Extracellular matrix protein N-glycosylation mediates immune self-tolerance in Drosophila melanogaster.

In order to respond to infection, hosts must distinguish pathogens from their own tissues. This allows for the precise targeting of immune responses against pathogens and also ensures self-tolerance, the ability of the host to protect self tissues from immune damage. One way to maintain self-tolerance is to evolve a self signal and suppress any immune response directed at tissues that carry this signal. Here, we characterize the Drosophila tuSz1 mutant strain, which mounts an aberrant immune response against its own fat body. We demonstrate that this autoimmunity is the result of two mutations: 1) a mutation in the GCS1 gene that disrupts N-glycosylation of extracellular matrix proteins covering the fat body, and 2) a mutation in the Drosophila Janus Kinase ortholog that causes precocious activation of hemocytes. Our data indicate that N-glycans attached to extracellular matrix proteins serve as a self signal and that activated hemocytes attack tissues lacking this signal. The simplicity of this invertebrate self-recognition system and the ubiquity of its constituent parts suggests it may have functional homologs across animals.

The Nutritional Environment Influences the Impact of Microbes on Drosophila melanogaster Life Span.

Microbes can extend Drosophila melanogaster life span by contributing to the nutritional value of malnourishing fly culture medium. The beneficial effect of microbes during malnutrition is dependent on their individual ability to proliferate in the fly environment and is mimicked by lifelong supplementation of equivalent levels of heat-killed microbes or dietary protein, suggesting that microbes can serve directly as a protein-rich food source. Here, we use nutritionally rich fly culture medium to demonstrate how changes in dietary composition influence monocolonized fly life span; microbes that extend fly life span on malnourishing diets can shorten life on rich diets. The mechanisms employed by microbes to affect host health likely differ on low- or high-nutrient diets. Our results demonstrate how Drosophila-associated microbes can positively or negatively influence fly life span depending on the nutritional environment. Although controlled laboratory environments allow focused investigations on the interaction between fly microbiota and nutrition, the relevance of these studies is not straightforward, because it is difficult to mimic the nutritional ecology of natural Drosophila-microbe interactions. As such, caution is needed in designing and interpreting fly-microbe experiments and before categorizing microbes into specific symbiotic roles based on results obtained from experiments testing limited conditions.IMPORTANCED. melanogaster ingests microorganisms growing within its rotting vegetation diet. Some of these microbes form associations with flies, while others pass through the gut with meals. Fly-microbe-diet interactions are dynamic, and changes to the fly culture medium can influence microbial growth in the overall environment. In turn, these alterations in microbial growth may not only impact the nutritional value of fly meals but also modulate behavior and health, at least in part due to direct contributions to fly nutrition. The interactive ecology between flies, microbes, and their environment can cause a specific microbe to be either beneficial or detrimental to fly life span, indicating that the environment should be considered a key influential factor in host-microbe interactions.

Microbial Quantity Impacts Drosophila Nutrition, Development, and Lifespan.

In Drosophila, microbial association can promote development or extend life. We tested the impact of microbial association during malnutrition and show that microbial quantity is a predictor of fly longevity. Although all tested microbes, when abundantly provided, can rescue lifespan on low-protein diet, the effect of a single inoculation seems linked to the ability of that microbial strain to thrive under experimental conditions. Microbes, dead or alive, phenocopy dietary protein, and the calculated dependence on microbial protein content is similar to the protein requirements determined from fly feeding studies, suggesting that microbes enhance host protein nutrition by serving as protein-rich food. Microbes that enhance larval growth are also associated with the ability to better thrive on fly culture medium. Our results suggest an unanticipated range of microbial species that promote fly development and longevity and highlight microbial quantity as an important determinant of effects on physiology and lifespan during undernutrition.

Evolutionary responses of Drosophila melanogaster under chronic malnutrition.

Drosophila species have successfully spread and adapted to diverse climates across the globe. For D. melanogaster, rotting vegetative matter provides the primary substrate for mating and oviposition, and also acts as a nutritional resource for developing larvae and adult flies. The transitory nature of decaying vegetation exposes D. melanogaster to rapidly changing nutrient availability. As evidenced by their successful global spread, flies are capable of dealing with fluctuating nutritional reserves within their respective ecological niches. Therefore, D. melanogaster populations might contain standing genetic variation to support survival during periods of nutrient scarcity. The natural history and genetic tractability of D. melanogaster make the fly an ideal model for studies on the genetic basis of resistance to nutritional stress. We review artificial selection studies on nutritionally-deprived D. melanogaster and summarize the phenotypic outcomes of selected animals. Many of the reported evolved traits phenocopy mutants of the nutrient-sensing PI3K/Akt pathway. Given that the PI3K/Akt pathway is also responsive to acute nutritional stress, the PI3K/Akt pathway might underlie traits evolved under chronic nutritional deprivation. Future studies that directly test for the genetic mechanisms driving evolutionary responses to nutritional stress will take advantage of the ease in manipulating fly nutrient availability in the laboratory.

Nutrition Influences Caffeine-Mediated Sleep Loss in Drosophila.

Study objectives: Plant-derived caffeine is regarded as a defensive compound produced to prevent herbivory. Caffeine is generally repellent to insects and often used to study the neurological basis for aversive responses in the model insect, Drosophila melanogaster. Caffeine is also studied for its stimulatory properties where sleep or drowsiness is suppressed across a range of species. Since limiting access to food also inhibits fly sleep-an effect known as starvation-induced sleep suppression-we tested whether aversion to caffeinated food results in reduced nutrient intake and assessed how this might influence fly studies on the stimulatory effects of caffeine. Methods: We measured sleep and total consumption during the first 24 hours of exposure to caffeinated diets containing a range of sucrose concentrations to determine the relative influence of caffeine and nutrient ingestion on sleep. Experiments were replicated using three fly strains. Results: Caffeine reduced total consumption and nighttime sleep, but only at intermediate sucrose concentrations. Although sleep can be modeled by an exponential dose response to nutrient intake, caffeine-mediated sleep loss cannot be explained by absolute caffeine or sucrose ingestion alone. Instead, reduced sleep strongly correlates with changes in total consumption due to caffeine. Other bitter compounds phenocopy the effect of caffeine on sleep and food intake. Conclusions: Our results suggest that a major effect of dietary caffeine is on fly feeding behavior. Changes in feeding behavior may drive caffeine-mediated sleep loss. Future studies using psychoactive compounds should consider the potential impact of nutrition when investigating effects on sleep.

Breaking Down Walls: Microbiota and the Aging Gut.

A range of animal species show increased inflammation with age. In this issue of Cell Host & Microbe, Thevaranjan et al. (2017) reveal that heightened inflammation is associated with deregulation of homeostatic interactions between intestinal microbes and the aging host.

Mifepristone Reduces Food Palatability and Affects Drosophila Feeding and Lifespan.

The Drosophila GeneSwitch system facilitates the spatial and temporal control of gene expression through dietary supplementation of mifepristone (RU486). Because experimental and control groups differ only by treatment with RU486, confounding results from using flies of different genetic backgrounds are eliminated, making GeneSwitch especially useful in studies of aging. However, the effect of RU486 itself on longevity has not been well characterized, particularly in relation to nutritional states known to affect lifespan. Here, we show that RU486 has dose- and diet-dependent effects on longevity in both sexes. On low nutrient diets, RU486 supplementation reduces total food consumption, perhaps exacerbating undernutrition to shorten life. RU486 also inhibits proboscis extension responses to low nutrient diets, suggesting that RU486 has an aversive taste which leads to decreased food consumption and diminished longevity. RU486 is not detrimental to fly lifespan on high nutrient food, correlating with reduced effects of the drug on palatability and total consumption on rich diets. Our results highlight the critical importance of considering how food palatability and nutrient intake might be altered by dietary or drug manipulations in studies of aging and behavior.

Microbes without Borders: Decompartmentalization of the Aging Gut.

The microbiota supports intestinal homeostasis in developing animals. With increased age, gut maintenance declines and microbes can stray from traditional zones, negatively impacting host health. In this issue of Cell Host & Microbe, Li et al. (2016) detail the mechanisms leading to the decline in intestinal health in aged flies.

Lactobacilli Modulate Epithelial Cytoprotection through the Nrf2 Pathway.

An optimal gut microbiota influences many beneficial processes in the metazoan host. However, the molecular mechanisms that mediate and function in symbiont-induced host responses have not yet been fully characterized. Here, we report that cellular ROS enzymatically generated in response to contact with lactobacilli in both mice and Drosophila has salutary effects against exogenous insults to the intestinal epithelium via the activation of Nrf2 responsive cytoprotective genes. These data show that the xenobiotic-inducible Nrf2 pathway participates as a signaling conduit between the prokaryotic symbiont and the eukaryotic host. Indeed, our data imply that the capacity of lactobacilli to induce redox signaling in epithelial cells is a highly conserved hormetic adaptation to impel cellular conditioning to exogenous biotic stimuli. These data also highlight the role the microbiota plays in eukaryotic cytoprotective pathways and may have significant implications in the characterization of a eubiotic microbiota.

EVOLUTION. Fruit flies diversify their offspring in response to parasite infection.

The evolution of sexual reproduction is often explained by Red Queen dynamics: Organisms must continually evolve to maintain fitness relative to interacting organisms, such as parasites. Recombination accompanies sexual reproduction and helps diversify an organism's offspring, so that parasites cannot exploit static host genotypes. Here we show that Drosophila melanogaster plastically increases the production of recombinant offspring after infection. The response is consistent across genetic backgrounds, developmental stages, and parasite types but is not induced after sterile wounding. Furthermore, the response appears to be driven by transmission distortion rather than increased recombination. Our study extends the Red Queen model to include the increased production of recombinant offspring and uncovers a remarkable ability of hosts to actively distort their recombination fraction in rapid response to environmental cues.

Insights from natural host-parasite interactions: the Drosophila model.

Immune responses against opportunistic pathogens have been extensively studied in Drosophila, leading to a detailed map of the genetics behind innate immunity networks including the Toll, Imd, Jak-Stat, and JNK pathways. However, immune mechanisms of other organisms, such as plants, have primarily been investigated using natural pathogens. It was the use of natural pathogens in plant research that revealed the plant R-Avr system, a specialized immune response derived from antagonistic coevolution between plant immune proteins and their natural pathogens' virulence proteins. Thus, we recommend that researchers begin to use natural Drosophila pathogens to identify novel immune strategies that may have arisen through antagonistic coevolution with common natural pathogens. In this review, we address the benefits of using natural pathogens in research, describe the known natural pathogens of Drosophila, and discuss the future prospects for research on natural pathogens of Drosophila.

Mgat1-dependent N-glycosylation of membrane components primes Drosophila melanogaster blood cells for the cellular encapsulation response.

In nature, larvae of the fruitfly Drosophila melanogaster are commonly infected by parasitoid wasps, and so have evolved a robust immune response to counter wasp infection. In this response, fly immune cells form a multilayered capsule surrounding the wasp egg, leading to death of the parasite. Many of the molecular mechanisms underlying this encapsulation response are conserved with human immune responses. Our findings suggest that protein N-glycosylation, a common protein post-translational modification of human immune proteins, may be one such conserved mechanism. We found that membrane proteins on Drosophila immune cells are N-glycosylated in a temporally specific manner following wasp infection. Furthermore we have identified mutations in eight genes encoding enzymes of the N-glycosylation pathway that decrease fly resistance to wasp infection. More specifically, loss of protein N-glycosylation in immune cells following wasp infection led to the formation of defective capsules, which disintegrated over time and were thereby unsuccessful at preventing wasp development. Interestingly, we also found that one species of Drosophila parasitoid wasp, Leptopilina victoriae, targets protein N-glycosylation as part of its virulence mechanism, and that overexpression of an N-glycosylation enzyme could confer resistance against this wasp species to otherwise susceptible flies. Taken together, these findings demonstrate that protein N-glycosylation is a key player in Drosophila cellular encapsulation and suggest that this response may provide a novel model to study conserved roles of protein glycosylation in immunity.

Adaptive evolution of a novel Drosophila lectin induced by parasitic wasp attack.

Drosophila melanogaster has long been used as a model for the molecular genetics of innate immunity. Such work has uncovered several immune receptors that recognize bacterial and fungal pathogens by binding unique components of their cell walls and membranes. Drosophila also act as hosts to metazoan pathogens such as parasitic wasps, which can infect a majority of individuals in natural populations, but many aspects of their immune responses against these more closely related pathogens are poorly understood. Here, we present data describing the transcriptional induction and molecular evolution of a candidate Drosophila anti-wasp immunity gene, lectin-24A. Lectin-24A has a secretion signal sequence and its lectin domain suggests a function in sugar group binding. Transcript levels of lectin-24A were induced significantly stronger and faster following wasp attack than following wounding or bacterial infection, demonstrating lectin-24A is not a general stress response or defense response gene but is instead part of a specific response against wasps. The major site of lectin-24A transcript production is the fat body, the main humoral immune tissue of flies. Interestingly, lectin-24A is a new gene of the D. melanogaster/Drosophila simulans clade, displaying very little homology to any other Drosophila lectins. Population genetic analyses of lectin-24A DNA sequence data from African and North American populations of D. melanogaster and D. simulans revealed gene length polymorphisms segregating at high frequencies as well as strong evidence of repeated and recent selective sweeps. Thus, lectin-24A is a rapidly evolving new gene that has seemingly developed functional importance for fly resistance against infection by parasitic wasps.

Decreased mtDNA, oxidative stress, cardiomyopathy, and death from transgenic cardiac targeted human mutant polymerase gamma.

POLG is the human gene that encodes the catalytic subunit of DNA polymerase gamma (Pol gamma), the replicase for human mitochondrial DNA (mtDNA). A POLG Y955C point mutation causes human chronic progressive external ophthalmoplegia (CPEO), a mitochondrial disease with eye muscle weakness and mtDNA defects. Y955C POLG was targeted transgenically (TG) to the murine heart. Survival was determined in four TG (+/-) lines and wild-type (WT) littermates (-/-). Left ventricle (LV) performance (echocardiography and MRI), heart rate (electrocardiography), mtDNA abundance (real time PCR), oxidation of mtDNA (8-OHdG), histopathology and electron microscopy defined the phenotype. Cardiac targeted Y955C POLG yielded a molecular signature of CPEO in the heart with cardiomyopathy (CM), mitochondrial oxidative stress, and premature death. Increased LV cavity size and LV mass, bradycardia, decreased mtDNA, increased 8-OHdG, and cardiac histopathological and mitochondrial EM defects supported and defined the phenotype. This study underscores the pathogenetic role of human mutant POLG and its gene product in mtDNA depletion, mitochondrial oxidative stress, and CM as it relates to the genetic defect in CPEO. The transgenic model pathophysiologically links human mutant Pol gamma, mtDNA depletion, and mitochondrial oxidative stress to the mtDNA replication apparatus and to CM.