Gut-derived peptidoglycan remotely inhibits bacteria dependent activation of SREBP by Drosophila adipocytes.

Bacteria that colonize eukaryotic gut have profound influences on the physiology of their host. In Drosophila, many of these effects are mediated by adipocytes that combine immune and metabolic functions. We show here that enteric infection with some bacteria species triggers the activation of the SREBP lipogenic protein in surrounding enterocytes but also in remote fat body cells and in ovaries, an effect that requires insulin signaling. We demonstrate that by activating the NF-κB pathway, the cell wall peptidoglycan produced by the same gut bacteria remotely, and cell-autonomously, represses SREBP activation in adipocytes. We finally show that by reducing the level of peptidoglycan, the gut born PGRP-LB amidase balances host immune and metabolic responses of the fat body to gut-associated bacteria. In the absence of such modulation, uncontrolled immune pathway activation prevents SREBP activation and lipid production by the fat body.

Bacterial peptidoglycan serves as a critical modulator of the gut-immune-brain axis in Drosophila.

Metabolites and compounds derived from gut-associated bacteria can modulate numerous physiological processes in the host, including immunity and behavior. Using a model of oral bacterial infection, we previously demonstrated that gut-derived peptidoglycan (PGN), an essential constituent of the bacterial cell envelope, influences female fruit fly egg-laying behavior by activating the NF-κB cascade in a subset of brain neurons. These findings underscore PGN as a potential mediator of communication between gut bacteria and the brain in Drosophila, prompting further investigation into its impact on all brain cells. Through high-resolution mass spectrometry, we now show that PGN fragments produced by gut bacteria can rapidly reach the central nervous system. In Addition, by employing a combination of whole-genome transcriptome analyses, comprehensive genetic assays, and reporter gene systems, we reveal that gut bacterial infection triggers a PGN dose-dependent NF-κB immune response in perineurial glia, forming the continuous outer cell layer of the blood-brain barrier. Furthermore, we demonstrate that persistent PGN-dependent NF-κB activation in perineurial glial cells correlates with a reduction in lifespan and early neurological decline. Overall, our findings establish gut-derived PGN as a critical mediator of the gut-immune-brain axis in Drosophila.

Bacteria-Derived Peptidoglycan Triggers a Noncanonical Nuclear Factor-κB-Dependent Response in Drosophila Gustatory Neurons.

Probing the external world is essential for eukaryotes to distinguish beneficial from pathogenic micro-organisms. If it is clear that the main part of this task falls to the immune cells, recent work shows that neurons can also detect microbes, although the molecules and mechanisms involved are less characterized. In Drosophila, detection of bacteria-derived peptidoglycan by pattern recognition receptors of the peptidoglycan recognition protein (PGRP) family expressed in immune cells triggers nuclear factor-κB (NF-κB)/immune deficiency (IMD)-dependent signaling. We show here that one PGRP protein, called PGRP-LB, is expressed in bitter gustatory neurons of proboscises. In vivo calcium imaging in female flies reveals that the PGRP/IMD pathway is cell-autonomously required in these neurons to transduce the peptidoglycan signal. We finally show that NF-κB/IMD pathway activation in bitter-sensing gustatory neurons influences fly behavior. This demonstrates that a major immune response elicitor and signaling module are required in the peripheral nervous system to sense the presence of bacteria in the environment.SIGNIFICANCE STATEMENT In addition to the classical immune response, eukaryotes rely on neuronally controlled mechanisms to detect microbes and engage in adapted behaviors. However, the mechanisms of microbe detection by the nervous system are poorly understood. Using genetic analysis and calcium imaging, we demonstrate here that bacteria-derived peptidoglycan can activate bitter gustatory neurons. We further show that this response is mediated by the PGRP-LC membrane receptor and downstream components of a noncanonical NF-κB signaling cascade. Activation of this signaling cascade triggers behavior changes. These data demonstrate that bitter-sensing neurons and immune cells share a common detection and signaling module to either trigger the production of antibacterial effectors or to modulate the behavior of flies that are in contact with bacteria. Because peptidoglycan detection doesn't mobilize the known gustatory receptors, it also demonstrates that taste perception is much more complex than anticipated.

Maintaining immune homeostasis in fly gut.

Like every metazoan species hosting a gut flora, drosophila tolerate commensal microbiota yet remain able to mount an efficient immune response to food-borne pathogens. New findings explain how the quantity of reactive oxygen species in the gut is 'tuned' to microbial burden and how intestinal immune homeostasis is thereby maintained

Mitofusin gain and loss of function drive pathogenesis in Drosophila models of CMT2A neuropathy.

Charcot-Marie-Tooth disease type 2A (CMT2A) is caused by dominant alleles of the mitochondrial pro-fusion factor Mitofusin 2 (MFN2). To address the consequences of these mutations on mitofusin activity and neuronal function, we generate Drosophila models expressing in neurons the two most frequent substitutions (R94Q and R364W, the latter never studied before) and two others localizing to similar domains (T105M and L76P). All alleles trigger locomotor deficits associated with mitochondrial depletion at neuromuscular junctions, decreased oxidative metabolism and increased mtDNA mutations, but they differently alter mitochondrial morphology and organization. Substitutions near or within the GTPase domain (R94Q, T105M) result in loss of function and provoke aggregation of unfused mitochondria. In contrast, mutations within helix bundle 1 (R364W, L76P) enhance mitochondrial fusion, as demonstrated by the rescue of mitochondrial alterations and locomotor deficits by over-expression of the fission factor DRP1. In conclusion, we show that both dominant negative and dominant active forms of mitofusin can cause CMT2A-associated defects and propose for the first time that excessive mitochondrial fusion drives CMT2A pathogenesis in a large number of patients.

Inhibition of a NF-κB/Diap1 Pathway by PGRP-LF Is Required for Proper Apoptosis during Drosophila Development.

NF-κB pathways are key signaling cascades of the Drosophila innate immune response. One of them, the Immune Deficiency (IMD) pathway, is under a very tight negative control. Although molecular brakes exist at each step of this signaling module from ligand availability to transcriptional regulation, it remains unknown whether repressors act in the same cells or tissues and if not, what is rationale behind this spatial specificity. We show here that the negative regulator of IMD pathway PGRP-LF is epressed in ectodermal derivatives. We provide evidence that, in the absence of any immune elicitor, PGRP-LF loss-of-function mutants, display a constitutive NF-κB/IMD activation specifically in ectodermal tissues leading to genitalia and tergite malformations. In agreement with previous data showing that proper development of these structures requires induction of apoptosis, we show that ectopic activation of NF-κB/IMD signaling leads to apoptosis inhibition in both genitalia and tergite primordia. We demonstrate that NF-κB/IMD signaling antagonizes apoptosis by up-regulating expression of the anti-apoptotic protein Diap1. Altogether these results show that, in the complete absence of infection, the negative regulation of NF-κB/IMD pathway by PGRP-LF is crucial to ensure proper induction of apoptosis and consequently normal fly development. These results highlight that IMD pathway regulation is controlled independently in different tissues, probably reflecting the different roles of this signaling cascade in both developmental and immune processes.

Gut-microbiota interactions in non-mammals: what can we learn from Drosophila?

Millions of people suffer from inflammatory diseases of the intestine, some of them potentiating gastrointestinal cancer. These gut-associated pathologies arise from imbalanced interactions between the host gut epithelia and resident or ingested microbes, interactions that are still poorly understood at the molecular level. Drosophila has been a very powerful model to study development and diseases. Its relatively simple tissue organization and sophisticated genetics are some of the advantages of using it as an experimental model to dissect gut-microbe interactions. Recent progress made in various research fields such as Drosophila microbiota composition, gut epithelium structure or gut immune reactions led us to believe that Drosophila is becoming an ad hoc model system to dissect the mechanisms that cooperate to maintain intestinal homeostasis in higher eukaryotes. It further may help us understand how an alteration of these finely tuned processes precipitates the inflammatory processes found in some inflammatory bowel diseases.

Polyglutamine Atrophin provokes neurodegeneration in Drosophila by repressing fat.

Large alterations in transcription accompany neurodegeneration in polyglutamine (polyQ) diseases. These pathologies manifest both general polyQ toxicity and mutant protein-specific effects. In this study, we report that the fat tumour suppressor gene mediates neurodegeneration induced by the polyQ protein Atrophin. We have monitored early transcriptional alterations in a Drosophila model of Dentatorubral-pallidoluysian Atrophy and found that polyQ Atrophins downregulate fat. Fat protects from neurodegeneration and Atrophin toxicity through the Hippo kinase cascade. Fat/Hippo signalling does not provoke neurodegeneration by stimulating overgrowth; rather, it alters the autophagic flux in photoreceptor neurons, thereby affecting cell homeostasis. Our data thus provide a crucial insight into the specific mechanism of a polyQ disease and reveal an unexpected neuroprotective role of the Fat/Hippo pathway.

SKIV2L mutations cause syndromic diarrhea, or trichohepatoenteric syndrome.

Syndromic diarrhea (or trichohepatoenteric syndrome) is a rare congenital bowel disorder characterized by intractable diarrhea and woolly hair, and it has recently been associated with mutations in TTC37. Although databases report TTC37 as being the human ortholog of Ski3p, one of the yeast Ski-complex cofactors, this lead was not investigated in initial studies. The Ski complex is a multiprotein complex required for exosome-mediated RNA surveillance, including the regulation of normal mRNA and the decay of nonfunctional mRNA. Considering the fact that TTC37 is homologous to Ski3p, we explored a gene encoding another Ski-complex cofactor, SKIV2L, in six individuals presenting with typical syndromic diarrhea without variation in TTC37. We identified mutations in all six individuals. Our results show that mutations in genes encoding cofactors of the human Ski complex cause syndromic diarrhea, establishing a link between defects of the human exosome complex and a Mendelian disease.

The Drosophila inner-membrane protein PMI controls crista biogenesis and mitochondrial diameter.

Cristae are mitochondrial inner-membrane structures that concentrate respiratory chain complexes and hence regulate ATP production. Mechanisms controlling crista morphogenesis are poorly understood and few crista determinants have been identified. Among them are the Mitofilins that are required to establish crista junctions and ATP-synthase subunits that bend the membrane at the tips of the cristae. We report here the phenotypic consequences associated with the in vivo inactivation of the inner-membrane protein Pantagruelian Mitochondrion I (PMI) both at the scale of the whole organism, and at the level of mitochondrial ultrastructure and function. We show that flies in which PMI is genetically inactivated experience synaptic defects and have a reduced life span. Electron microscopy analysis of the inner-membrane morphology demonstrates that loss of PMI function increases the average length of mitochondrial cristae in embryonic cells. This phenotype is exacerbated in adult neurons in which cristae form a dense tangle of elongated membranes. Conversely, we show that PMI overexpression is sufficient to reduce crista length in vivo. Finally, these crista defects are associated with impaired respiratory chain activity and increases in the level of reactive oxygen species. Since PMI and its human orthologue TMEM11 are regulators of mitochondrial morphology, our data suggest that, by controlling crista length, PMI influences mitochondrial diameter and tubular shape.

Larval microbiota primes the Drosophila adult gustatory response.

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.

Toll-8/Tollo negatively regulates antimicrobial response in the Drosophila respiratory epithelium.

Barrier epithelia that are persistently exposed to microbes have evolved potent immune tools to eliminate such pathogens. If mechanisms that control Drosophila systemic responses are well-characterized, the epithelial immune responses remain poorly understood. Here, we performed a genetic dissection of the cascades activated during the immune response of the Drosophila airway epithelium i.e. trachea. We present evidence that bacteria induced-antimicrobial peptide (AMP) production in the trachea is controlled by two signalling cascades. AMP gene transcription is activated by the inducible IMD pathway that acts non-cell autonomously in trachea. This IMD-dependent AMP activation is antagonized by a constitutively active signalling module involving the receptor Toll-8/Tollo, the ligand Spätzle2/DNT1 and Ect-4, the Drosophila ortholog of the human Sterile alpha and HEAT/ARMadillo motif (SARM). Our data show that, in addition to Toll-1 whose function is essential during the systemic immune response, Drosophila relies on another Toll family member to control the immune response in the respiratory epithelium.

The Drosophila peptidoglycan-recognition protein LF interacts with peptidoglycan-recognition protein LC to downregulate the Imd pathway.

The peptidoglycan (PGN)-recognition protein LF (PGRP-LF) is a specific negative regulator of the immune deficiency (Imd) pathway in Drosophila. We determine the crystal structure of the two PGRP domains constituting the ectodomain of PGRP-LF at 1.72 and 1.94 Å resolution. The structures show that the LFz and LFw domains do not have a PGN-docking groove that is found in other PGRP domains, and they cannot directly interact with PGN, as confirmed by biochemical-binding assays. By using surface plasmon resonance analysis, we show that the PGRP-LF ectodomain interacts with the PGRP-LCx ectodomain in the absence and presence of tracheal cytotoxin. Our results suggest a mechanism for downregulation of the Imd pathway on the basis of the competition between PRGP-LCa and PGRP-LF to bind to PGRP-LCx.

Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways.

Mitochondria are highly dynamic organelles that can change in number and morphology during cell cycle, development or in response to extracellular stimuli. These morphological dynamics are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Genetic approaches have identified a cohort of conserved proteins that form the core of mitochondrial remodelling machineries. Mitofusins (MFNs) and OPA1 proteins are dynamin-related GTPases that are required for outer- and inner-mitochondrial membrane fusion respectively whereas dynamin-related protein 1 (DRP1) is the master regulator of mitochondrial fission. We demonstrate here that the Drosophila PMI gene and its human orthologue TMEM11 encode mitochondrial inner-membrane proteins that regulate mitochondrial morphogenesis. PMI-mutant cells contain a highly condensed mitochondrial network, suggesting that PMI has either a pro-fission or an anti-fusion function. Surprisingly, however, epistatic experiments indicate that PMI shapes the mitochondria through a mechanism that is independent of drp1 and mfn. This shows that mitochondrial networks can be shaped in higher eukaryotes by at least two separate pathways: one PMI-dependent and one DRP1/MFN-dependent.

Role of Rab5 early endosomes in regulating Drosophila gut antibacterial response.

Interactions between prokaryotes and eukaryotes require a dialogue between MAMPs and PRRs. In Drosophila, bacterial peptidoglycan is detected by PGRP receptors. While the components of the signaling cascades activated upon PGN/PGRP interactions are well characterized, little is known about the subcellular events that translate these early signaling steps into target gene transcription. Using a Drosophila enteric infection model, we show that gut-associated bacteria can induce the formation of intracellular PGRP-LE aggregates which colocalized with the early endosome marker Rab5. Combining microscopic and RNA-seq analysis, we demonstrate that RNAi inactivation of the endocytosis pathway in the Drosophila gut affects the expression of essential regulators of the NF-κB response leading not only to a disruption of the immune response locally in the gut but also at the systemic level. This work sheds new light on the involvement of the endocytosis pathway in the control of the gut response to intestinal bacterial infection.

Epithelial homeostasis and the underlying molecular mechanisms in the gut of the insect model Drosophila melanogaster.

Insects mostly develop on decaying and contaminated organic matter and often serve as vectors of biologically transmitted diseases by transporting microorganisms to the plant and animal hosts. As such, insects are constantly ingesting microorganisms, a small fraction of which reach their epithelial surfaces, mainly their digestive tract, where they can establish relationships ranging from symbiosis to mutualism or even parasitism. Understanding the tight physical, genetic, and biochemical interactions that takes place between intestinal epithelia and either resident or infectious microbes has been a long-lasting objective of the immunologist. Research in this field has recently been re-vitalized with the development of deep sequencing techniques, which allow qualitative and quantitative characterization of gut microbiota. Interestingly, the recent identification of regenerative stem cells in the Drosophila gut together with the initial characterization of Drosophila gut microbiota have opened up new avenues of study aimed at understanding the mechanisms that regulate the dialog between the Drosophila gut epithelium and its microbiota of this insect model. The fact that some of the responses are conserved across species combined with the power of Drosophila genetics could make this organism model a useful tool to further elucidate some aspects of the interaction occurring between the microbiota and the human gut.

Elimination of plasmatocytes by targeted apoptosis reveals their role in multiple aspects of the Drosophila immune response.

Drosophila hemocytes have strong phagocytic capacities and produce antimicrobial peptides (AMPs). However, the precise role of blood cells during immune responses and developmental processes has only been studied using indirect means. To overcome this limitation, we generated plasmatocyte-depleted flies by specifically overexpressing the proapoptotic protein Hid into plasmatocytes. Unexpectedly, these plasmatocyte-depleted animals have a normal larval and pupal development and do not exhibit any obvious defect after birth. Remarkably, plasmatocyte-depleted adults show a strong susceptibility to infections by various microorganisms, although activation of systemic AMP gene transcription via the Toll and immune deficiency (IMD) pathways is wild-type. Our data show that this susceptibility, which correlates with overproliferation of bacteria, is likely due to the absence of phagocytosis. We also demonstrate that during larval stages, plasmatocytes play an essential role in mediating AMP production by the fat body after oral bacterial infection. Finally, we show that plasmatocytes are involved in immune surveillance during pupal development, because they prevent bacterial infection that causes pupal lethality.

Impact of Microorganisms and Parasites on Neuronally Controlled Drosophila Behaviours.

Like all invertebrates, flies such as Drosophila lack an adaptive immune system and depend on their innate immune system to protect them against pathogenic microorganisms and parasites. In recent years, it appears that the nervous systems of eucaryotes not only control animal behavior but also cooperate and synergize very strongly with the animals' immune systems to detect and fight potential pathogenic threats, and allow them to adapt their behavior to the presence of microorganisms and parasites that coexist with them. This review puts into perspective the latest progress made using the Drosophila model system, in this field of research, which remains in its infancy.

Protocol for a Binary Choice Feeding Assay Using Adult, Axenic Drosophila.

This protocol is designed to prepare adult axenic Drosophila before monitoring their behavior in a two-choice feeding assay, where flies are confronted with an axenic versus a dead or alive bacteria-contaminated feeding solution. Several aspects of the procedure, including raising and aging flies in axenic conditions, starving adult flies, and composing feeding solutions, are detailed. The bacterium used in this protocol, Erwinia carotovora carotovora-15 2141 (Ecc-15 2141 ), is commonly used to decipher the mechanisms controlling host-pathogen interactions in the Drosophila model. For complete details on the use and execution of this protocol, please refer to Charroux et al. (2020).