A humoral stress response protects Drosophila tissues from antimicrobial peptides.

7An efficient immune system must provide protection against a broad range of pathogens without causing excessive collateral tissue damage. While immune effectors have been well characterized, we know less about the resilience mechanisms protecting the host from its own immune response. Antimicrobial peptides (AMPs) are small, cationic peptides that contribute to innate defenses by targeting negatively charged membranes of microbes. While protective against pathogens, AMPs can be cytotoxic to host cells. Here, we reveal that a family of stress-induced proteins, the Turandots, protect the Drosophila respiratory system from AMPs, increasing resilience to stress. Flies lacking Turandot genes are susceptible to environmental stresses due to AMP-induced tracheal apoptosis. Turandot proteins bind to host cell membranes and mask negatively charged phospholipids, protecting them from cationic pore-forming AMPs. Collectively, these data demonstrate that Turandot stress proteins mitigate AMP cytotoxicity to host tissues and therefore improve their efficacy.

DNA damage signaling in Drosophila macrophages modulates systemic cytokine levels in response to oxidative stress.

Environmental factors, infection, or injury can cause oxidative stress in diverse tissues and loss of tissue homeostasis. Effective stress response cascades, conserved from invertebrates to mammals, ensure reestablishment of homeostasis and tissue repair. Hemocytes, the Drosophila blood-like cells, rapidly respond to oxidative stress by immune activation. However, the precise signals how they sense oxidative stress and integrate these signals to modulate and balance the response to oxidative stress in the adult fly are ill-defined. Furthermore, hemocyte diversification was not explored yet on oxidative stress. Here, we employed high-throughput single nuclei RNA-sequencing to explore hemocytes and other cell types, such as fat body, during oxidative stress in the adult fly. We identified distinct cellular responder states in plasmatocytes, the Drosophila macrophages, associated with immune response and metabolic activation upon oxidative stress. We further define oxidative stress-induced DNA damage signaling as a key sensor and a rate-limiting step in immune-activated plasmatocytes controlling JNK-mediated release of the pro-inflammatory cytokine unpaired-3. We subsequently tested the role of this specific immune activated cell stage during oxidative stress and found that inhibition of DNA damage signaling in plasmatocytes, as well as JNK or upd3 overactivation, result in a higher susceptibility to oxidative stress. Our findings uncover that a balanced composition and response of hemocyte subclusters is essential for the survival of adult Drosophila on oxidative stress by regulating systemic cytokine levels and cross-talk to other organs, such as the fat body, to control energy mobilization.

TECPR1 conjugates LC3 to damaged endomembranes upon detection of sphingomyelin exposure.

Invasive bacteria enter the cytosol of host cells through initial uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the BCV membrane, thereby exposing to the cytosol intraluminal, otherwise shielded danger signals such as glycans and sphingomyelin. The detection of glycans by galectin-8 triggers anti-bacterial autophagy, but how cells sense and respond to cytosolically exposed sphingomyelin remains unknown. Here, we identify TECPR1 (tectonin beta-propeller repeat containing 1) as a receptor for cytosolically exposed sphingomyelin, which recruits ATG5 into an E3 ligase complex that mediates lipid conjugation of LC3 independently of ATG16L1. TECPR1 binds sphingomyelin through its N-terminal DysF domain (N'DysF), a feature not shared by other mammalian DysF domains. Solving the crystal structure of N'DysF, we identified key residues required for the interaction, including a solvent-exposed tryptophan (W154) essential for binding to sphingomyelin-positive membranes and the conjugation of LC3 to lipids. Specificity of the ATG5/ATG12-E3 ligase responsible for the conjugation of LC3 is therefore conferred by interchangeable receptor subunits, that is, the canonical ATG16L1 and the sphingomyelin-specific TECPR1, in an arrangement reminiscent of certain multi-subunit ubiquitin E3 ligases.

Infection increases activity via Toll dependent and independent mechanisms in Drosophila melanogaster.

Host behavioural changes are among the most apparent effects of infection. 'Sickness behaviour' can involve a variety of symptoms, including anorexia, depression, and changed activity levels. Here, using a real-time tracking and behavioural profiling platform, we show that in Drosophila melanogaster, several systemic bacterial infections cause significant increases in physical activity, and that the extent of this activity increase is a predictor of survival time in some lethal infections. Using multiple bacteria and D. melanogaster immune and activity mutants, we show that increased activity is driven by at least two different mechanisms. Increased activity after infection with Micrococcus luteus, a Gram-positive bacterium rapidly cleared by the immune response, strictly requires the Toll ligand spätzle. In contrast, increased activity after infection with Francisella novicida, a Gram-negative bacterium that cannot be cleared by the immune response, is entirely independent of both Toll and the parallel IMD pathway. The existence of multiple signalling mechanisms by which bacterial infections drive increases in physical activity implies that this effect may be an important aspect of the host response.

Mycobacterium abscessus pathogenesis identified by phenogenomic analyses.

The medical and scientific response to emerging and established pathogens is often severely hampered by ignorance of the genetic determinants of virulence, drug resistance and clinical outcomes that could be used to identify therapeutic drug targets and forecast patient trajectories. Taking the newly emergent multidrug-resistant bacteria Mycobacterium abscessus as an example, we show that combining high-dimensional phenotyping with whole-genome sequencing in a phenogenomic analysis can rapidly reveal actionable systems-level insights into bacterial pathobiology. Through phenotyping of 331 clinical isolates, we discovered three distinct clusters of isolates, each with different virulence traits and associated with a different clinical outcome. We combined genome-wide association studies with proteome-wide computational structural modelling to define likely causal variants, and employed direct coupling analysis to identify co-evolving, and therefore potentially epistatic, gene networks. We then used in vivo CRISPR-based silencing to validate our findings and discover clinically relevant M. abscessus virulence factors including a secretion system, thus illustrating how phenogenomics can reveal critical pathways within emerging pathogenic bacteria.

Pre-copulatory reproductive behaviours are preserved in Drosophila melanogaster infected with bacteria.

The activation of the immune system upon infection exerts a huge energetic demand on an individual, likely decreasing available resources for other vital processes, like reproduction. The factors that determine the trade-off between defensive and reproductive traits remain poorly understood. Here, we exploit the experimental tractability of the fruit fly Drosophila melanogaster to systematically assess the impact of immune system activation on pre-copulatory reproductive behaviour. Contrary to expectations, we found that male flies undergoing an immune activation continue to display high levels of courtship and mating success. Similarly, immune-challenged female flies remain highly sexually receptive. By combining behavioural paradigms, a diverse panel of pathogens and genetic strategies to induce the fly immune system, we show that pre-copulatory reproductive behaviours are preserved in infected flies, despite the significant metabolic cost of infection.

Bacterial Toxin-Triggered Release of Antibiotics from Capsosomes Protects a Fly Model from Lethal Methicillin-Resistant Staphylococcus aureus (MRSA) Infection.

Antibiotic resistance is a severe global health threat and hence demands rapid action to develop novel therapies, including microscale drug delivery systems. Herein, a hierarchical microparticle system is developed to achieve bacteria-activated single- and dual-antibiotic drug delivery for preventing methicillin-resistant Staphylococcus aureus (MRSA) bacterial infections. The designed system is based on a capsosome structure, which consists of a mesoporous silica microparticle coated in alternating layers of oppositely charged polymers and antibiotic-loaded liposomes. The capsosomes are engineered and shown to release their drug payloads in the presence of MRSA toxins controlled by the Agr quorum sensing system. MRSA-activated single drug delivery of vancomycin and synergistic dual delivery of vancomycin together with an antibacterial peptide successfully kills MRSA in vitro. The capability of capsosomes to selectively deliver their cargo in the presence of bacteria, producing a bactericidal effect to protect the host organism, is confirmed in vivo using a Drosophila melanogaster MRSA infection model. Thus, the capsosomes serve as a versatile multidrug, subcompartmentalized microparticle system for preventing antibiotic-resistant bacterial infections, with potential applications to protect wounds or medical device implants from infections.

Drosophila versus Mycobacteria: A model for mycobacterial host-pathogen interactions.

Animal models have played an essential role in understanding the host-pathogen interactions of pathogenic mycobacteria, including the Mycobacterium tuberculosis and emerging nontuberculous mycobacteria (NTM) species such as M. avium and M. abscessus. Drosophila melanogaster has become a well-established model for the study of innate immunity and is increasingly being used as a tool to study host-pathogen interactions, in part due to its genetic tractability. The use of D. melanogaster has led to greater understanding of the role of the innate immune system in response to mycobacterial infection, including in vitro RNAi screens and in vivo studies. These studies have identified processes and host factors involved in mycobacterial infection, such as those required for cellular entry, those required to control or resist non-pathogenic mycobacteria, or factors that become dysregulated as a result of mycobacterial infection. Developments in genetic tools for manipulating mycobacterial genomes will allow for more detailed studies into how specific host and pathogen factors interact with one another by using D. melanogaster; however, the full potential of this model has not yet been reached. Here we provide an overview of how D. melanogaster has been used to study mycobacterial infection and discuss the current gaps in our understanding.

Disparate regulation of IMD signaling drives sex differences in infection pathology in Drosophila melanogaster.

Male and female animals exhibit differences in infection outcomes. One possible source of sexually dimorphic immunity is the sex-specific costs of immune activity or pathology, but little is known about the independent effects of immune- versus microbe-induced pathology and whether these may differ for the sexes. Here, by measuring metabolic and physiological outputs in Drosophila melanogaster with wild-type and mutant immune responses, we test whether the sexes are differentially impacted by these various sources of pathology and identify a critical regulator of this difference. We find that the sexes exhibit differential immune activity but similar bacteria-derived metabolic pathology. We show that female-specific immune-inducible expression of PGRP-LB, a negative regulator of the immune deficiency (IMD) pathway, enables females to reduce immune activity in response to reductions in bacterial numbers. In the absence of PGRP-LB, females are more resistant to infection, confirming the functional importance of this regulation and suggesting that female-biased immune restriction comes at a cost.

EslB Is Required for Cell Wall Biosynthesis and Modification in Listeria monocytogenes.

Lysozyme is an important component of the innate immune system. It functions by hydrolyzing the peptidoglycan (PG) layer of bacteria. The human pathogen Listeria monocytogenes is intrinsically lysozyme resistant. The peptidoglycan N-deacetylase PgdA and O-acetyltransferase OatA are two known factors contributing to its lysozyme resistance. Furthermore, it was shown that the absence of components of an ABC transporter, referred to here as EslABC, leads to reduced lysozyme resistance. How its activity is linked to lysozyme resistance is still unknown. To investigate this further, a strain with a deletion in eslB, coding for a membrane component of the ABC transporter, was constructed in L. monocytogenes strain 10403S. The eslB mutant showed a 40-fold reduction in the MIC to lysozyme. Analysis of the PG structure revealed that the eslB mutant produced PG with reduced levels of O-acetylation. Using growth and autolysis assays, we showed that the absence of EslB manifests in a growth defect in media containing high concentrations of sugars and increased endogenous cell lysis. A thinner PG layer produced by the eslB mutant under these growth conditions might explain these phenotypes. Furthermore, the eslB mutant had a noticeable cell division defect and formed elongated cells. Microscopy analysis revealed that an early cell division protein still localized in the eslB mutant, indicating that a downstream process is perturbed. Based on our results, we hypothesize that EslB affects the biosynthesis and modification of the cell wall in L. monocytogenes and is thus important for the maintenance of cell wall integrity.IMPORTANCE The ABC transporter EslABC is associated with the intrinsic lysozyme resistance of Listeria monocytogenes However, the exact role of the transporter in this process and in the physiology of L. monocytogenes is unknown. Using different assays to characterize an eslB deletion strain, we found that the absence of EslB affects not only lysozyme resistance but also endogenous cell lysis, cell wall biosynthesis, cell division, and the ability of the bacterium to grow in media containing high concentrations of sugars. Our results indicate that EslB is, by means of a yet-unknown mechanism, an important determinant for cell wall integrity in L. monocytogenes.

Origins of Metabolic Pathology in Francisella-Infected Drosophila.

The origins and causes of infection pathologies are often not understood. Despite this, the study of infection and immunity relies heavily on the ability to discern between potential sources of pathology. Work in the fruit fly has supported the assumption that mortality resulting from bacterial invasion is largely due to direct host-pathogen interactions, as lower pathogen loads are often associated with reduced pathology, and bacterial load upon death is predictable. However, the mechanisms through which these interactions bring about host death are complex. Here we show that infection with the bacterium Francisella novicida leads to metabolic dysregulation and, using treatment with a bacteriostatic antibiotic, we show that this pathology is the result of direct interaction between host and pathogen. We show that mutants of the immune deficiency immune pathway fail to exhibit similar metabolic dysregulation, supporting the idea that the reallocation of resources for immune-related activities contributes to metabolic dysregulation. Targeted investigation into the cross-talk between immune and metabolic pathways has the potential to illuminate some of this interaction.

Muscle function and homeostasis require cytokine inhibition of AKT activity in Drosophila.

Unpaired ligands are secreted signals that act via a GP130-like receptor, domeless, to activate JAK/STAT signalling in Drosophila. Like many mammalian cytokines, unpaireds can be activated by infection and other stresses and can promote insulin resistance in target tissues. However, the importance of this effect in non-inflammatory physiology is unknown. Here, we identify a requirement for unpaired-JAK signalling as a metabolic regulator in healthy adult Drosophila muscle. Adult muscles show basal JAK-STAT signalling activity in the absence of any immune challenge. Plasmatocytes (Drosophila macrophages) are an important source of this tonic signal. Loss of the dome receptor on adult muscles significantly reduces lifespan and causes local and systemic metabolic pathology. These pathologies result from hyperactivation of AKT and consequent deregulation of metabolism. Thus, we identify a cytokine signal that must be received in muscle to control AKT activity and metabolic homeostasis.

fs(1)h controls metabolic and immune function and enhances survival via AKT and FOXO in Drosophila.

The Drosophila fat body is the primary organ of energy storage as well as being responsible for the humoral response to infection. Its physiological function is of critical importance to the survival of the organism; however, many molecular regulators of its function remain ill-defined. Here, we show that the Drosophila melanogaster bromodomain-containing protein FS(1)H is required in the fat body for normal lifespan as well as metabolic and immune homeostasis. Flies lacking fat body fs(1)h exhibit short lifespan, increased expression of immune target genes, an inability to metabolize triglyceride, and low basal AKT activity, mostly resulting from systemic defects in insulin signalling. Removal of a single copy of the AKT-responsive transcription factor foxo normalises lifespan, metabolic function, uninduced immune gene expression and AKT activity. We suggest that the promotion of systemic insulin signalling activity is a key in vivo function of fat body fs(1)h This article has an associated First Person interview with the first author of the paper.

α-actinin accounts for the bioactivity of actin preparations in inducing STAT target genes in Drosophila melanogaster.

Damage-associated molecular patterns (DAMPs) are molecules exposed or released by dead cells that trigger or modulate immunity and tissue repair. In vertebrates, the cytoskeletal component F-actin is a DAMP specifically recognised by DNGR-1, an innate immune receptor. Previously we suggested that actin is also a DAMP in Drosophila melanogaster by inducing STAT-dependent genes (Srinivasan et al., 2016). Here, we revise that conclusion and report that α-actinin is far more potent than actin at inducing the same STAT response and can be found in trace amounts in actin preparations. Recombinant expression of actin or α-actinin in bacteria demonstrated that only α-actinin could drive the expression of STAT target genes in Drosophila. The response to injected α-actinin required the same signalling cascade that we had identified in our previous work using actin preparations. Taken together, these data indicate that α-actinin rather than actin drives STAT activation when injected into Drosophila.

Regulation of phagocyte triglyceride by a STAT-ATG2 pathway controls mycobacterial infection.

Mycobacterium tuberculosis remains a global threat to human health, yet the molecular mechanisms regulating immunity remain poorly understood. Cytokines can promote or inhibit mycobacterial survival inside macrophages and the underlying mechanisms represent potential targets for host-directed therapies. Here we show that cytokine-STAT signalling promotes mycobacterial survival within macrophages by deregulating lipid droplets via ATG2 repression. In Drosophila infected with Mycobacterium marinum, mycobacterium-induced STAT activity triggered by unpaired-family cytokines reduces Atg2 expression, permitting deregulation of lipid droplets. Increased Atg2 expression or reduced macrophage triglyceride biosynthesis, normalizes lipid deposition in infected phagocytes and reduces numbers of viable intracellular mycobacteria. In human macrophages, addition of IL-6 promotes mycobacterial survival and BCG-induced lipid accumulation by a similar, but probably not identical, mechanism. Our results reveal Atg2 regulation as a mechanism by which cytokines can control lipid droplet homeostasis and consequently resistance to mycobacterial infection in Drosophila.

Actin is an evolutionarily-conserved damage-associated molecular pattern that signals tissue injury in Drosophila melanogaster.

Damage-associated molecular patterns (DAMPs) are molecules released by dead cells that trigger sterile inflammation and, in vertebrates, adaptive immunity. Actin is a DAMP detected in mammals by the receptor, DNGR-1, expressed by dendritic cells (DCs). DNGR-1 is phosphorylated by Src-family kinases and recruits the tyrosine kinase Syk to promote DC cross-presentation of dead cell-associated antigens. Here we report that actin is also a DAMP in invertebrates that lack DCs and adaptive immunity. Administration of actin to Drosophila melanogaster triggers a response characterised by selective induction of STAT target genes in the fat body through the cytokine Upd3 and its JAK/STAT-coupled receptor, Domeless. Notably, this response requires signalling via Shark, the Drosophila orthologue of Syk, and Src42A, a Drosophila Src-family kinase, and is dependent on Nox activity. Thus, extracellular actin detection via a Src-family kinase-dependent cascade is an ancient means of detecting cell injury that precedes the evolution of adaptive immunity.

The Software and Hardware of Macrophages: A Diversity of Options.

Macrophages play important immune and homeostatic roles that depend on the ability to receive and interpret specific signals from environmental stimuli. Here we describe the different activation states these cells can exhibit in response to signals and how these states affect and can be affected by bacterial pathogens.

Mitochondrial retrograde signaling regulates neuronal function.

Mitochondria are key regulators of cellular homeostasis, and mitochondrial dysfunction is strongly linked to neurodegenerative diseases, including Alzheimer's and Parkinson's. Mitochondria communicate their bioenergetic status to the cell via mitochondrial retrograde signaling. To investigate the role of mitochondrial retrograde signaling in neurons, we induced mitochondrial dysfunction in the Drosophila nervous system. Neuronal mitochondrial dysfunction causes reduced viability, defects in neuronal function, decreased redox potential, and reduced numbers of presynaptic mitochondria and active zones. We find that neuronal mitochondrial dysfunction stimulates a retrograde signaling response that controls the expression of several hundred nuclear genes. We show that the Drosophila hypoxia inducible factor alpha (HIFα) ortholog Similar (Sima) regulates the expression of several of these retrograde genes, suggesting that Sima mediates mitochondrial retrograde signaling. Remarkably, knockdown of Sima restores neuronal function without affecting the primary mitochondrial defect, demonstrating that mitochondrial retrograde signaling is partly responsible for neuronal dysfunction. Sima knockdown also restores function in a Drosophila model of the mitochondrial disease Leigh syndrome and in a Drosophila model of familial Parkinson's disease. Thus, mitochondrial retrograde signaling regulates neuronal activity and can be manipulated to enhance neuronal function, despite mitochondrial impairment.

Macrophage-derived upd3 cytokine causes impaired glucose homeostasis and reduced lifespan in Drosophila fed a lipid-rich diet.

Long-term consumption of fatty foods is associated with obesity, macrophage activation and inflammation, metabolic imbalance, and a reduced lifespan. We took advantage of Drosophila genetics to investigate the role of macrophages and the pathway(s) that govern their response to dietary stress. Flies fed a lipid-rich diet presented with increased fat storage, systemic activation of JAK-STAT signaling, reduced insulin sensitivity, hyperglycemia, and a shorter lifespan. Drosophila macrophages produced the JAK-STAT-activating cytokine upd3, in a scavenger-receptor (crq) and JNK-dependent manner. Genetic depletion of macrophages or macrophage-specific silencing of upd3 decreased JAK-STAT activation and rescued insulin sensitivity and the lifespan of Drosophila, but did not decrease fat storage. NF-κB signaling made no contribution to the phenotype observed. These results identify an evolutionarily conserved "scavenger receptor-JNK-type 1 cytokine" cassette in macrophages, which controls glucose metabolism and reduces lifespan in Drosophila maintained on a lipid-rich diet via activation of the JAK-STAT pathway.