophagy in mouse and human cells, and viral replication was increased in the absence of autophagy genes. The mammalian Toll-like receptor adaptor, MyD88, was required for anti-RVFV autophagy, revealing an evolutionarily conserved requirement for pattern-recognition receptors in antiviral autophagy. Pharmacologic activation of autophagy inhibited RVFV infection in mammalian cells, including primary hepatocytes and neurons. Thus, autophagy modulation might be an effective strategy for treating RVFV infection, which lacks approved vaccines and therapeutics.
While RNA interference (RNAi) functions as an antiviral response in plants, nematodes, and arthropods, a similar antiviral role in mammals has remained controversial. Three recent papers provide evidence that either favors or challenges this hypothesis. Here, we discuss these new findings in the context of previous research.
Significance
A unique facet of arthropod-borne virus infection is that the pathogens are orally acquired by insects during the taking of a blood meal. Hence, there is a direct link between nutrient acquisition and pathogen challenge. We found that the classic nutrient-responsive ERK pathway is a molecular determinant of this “midgut barrier”; ERK signaling is essential for antiviral defense in the insect intestinal epithelial cells. Surprisingly, we also found vertebrate insulin, which activates ERK signaling during a blood meal, both restricts viral infection in insect cells and protects against viral invasion of the gut epithelium. ERK signaling in the insect intestines restricts viral infection, suggesting that insects may take advantage of cross-species signals in the meal to preemptively activate antiviral immunity.
During the last century, insect model systems have provided fascinating insights into the endocrinology and developmental biology of all animals. During the insect life cycle, molts and metamorphosis delineate transitions from one developmental stage to the next. In most insects, pulses of the steroid hormone ecdysone drive these developmental transitions by activating signaling cascades in target tissues. In holometabolous insects, ecdysone triggers metamorphosis, the remarkable remodeling of an immature larva into a sexually mature adult. The input from another developmental hormone, juvenile hormone (JH), is required to repress metamorphosis by promoting juvenile fates until the larva has acquired sufficient nutrients to survive metamorphosis. Ecdysone and JH act together as key endocrine timers to precisely control the onset of developmental transitions such as the molts, pupation, or eclosion. In this review, we will focus on the role of the endocrine system and the circadian clock, both individually and together, in temporally regulating insect development. Since this is not a coherent field, we will review recent developments that serve as examples to illuminate this complex topic. First, we will consider studies conducted in Rhodnius that revealed how circadian pathways exert temporal control over the production and release of ecdysone. We will then take a look at molecular and genetic data that revealed the presence of two circadian clocks, located in the brain and the prothoracic gland, that regulate eclosion rhythms in Drosophila. In this context, we will also review recent developments that examined how the ecdysone hierarchy delays the differentiation of the crustacean cardioactive peptide (CCAP) neurons, an event that is critical for the timing of ecdysis and eclosion. Finally, we will discuss some recent findings that transformed our understanding of JH function.
The capa peptide family exists in a very wide range of insects including species of medical, veterinary and agricultural importance. Capa peptides act via a cognate G-protein coupled receptor (capaR) and have a diuretic action on the Malpighian tubules of Dipteran and Lepidopteran species. Capa signaling is critical for fluid homeostasis and has been associated with desiccation tolerance in the fly, D. melanogaster. The mode of capa signaling is highly complex, affecting calcium, nitric oxide and cyclic GMP pathways. Such complex physiological regulation by cell signaling pathways may occur ultimately for optimal organismal stress tolerance to multiple stressors. Here we show that D. melanogaster capa-1 (Drome-capa-1) acts via the Nuclear Factor kappa B (NF-kB) stress signaling network. Human PCR gene arrays of capaR-transfected Human Embryonic Kidney (HEK) 293 cells showed that Drome-capa-1 increases expression of NF-kB, NF-kB regulated genes including IL8, TNF and PTGS2, and NF-kB pathway-associated transcription factors i.e EGR1, FOS, cJUN. Furthermore, desiccated HEK293 cells show increased EGR1, EGR3 and PTGS2 - but not IL8 - expression. CapaR-transfected NF-kB reporter cells showed that Drome-capa-1 increased NF-kB promoter activity via increased calcium. In Malpighian tubules, both Drome-capa-1 stimulation and desiccation result in increased gene expression of the D. melanogaster NF-kB orthologue, Relish; as well as EGR-like stripe and klumpfuss. Drome-capa-1 also induces Relish translocation in tubule principal cells. Targeted knockdown of Relish in only tubule principal cells reduces desiccation stress tolerance of adult flies. Together, these data suggest that Drome-capa-1 acts in desiccation stress tolerance, by activating NF-kB signaling.
Dual oxidases (DUOX) are conserved NADPH oxidases that produce H2O2 at the epithelial cell surface. The DUOX enzyme comprises the DUOX and DUOXA (DUOX maturation factor) subunits. Mammalian genomes encode 2 DUOX isoenzymes (DUOX1-DUOXA1 and DUOX2-DUOXA2). Expression of these genes is upregulated during bacterial infection and chronic inflammatory diseases of the luminal gastrointestinal tract. The roles of DUOX in cellular interactions with microbes have not been determined in higher vertebrates.
Mice with disruptions of Duoxa1 and Duoxa2 genes (Duoxa(-/-) mice) and control mice were infected with Helicobacter felis to create a model of Helicobacter pylori infection-the most common human chronic infection.
Infection with H felis induced expression of Duox2 and Duoxa2 in the stomachs of wild-type mice, and DUOX protein specifically localized to the apical surface of epithelial cells. H felis colonized the mucus layer in the stomachs of Duoxa(-/-) mice to a greater extent than in control mice. The increased colonization persisted into the chronic phase of infection and correlated with an increased, yet ineffective, inflammatory response. H felis colonization was also increased in Duoxa(+/-) mice, compared with controls. We observed reduced expression of the H2O2-inducible katA gene in H felis that colonized Duoxa(-/-) mice, compared with that found in controls (P=.0002), indicating that Duox causes oxidative stress in these bacteria. In vitro, induction of oxidative defense by H felis failed to prevent a direct bacteriostatic effect, at sustained levels of H2O2 as low as 30 μM.
Based on studies of Duoxa(-/-) mice, the DUOX enzyme complex prevents gastric colonization by H felis and the inflammatory response. These findings indicate the non-redundant function of epithelial production of H2O2 in restricting microbial colonization.
Detection of pathogen-derived nucleic acids by pattern recognition receptors (PRRs) is essential for the host to mount an appropriate immune response, which for viruses involves the induction of type I interferons (IFNs). By contrast, inappropriate activation of PRRs by self nucleic acids can lead to autoimmunity. Recent developments in PRR research have uncovered important new molecular details as to how Toll-like receptors (TLRs) and retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) distinguish pathogen from self RNA, while the discovery of cytosolic DNA sensing pathways for IFN induction has revealed completely new innate signaling mechanisms, and also questions how innate immunity discriminates between self and non-self DNA, if at all.
Viral pathogens present many challenges to organisms, driving the evolution of a myriad of antiviral strategies to combat infections. A wide variety of viruses infect invertebrates, including both natural pathogens that are insect-restricted, and viruses that are transmitted to vertebrates. Studies using the powerful tools available in the model organism Drosophila have expanded our understanding of antiviral defenses against diverse viruses. In this review, we will cover three major areas. First, we will describe the tools used to study viruses in Drosophila. Second, we will survey the major viruses that have been studied in Drosophila. And lastly, we will discuss the well-characterized mechanisms that are active against these diverse pathogens, focusing on non-RNAi mediated antiviral mechanisms. Antiviral RNAi is discussed in another paper in this issue.
All metazoan guts are subjected to immunologically unique conditions in which an efficient antimicrobial system operates to eliminate pathogens while tolerating symbiotic commensal microbiota. However, the molecular mechanisms controlling this process are only partially understood. Here, we show that bacterial-derived uracil acts as a ligand for dual oxidase (DUOX)-dependent reactive oxygen species generation in Drosophila gut and that the uracil production in bacteria causes inflammation in the gut. The acute and controlled uracil-induced immune response is required for efficient elimination of bacteria, intestinal cell repair, and host survival during infection of nonresident species. Among resident gut microbiota, uracil production is absent in symbionts, allowing harmonious colonization without DUOX activation, whereas uracil release from opportunistic pathobionts provokes chronic inflammation. These results reveal that bacteria with distinct abilities to activate uracil-induced gut inflammation, in terms of intensity and duration, act as critical factors that determine homeostasis or pathogenesis in gut-microbe interactions.
Neurodegeneration is a hallmark of the human disease Ataxia-telangiectasia (A-T) that is caused by mutation of the A-T mutated (ATM) gene. We have analyzed Drosophila melanogaster ATM mutants to determine the molecular mechanisms underlying neurodegeneration in A-T. Previously, we found that ATM mutants upregulate the expression of innate immune response (IIR) genes and undergo neurodegeneration in the central nervous system. Here, we present evidence that activation of the IIR is a cause of neurodegeneration in ATM mutants. Three lines of evidence indicate that ATM mutations cause neurodegeneration by activating the NF-κB transcription factor Relish, a key regulator of the Imd IIR signaling pathway. First, the level of upregulation of IIR genes, including Relish target genes, was directly correlated with the level of neurodegeneration in ATM mutants. Second, Relish mutations inhibited upregulation of IIR genes and neurodegeneration in ATM mutants. Third, overexpression of constitutively active Relish in glial cells activated the IIR and caused neurodegeneration. In contrast, we found that Imd and Dif mutations did not affect neurodegeneration in ATM mutants. Imd encodes an activator of Relish in the response to gram-negative bacteria, and Dif encodes an immune responsive NF-κB transcription factor in the Toll signaling pathway. These data indicate that the signal that causes neurodegeneration in ATM mutants activates a specific NF-κB protein and does so through an unknown activator. In summary, these findings suggest that neurodegeneration in human A-T is caused by activation of a specific NF-κB protein in glial cells.