Effect of aminergic signaling on the humoral innate immunity response of Drosophila.

Biogenic amines are crucial signaling molecules that modulate various physiological life functions both in vertebrates and invertebrates. In humans, these neurotransmitters influence the innate and adaptive immunity systems. In this work, we analyzed whether the aminergic neurotransmission of dopamine, serotonin, and octopamine could have an impact on the humoral innate immune response of Drosophila melanogaster. This is a powerful model system widely used to uncover the insect innate immunity mechanisms which are also conserved in mammals. We found that the neurotransmission of all these amines positively modulates the Toll-responsive antimicrobial peptide (AMP) drosomycin (drs) gene in adult flies infected with the Micrococcus luteus bacterium. Indeed, we showed that either blocking the neurotransmission in their specific aminergic neurons by expressing shibirets (Shits) or silencing the vesicular monoamine transporter gene (dVMAT) by RNAi caused a significantly reduced expression of the Toll-responsive drs gene. However, upon M. luteus infection, the block of aminergic transmission did not alter the expression of AMP attacin genes responding to the immune deficiency (Imd) and Toll pathways. Overall, our results not only reveal a neuroimmune function for biogenic amines in humoral immunity but also further highlight the complexity of the network controlling AMP gene regulation.

Mutation-driven evolution of antibacterial function in an ancestral antifungal scaffold: Significance for peptide engineering.

Mutation-driven evolution of novel function on an old gene has been documented in many development- and adaptive immunity-related genes but is poorly understood in immune effector molecules. Drosomycin-type antifungal peptides (DTAFPs) are a family of defensin-type effectors found in plants and ecdysozoans. Their primitive function was to control fungal infection and then co-opted for fighting against bacterial infection in plants, insects, and nematodes. This provides a model to study the structural and evolutionary mechanisms behind such functional diversification. In the present study, we determined the solution structure of mehamycin, a DTAFP from the Northern root-knot nematode Meloidogyne hapla with antibacterial activity and an 18-mer insert, and studied the mutational effect through using a mutant with the insert deleted. Mehamycin adopts an expected cysteine-stabilized α-helix and ß-sheet fold in its core scaffold and the inserted region, called single Disulfide Bridge-linked Domain (abbreviated as sDBD), forms an extended loop protruding from the scaffold. The latter folds into an amphipathic architecture stabilized by one disulfide bridge, which likely confers mehamycin a bacterial membrane permeability. Deletion of the sDBD remarkably decreased the ability but accompanying an increase in thermostability, indicative of a structure-function trade-off in the mehamycin evolution. Allosteric analysis revealed an interior interaction between the two domains, which might promote point mutations at some key sites of the core domain and ultimately give rise to the emergence of antibacterial function. Our work may be valuable in guiding protein engineering of mehamycin to improve its activity and stability.

Tissue-autonomous immune response regulates stress signaling during hypertrophy.

Postmitotic tissues are incapable of replacing damaged cells through proliferation, but need to rely on buffering mechanisms to prevent tissue disintegration. By constitutively activating the Ras/MAPK-pathway via RasV12-overexpression in the postmitotic salivary glands (SGs) of Drosophila larvae, we overrode the glands adaptability to growth signals and induced hypertrophy. The accompanied loss of tissue integrity, recognition by cellular immunity, and cell death are all buffered by blocking stress signaling through a genuine tissue-autonomous immune response. This novel, spatio-temporally tightly regulated mechanism relies on the inhibition of a feedback-loop in the JNK-pathway by the immune effector and antimicrobial peptide Drosomycin. While this interaction might allow growing SGs to cope with temporary stress, continuous Drosomycin expression in RasV12-glands favors unrestricted hypertrophy. These findings indicate the necessity to refine therapeutic approaches that stimulate immune responses by acknowledging their possible, detrimental effects in damaged or stressed tissues.

Tissues and organs work hard to maintain balance in everything from taking up nutrients to controlling their growth. Ageing, wounding, sickness, and changes in the genetic code can all alter this balance, and cause the tissue or organ to lose some of its cells. Many tissues restore this loss by dividing their remaining cells to fill in the gaps. But some ­ like the salivary glands of fruit fly larvae ­ have lost this ability. Tissues like these rely on being able to sense and counteract problems as they arise so as to not lose their balance in the first place. The immune system and stress responses are crucial for this process. They trigger steps to correct the problem and interact with each other to find a common decision about the fate of the affected tissue. To better understand how the immune system and stress response work together, Krautz, Khalili and Theopold genetically manipulated cells in the salivary gland of fruit fly larvae. These modifications switched on signals that stimulate cells to keep growing, causing the salivary gland's tissue to slowly lose its balance and trigger the stress and immune response. The experiments showed that while the stress response instructed the cells in the gland to die, a peptide released by the immune system called Drosomycin blocked this response and prevented the tissue from collapsing. The cells in the part of the gland not producing this immune peptide were consequently killed by the stress response. When all the cells in the salivary gland were forced to produce Drosomycin, none of the cells died and the whole tissue survived. But it also allowed the cells in the gland to grow uncontrollably, like a tumor, threatening the health of the entire organism. Mapping the interactions between immune and stress pathways could help to fine-tune treatments that can prevent tissue damage. Fruit flies share many genetic features and molecular pathways with humans. So, the next step towards these kinds of treatments would be to screen for similar mechanisms that block stress activation in damaged human tissues. But this research carries a warning: careless activation of the immune system to protect stressed tissues could lead to uncontrolled tissue growth, and might cause more harm than good.

CDKI-73 is a Novel Pharmacological Inhibitor of Rab11 Cargo Delivery and Innate Immune Secretion.

Innate immunity is critical for host defence against pathogen and environmental challenge and this involves the production and secretion of immune mediators, such as antimicrobial peptides and pro-inflammatory cytokines. However, when dysregulated, innate immunity can contribute to multifactorial diseases, including inflammatory rheumatic disorders, type 2 diabetes, cancer, neurodegenerative and cardiovascular diseases and even septic shock. During an innate immune response, antimicrobial peptides and cytokines are trafficked via Rab11 multivesicular endosomes, and then sorted into Rab11 vesicles for traffic to the plasma membrane and secretion. In this study, a cyclin-dependent kinase inhibitor CDKI-73 was used to determine its effect on the innate immune response, based on previously identified targets for this compound. Our results showed that CDKI-73 inhibited the delivery of Rab11 vesicles to the plasma membrane, resulting in the accumulation of large multivesicular Rab11 endosomes near the cell periphery. In addition to the effect on endosome delivery, CDKI-73 down-regulated the amount of innate immune cargo, including the antimicrobial peptide Drosomycin and pro-inflammatory cytokines interleukin-6 (IL-6) and tumour necrosis factor alpha (TNFα). We concluded that CDKI-73 has the potential to regulate the delivery and secretion of certain innate immune cargo, which could be used to control inflammation.

Characterization of bi-domain drosomycin-type antifungal peptides in nematodes: An example of convergent evolution.

Drosomycin-type antifungal peptides (DTAFPs) are natural effectors of the innate immune system, which are restrictedly distributed in plants and ecdysozoans. Mehamycin is a bi-domain DTAFP (abbreviated as bDTAFP) firstly found in the Northern root-knot nematode Meloidogyne hapla. Here, we report its structural and functional features and the evolution of bDTAFPs in nematodes. Different from classical DTAFPs, mehamycin contains an insertion, called single Disulfide Bridge-linked Domain (abbreviated as sDBD), located in a loop region of the drosomycin scaffold. Despite this, recombinant mehamycin likely adopts a similar fold to drosomycin, as revealed by the circular dichroism spectral analysis. Functionally, it showed some weak activity against three species of fungi but relatively stronger activity against seven species of Gram-positive bacteria, indicative of functional diversification between mehamycin and classical DTAFPs. By computational data mining of the nematode databases, we identified polymorphic genes encoding mehamycin and a new multigene family of bDTAFPs (named roremycins) from Rotylenchulus reniformis. A combination of data suggests that the origination of sDBDs from M. hapla and R. reniformis is a consequence of convergent evolution, in which some probably suffered positive selection during evolution. Our study may be valuable in understanding the role of these unique antimicrobial peptides in the innate immunity of nematodes.

Rapid seasonal evolution in innate immunity of wild Drosophila melanogaster.

Understanding the rate of evolutionary change and the genetic architecture that facilitates rapid adaptation is a current challenge in evolutionary biology. Comparative studies show that genes with immune function are among the most rapidly evolving genes across a range of taxa. Here, we use immune defence in natural populations of Drosophila melanogaster to understand the rate of evolution in natural populations and the genetics underlying rapid change. We probed the immune system using the natural pathogens Enterococcus faecalis and Providencia rettgeri to measure post-infection survival and bacterial load of wild D. melanogaster populations collected across seasonal time along a latitudinal transect along eastern North America (Massachusetts, Pennsylvania and Virginia). There are pronounced and repeatable changes in the immune response over the approximately 10 generations between spring and autumn collections, with a significant but less distinct difference observed among geographical locations. Genes with known immune function are not enriched among alleles that cycle with seasonal time, but the immune function of a subset of seasonally cycling alleles in immune genes was tested using reconstructed outbred populations. We find that flies containing seasonal alleles in Thioester-containing protein 3 (Tep3) have different functional responses to infection and that epistatic interactions among seasonal Tep3 and Drosomycin-like 6 (Dro6) alleles underlie the immune phenotypes observed in natural populations. This rapid, cyclic response to seasonal environmental pressure broadens our understanding of the complex ecological and genetic interactions determining the evolution of immune defence in natural populations.

Drosophila miR-964 modulates Toll signaling pathway in response to bacterial infection.

Recent studies suggest that microRNA (miRNA) plays important roles in the control of immune response and tolerance. We previously found that the expression level of antimicrobial peptide gene Drosomycin (Drs) is decreased in miR-964 overexpressing flies. Here, we further verified that miR-964 deficiency leads to hyper-activation of Drs. In addition, we employed three widely-used bioinformatic algorithms to screen potential miR-964 targets. Finally, we identified that miR-964 modulates Toll signaling pathway, at least in part, by repressing the expression of Drs. Taken together, our study identifies miR-964 as a modulator of Toll signaling and enriches the repertoire of immune-modulating miRNAs in Drosophila.

Antifungal Activity against Filamentous Fungi of Ts1, a Multifunctional Toxin from Tityus serrulatus Scorpion Venom.

Antimicrobial peptides (AMPs) are ubiquitous and multipotent components of the innate immune defense arsenal used by both prokaryotic and eukaryotic organisms. The search for new AMPs has increased in recent years, due to the growing development of microbial resistance to therapeutical drugs. In this work, we evaluate the effects of Tityus serrulatus venom (Tsv), its fractions and its major toxin Ts1, a beta-neurotoxin, on fungi growth. The fractions were obtained by ion-exchange chromatography of Tsv. The growth inhibition of 11 pathogenic and non-pathogenic filamentous fungi (Aspergillus fumigatus, A. nidulans, A. niger, A. terreus, Neurospora crassa, Penicillium corylophilum, P. ochrochloron, P. verrucosum, P. viridicatum, P. waksmanii, and Talaromyces flavus) was evaluated by quantitative microplate reader assay. Tsv (100 and 500 µg/well, which correspond to 1 and 5 mg/mL, respectively, of total soluble protein) was active in inhibiting growth of A. nidulans, A. terreus, P. corylophilum, and P. verrucosum, especially in the higher concentration used and at the first 30 h. After this period, fungi might have used Tsv components as alternative sources of nutrients, and therefore, increased their growth tax. Only fractions IX, X, XI, XIIA, XIIB (3 and 7.5 µg/well, which correspond to 30 and 75 µg/mL, respectively, of total soluble protein) and Ts1 (1.5, 3, and 6 µg/well, which correspond to 2.18, 4.36, and 8.72 µM, respectively) showed antifungal activity. Ts1 showed to be a non-morphogenic toxin with dose-dependent activity against A. nidulans, inhibiting 100% of fungal growth from 3 µg/well (4.36 µM). The inhibitory effect of Ts1 against A. nidulans growth was accompanied by fungistatic effects and was not amended by 1 mM CaCl2 or tetrodotoxin (46.98 and 93.96 µM). The structural differences between Ts1 and drosomycin, a potent cysteine-rich antifungal peptide, are discussed here. Our results highlight the antifungal potential of the first cysteine-containing scorpion toxin. Since Ts1 is a multifunctional toxin, we suggest that it could be used as a template in the design of engineered scorpion AMPs and in the search for new mechanisms of action of antifungal drugs.

Genome-wide miRNA screening reveals miR-310 family members negatively regulate the immune response in Drosophila melanogaster via co-targeting Drosomycin.

Although innate immunity mediated by Toll signaling has been extensively studied in Drosophila melanogaster, the role of miRNAs in regulating the Toll-mediated immune response remains largely unknown. In this study, following Gram-positive bacterial challenge, we identified 93 differentially expressed miRNAs via genome-wide miRNA screening. These miRNAs were regarded as immune response related (IRR). Eight miRNAs were confirmed to be involved in the Toll-mediated immune response upon Gram-positive bacterial infection through genetic screening of 41 UAS-miRNA lines covering 60 miRNAs of the 93 IRR miRNAs. Interestingly, four out of these eight miRNAs, miR-310, miR-311, miR-312 and miR-313, are clustered miRNAs and belong to the miR-310 family. These miR-310 family members were shown to target and regulate the expression of Drosomycin, an antimicrobial peptide produced by Toll signaling. Taken together, our study implies important regulatory roles of miRNAs in the Toll-mediated innate immune response of Drosophila upon Gram-positive bacterial infection.

Drosophila Pkaap regulates Rab4/Rab11-dependent traffic and Rab11 exocytosis of innate immune cargo.

The secretion of immune-mediators is a critical step in the host innate immune response to pathogen invasion, and Rab GTPases have an important role in the regulation of this process. Rab4/Rab11 recycling endosomes are involved in the sorting of immune-mediators into specialist Rab11 vesicles that can traffic this cargo to the plasma membrane; however, how this sequential delivery process is regulated has yet to be fully defined. Here, we report that Drosophila Pkaap, an orthologue of the human dual-specific A-kinase-anchoring protein 2 or D-AKAP2 (also called AKAP10), appeared to have a nucleotide-dependent localisation to Rab4 and Rab11 endosomes. RNAi silencing of pkaap altered Rab4/Rab11 recycling endosome morphology, suggesting that Pkaap functions in cargo sorting and delivery in the secretory pathway. The depletion of pkaap also had a direct effect on Rab11 vesicle exocytosis and the secretion of the antimicrobial peptide Drosomycin at the plasma membrane. We propose that Pkaap has a dual role in antimicrobial peptide traffic and exocytosis, making it an essential component for the secretion of inflammatory mediators and the defence of the host against pathogens.

Cost of surviving sepsis: a novel model of recovery from sepsis in Drosophila melanogaster.

BACKGROUND: Multiple organ failure, wasting, increased morbidity, and mortality following acute illness complicates the health span of patients surviving sepsis. Persistent inflammation has been implicated, and it is proposed that insulin signaling contributes to persistent inflammatory signaling during the recovery phase after sepsis. However, mechanisms are unknown and suitable pre-clinical models are lacking. We therefore developed a novel Drosophila melanogaster model of sepsis to recapitulate the clinical course of sepsis, explored inflammation over time, and its relation to impaired mobility, metabolic disturbance, and changes in lifespan. METHODS: We used wild-type (WT), Drosomycin-green fluorescent protein (GFP), and NF-κB-luc reporter male Drosophila melanogaster 4-5 days of age (unmanipulated). We infected Drosophila with Staphylococcus aureus (infected without treatment) or pricked with aseptic needles (sham). Subsets of insects were treated with oral linezolid after the infection (infected with antibiotics). We assessed rapid iterative negative geotaxis (RING) in all the groups as a surrogate for neuromuscular functional outcome up to 96 h following infection. We harvested the flies over the 7-day course to evaluate bacterial burden, inflammatory and metabolic pathway gene expression patterns, NF-κB translation, and metabolic reserve. We also followed the lifespan of the flies. RESULTS: Our results showed that when treated with antibiotics, flies had improved survival compared to infected without treatment flies in the early phase of sepsis up to 1 week (81 %, p = 0.001). However, the lifespan of infected with antibiotics flies was significantly shorter than that of sham controls (p = 0.001). Among infected with antibiotic sepsis survivors, we observed persistent elevation of NF-κB in the absence of any obvious infection as shown by culturing flies surviving sepsis. In the same group, geotaxis had an early (18 h) and sustained decline compared to its baseline. Geotaxis in infected with antibiotics sepsis survivors was significantly lower than that in sham and age-matched unmanipulated flies at 18 and 48 h. Expression of antimicrobial peptides (AMP) remained significantly elevated over the course of 7 days after sepsis, especially drosomycin (5.7-fold, p = 0.0145) on day 7 compared to that of sham flies. Infected with antibiotics flies had a trend towards decreased Akt activation, yet their glucose stores were significantly lower than those of sham flies (p = 0.001). Sepsis survivors had increased lactate levels and LDH activity by 1 week, whereas ATP and pyruvate content was similar to that of the sham group. CONCLUSIONS: In summary, our model mimics human survivors of sepsis with persistent inflammation, impaired motility, dysregulated glucose metabolism, and shortened lifespan.

Innate immunity and exocytosis of antimicrobial peptides.

In Drosophila, anti-microbial peptides are activated and secreted in response to microbial challenge, but the intracellular route of anti-microbial peptide trafficking and the regulatory mechanism controlling their secretion are yet to be fully characterized. We have demonstrated that in Drosophila immune response cells (i.e., fat body cells and hemocytes) the anti-microbial peptide Drosomycin is localized within Rab4 and Rab11 intracellular vesicles. Moreover, both of these small GTPases were required for the delivery of this Drosomycin cargo to the plasma membrane. At the plasma membrane, exocytosis and Drosomycin secretion depend on the SNARE protein Syntaxin1A. Thus, the depletion of Syntaxin1A impaired the release of this antimicrobial peptide, and resulted in the accumulation of Drosomycin and Rab11 carrier vesicles near the plasma membrane. Intriguingly, a similar phenotype was generated by the loss of the adaptor protein 14-3-3ε; there was accumulation of Rab11 vesicles and Drosomycin containing vesicles near the plasma membrane, and a concomitant increase in the susceptibility of 14-3-3ε mutant Drosophila to acute bacterial infection. This suggested that 14-3-3ε, possibly via interaction with Syntaxin1A, is required to promote exocytosis of immune-mediators, thereby regulating innate immune secretion and organism survival under conditions of immune stress.