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.

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.

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.

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.

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.

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).

How Bacteria Impact Host Nervous System and Behaviors: Lessons from Flies and Worms.

Behavior is the neuronally controlled, voluntary or involuntary response of an organism to its environment. An increasing body of evidence indicates that microbes, which live closely associated with animals or in their immediate surroundings, significantly influence animals' behavior. The extreme complexity of the nervous system of animals, combined with the extraordinary microbial diversity, are two major obstacles to understand, at the molecular level, how microbes modulate animal behavior. In this review, we discuss recent advances in dissecting the impact that bacteria have on the nervous system of two genetically tractable invertebrate models, Drosophila melanogaster and Caenorhabditis elegans.

Gut bacteria-derived peptidoglycan induces a metabolic syndrome-like phenotype via NF-κB-dependent insulin/PI3K signaling reduction in Drosophila renal system.

Although microbiome-host interactions are usual at steady state, gut microbiota dysbiosis can unbalance the physiological and behavioral parameters of the host, mostly via yet not understood mechanisms. Using the Drosophila model, we investigated the consequences of a gut chronic dysbiosis on the host physiology. Our results show that adult flies chronically infected with the non-pathogenic Erwinia carotorova caotovora bacteria displayed organ degeneration resembling wasting-like phenotypes reminiscent of Metabolic Syndrome associated pathologies. Genetic manipulations demonstrate that a local reduction of insulin signaling consecutive to a peptidoglycan-dependent NF-κB activation in the excretory system of the flies is responsible for several of the observed phenotypes. This work establishes a functional crosstalk between bacteria-derived peptidoglycan and the immune NF-κB cascade that contributes to the onset of metabolic disorders by reducing insulin signal transduction. Giving the high degree of evolutionary conservation of the mechanisms and pathways involved, this study is likely to provide a helpful model to elucidate the contribution of altered intestinal microbiota in triggering human chronic kidney diseases.

Drosophila Aversive Behavior toward Erwinia carotovora carotovora Is Mediated by Bitter Neurons and Leukokinin.

The phytopathogen Erwinia carotovora carotovora (Ecc) has been used successfully to decipher some of the mechanisms that regulate the interactions between Drosophila melanogaster and bacteria, mostly following forced association between the two species. How do Drosophila normally perceive and respond to the presence of Ecc is unknown. Using a fly feeding two-choice assay and video tracking, we show that Drosophila are first attracted but then repulsed by an Ecc-contaminated solution. The initial attractive phase is dependent on the olfactory Gr63a and Gαq proteins, whereas the second repulsive phase requires a functional gustatory system. Genetic manipulations and calcium imaging indicate that bitter neurons and gustatory receptors Gr66a and Gr33a are needed for the aversive phase and that the neuropeptide leukokinin is also involved. We also demonstrate that these behaviors are independent of the NF-κB cascade that controls some of the immune, metabolic, and behavioral responses to bacteria.

Uridine Catabolism Breaks the Bonds of Commensalism.

How gut cells distinguish between beneficial symbionts and deleterious pathobionts is a central question. In this issue of Cell Host & Microbe, Kim et al. (2020) demonstrate that the nucleoside catabolism pathway controlling bacterial uracil and ribose production is an essential trigger of the commensal to pathogen transition.

Peptidoglycan-dependent NF-κB activation in a small subset of brain octopaminergic neurons controls female oviposition.

When facing microbes, animals engage in behaviors that lower the impact of the infection. We previously demonstrated that internal sensing of bacterial peptidoglycan reduces Drosophila female oviposition via NF-κB pathway activation in some neurons (Kurz et al., 2017). Although we showed that the neuromodulator octopamine is implicated, the identity of the involved neurons, as well as the physiological mechanism blocking egg-laying, remained unknown. In this study, we identified few ventral nerve cord and brain octopaminergic neurons expressing an NF-κB pathway component. We functionally demonstrated that NF-κB pathway activation in the brain, but not in the ventral nerve cord octopaminergic neurons, triggers an egg-laying drop in response to infection. Furthermore, we demonstrated via calcium imaging that the activity of these neurons can be directly modulated by peptidoglycan and that these cells do not control other octopamine-dependent behaviors such as female receptivity. This study shows that by sensing peptidoglycan and hence activating NF-κB cascade, a couple of brain neurons modulate a specific octopamine-dependent behavior to adapt female physiology status to their infectious state.

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.

Lipid Catabolism Fuels Drosophila Gut Immunity.

Immune responses and metabolic regulation are tightly coupled in animals, but the underlying mechanistic connections are not fully understood. In this issue of Cell Host & Microbe, Lee et al. (2018) reveal how sustained ROS production in the gut depends on an upstream metabolic switch.

Cytosolic and Secreted Peptidoglycan-Degrading Enzymes in Drosophila Respectively Control Local and Systemic Immune Responses to Microbiota.

Gut-associated bacteria produce metabolites that both have a local influence on the intestinal tract and act at a distance on remote organs. In Drosophila, bacteria-derived peptidoglycan (PGN) displays such a dual role. PGN triggers local antimicrobial peptide production by enterocytes; it also activates systemic immune responses in fat-body cells and modulates fly behavior by acting on neurons. How these responses to a single microbiota-derived compound are simultaneously coordinated is not understood. We show here that the PGRP-LB locus generates both cytosolic and secreted PGN-cleaving enzymes. Through genetic analysis, we demonstrate that the cytosolic PGRP-LB isoforms cell-autonomously control the intensity of NF-κB activation in enterocytes, whereas the secreted isoform prevents massive and detrimental gut-derived PGN dissemination throughout the organism. This study explains how Drosophila are able to uncouple the modulation of local versus systemic responses to a single gut-bacteria-derived product by using isoform-specific enzymes.

Oligopeptide Transporters of the SLC15 Family Are Dispensable for Peptidoglycan Sensing and Transport in Drosophila.

Peptidoglycan (PGN) detection by PGN recognition proteins (PGRP) is the main trigger of the antibacterial immune response in Drosophila. Depending on the type of immune cell, PGN can be sensed either at the cell membrane by PGRP-LC or inside the cell by PGRP-LE, which plays a role similar to that of Nod2 in mammals. Previous work, mainly in cell cultures, has shown that oligopeptide transporters of the SLC15 family are essential for the delivery of PGN for Nod2 detection inside of the cells, and that this function might be conserved in flies. By generating and analyzing the immune phenotypes of loss-of-function mutations in 3 SLC15 Drosophila family members, we tested their role in mediating PGRP-LE-dependent PGN activation. Our results show that Yin, CG2930, and CG9444 are required neither for PGRP-LE activation by PGN nor for PGN transport from the gut lumen to the insect blood. These data show that, while intracellular PGN detection is an essential step of the antibacterial response in both insects and mammals, the types of PGN transporters and sensors are different in these animals.

Peptidoglycan sensing by octopaminergic neurons modulates Drosophila oviposition.

As infectious diseases pose a threat to host integrity, eukaryotes have evolved mechanisms to eliminate pathogens. In addition to develop strategies reducing infection, animals can engage in behaviors that lower the impact of the infection. The molecular mechanisms by which microbes impact host behavior are not well understood. We demonstrate that bacterial infection of Drosophila females reduces oviposition and that peptidoglycan, the component that activates Drosophila antibacterial response, is also the elicitor of this behavioral change. We show that peptidoglycan regulates egg-laying rate by activating NF-κB signaling pathway in octopaminergic neurons and that, a dedicated peptidoglycan degrading enzyme acts in these neurons to buffer this behavioral response. This study shows that a unique ligand and signaling cascade are used in immune cells to mount an immune response and in neurons to control fly behavior following infection. This may represent a case of behavioral immunity.

Drosophila larvae food intake cessation following exposure to Erwinia contaminated media requires odor perception, Trpa1 channel and evf virulence factor.

When exposed to microorganisms, animals use several protective strategies. On one hand, as elegantly exemplified in Drosophila melanogaster, the innate immune system recognizes microbial compounds and triggers an antimicrobial response. On the other hand, behaviors preventing an extensive contact with the microbes and thus reducing the risk of infection have been described. However, these reactions ranging from microbes aversion to intestinal transit increase or food intake decrease have been rarely defined at the molecular level. In this study, we set up an experimental system that allowed us to rapidly identify and quantify food intake decreases in Drosophila larvae exposed to media contaminated with bacteria. Specifically, we report a robust dose-dependent food intake decrease following exposure to the bacteria Erwinia carotovora carotovora strain Ecc15. We demonstrate that this response does not require Imd innate immune pathway, but rather the olfactory neuronal circuitry, the Trpa1 receptor and the evf virulence factor. Finally, we show that Ecc15 induce the same behavior in the invasive pest insect Drosophila suzukii.

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.