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.

Protein target highlights in CASP15: Analysis of models by structure providers.

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.

CERT1 mutations perturb human development by disrupting sphingolipid homeostasis.

Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.

Disproportionate investment in Spiralin B production limits in-host growth and favors the vertical transmission of Spiroplasma insect endosymbionts.

Insects frequently harbor endosymbionts, which are bacteria housed within host tissues. These associations are stably maintained over evolutionary timescales through vertical transmission of endosymbionts from host mothers to their offspring. Some endosymbionts manipulate host reproduction to facilitate spread within natural populations. Consequently, such infections have major impacts on insect physiology and evolution. However, technical hurdles have limited our understanding of the molecular mechanisms underlying such insect-endosymbiont interactions. Here, we investigate the nutritional interactions between endosymbiotic partners using the tractable insect Drosophila melanogaster and its natural endosymbiont Spiroplasma poulsonii. Using a combination of functional assays, metabolomics, and proteomics, we show that the abundance and amino acid composition of a single Spiroplasma membrane lectin, Spiralin B (SpiB), dictates the amino acid requirements of the endosymbiont and determines its proliferation within host tissues. Ectopically increasing SpiB levels in host tissues disrupts localization of endosymbionts in the fly egg chambers and decreases vertical transmission. We find that SpiB is likely to be required by the endosymbiont to enter host oocytes, which may explain the massive investment of S. poulsonii in SpiB synthesis. SpiB both permits vertical transmission of the symbiont and limits its growth in nutrient-limiting conditions for the host; therefore, a single protein plays a pivotal role in ensuring durability of the interaction in a variable environment.

A secreted factor NimrodB4 promotes the elimination of apoptotic corpses by phagocytes in Drosophila.

Programmed cell death plays a fundamental role in development and tissue homeostasis. Professional and non-professional phagocytes achieve the proper recognition, uptake, and degradation of apoptotic cells, a process called efferocytosis. Failure in efferocytosis leads to autoimmune and neurodegenerative diseases. In Drosophila, two transmembrane proteins of the Nimrod family, Draper and SIMU, mediate the recognition and internalization of apoptotic corpses. Beyond this early step, little is known about how apoptotic cell degradation is regulated. Here, we study the function of a secreted member of the Nimrod family, NimB4, and reveal its crucial role in the clearance of apoptotic cells. We show that NimB4 is expressed by macrophages and glial cells, the two main types of phagocytes in Drosophila. Similar to draper mutants, NimB4 mutants accumulate apoptotic corpses during embryogenesis and in the larval brain. Our study points to the role of NimB4 in phagosome maturation, more specifically in the fusion between the phagosome and lysosomes. We propose that similar to bridging molecules, NimB4 binds to apoptotic corpses to engage a phagosome maturation program dedicated to efferocytosis.

Dual proteomics of Drosophila melanogaster hemolymph infected with the heritable endosymbiont Spiroplasma poulsonii.

Insects are frequently infected with heritable bacterial endosymbionts. Endosymbionts have a dramatic impact on their host physiology and evolution. Their tissue distribution is variable with some species being housed intracellularly, some extracellularly and some having a mixed lifestyle. The impact of extracellular endosymbionts on the biofluids they colonize (e.g. insect hemolymph) is however difficult to appreciate because biofluid composition can depend on the contribution of numerous tissues. Here we investigate Drosophila hemolymph proteome changes in response to the infection with the endosymbiont Spiroplasma poulsonii. S. poulsonii inhabits the fly hemolymph and gets vertically transmitted over generations by hijacking the oogenesis in females. Using dual proteomics on infected hemolymph, we uncovered a weak, chronic activation of the Toll immune pathway by S. poulsonii that was previously undetected by transcriptomics-based approaches. Using Drosophila genetics, we also identified candidate proteins putatively involved in controlling S. poulsonii growth. Last, we also provide a deep proteome of S. poulsonii, which, in combination with previously published transcriptomics data, improves our understanding of the post-transcriptional regulations operating in this bacterium.

Renal Purge of Hemolymphatic Lipids Prevents the Accumulation of ROS-Induced Inflammatory Oxidized Lipids and Protects Drosophila from Tissue Damage.

Animals require complex metabolic and physiological adaptations to maintain the function of vital organs in response to environmental stresses and infection. Here, we found that infection or injury in Drosophila induced the excretion of hemolymphatic lipids by Malpighian tubules, the insect kidney. This lipid purge was mediated by a stress-induced lipid-binding protein, Materazzi, which was enriched in Malpighian tubules. Flies lacking materazzi had higher hemolymph concentrations of reactive oxygen species (ROS) and increased lipid peroxidation. These flies also displayed Malpighian tubule dysfunction and were susceptible to infections and environmental stress. Feeding flies with antioxidants rescued the materazzi phenotype, indicating that the main role of Materazzi is to protect the organism from damage caused by stress-induced ROS. Our findings suggest that purging hemolymphatic lipids presents a physiological adaptation to protect host tissues from excessive ROS during immune and stress responses, a process that is likely to apply to other organisms.

Blind killing of both male and female Drosophila embryos by a natural variant of the endosymbiotic bacterium Spiroplasma poulsonii.

Spiroplasma poulsonii is a vertically transmitted endosymbiont of Drosophila melanogaster that causes male-killing, that is the death of infected male embryos during embryogenesis. Here, we report a natural variant of S. poulsonii that is efficiently vertically transmitted yet does not selectively kill males, but kills rather a subset of all embryos regardless of their sex, a phenotype we call 'blind-killing'. We show that the natural plasmid of S. poulsonii has an altered structure: Spaid, the gene coding for the male-killing toxin, is deleted in the blind-killing strain, confirming its function as a male-killing factor. Then we further investigate several hypotheses that could explain the sex-independent toxicity of this new strain on host embryos. As the second non-male-killing variant isolated from a male-killing original population, this new strain raises questions on how male-killing is maintained or lost in fly populations. As a natural knock-out of Spaid, which is unachievable yet by genetic engineering approaches, this variant also represents a valuable tool for further investigations on the male-killing mechanism.

The Exchangeable Apolipoprotein Nplp2 Sustains Lipid Flow and Heat Acclimation in Drosophila.

In ectotherms, increased ambient temperature requires the organism to consume substantial amounts of energy to sustain a higher metabolic rate, prevent cellular damage, and respond to heat stress. Here, we identify a heat-inducible apolipoprotein required for thermal acclimation in Drosophila. Neuropeptide-like precursor 2 (Nplp2) is an abundant hemolymphatic protein thought to be a neuropeptide. In contrast, we show that Nplp2 contributes to lipid transport, functioning as an exchangeable apolipoprotein. More precisely, Nplp2-deficient flies accumulate lipids in their gut, have reduced fat stores, and display a dyslipoproteinemia, showing that Nplp2 is required for dietary lipid assimilation. Importantly, Nplp2 is induced upon thermal stress and contributes to survival upon heat stress. We propose that Nplp2 associates with lipoprotein particles under homeostatic and high energy-demand conditions to optimize fat transport and storage. Our study also shows that modulation of the lipid uptake and transport machinery is part of an integrated cytoprotective response.

Thioester-containing proteins regulate the Toll pathway and play a role in Drosophila defence against microbial pathogens and parasitoid wasps.

BACKGROUND: Members of the thioester-containing protein (TEP) family contribute to host defence in both insects and mammals. However, their role in the immune response of Drosophila is elusive. In this study, we address the role of TEPs in Drosophila immunity by generating a mutant fly line, referred to as TEPq Δ , lacking the four immune-inducible TEPs, TEP1, 2, 3 and 4. RESULTS: Survival analyses with TEPq Δ flies reveal the importance of these proteins in defence against entomopathogenic fungi, Gram-positive bacteria and parasitoid wasps. Our results confirm that TEPs are required for efficient phagocytosis of bacteria, notably for the two Gram-positive species tested, Staphylococcus aureus and Enterococcus faecalis. Furthermore, we show that TEPq Δ flies have reduced Toll pathway activation upon microbial infection, resulting in lower expression of antimicrobial peptide genes. Epistatic analyses suggest that TEPs function upstream or independently of the serine protease ModSP at an initial stage of Toll pathway activation. CONCLUSIONS: Collectively, our study brings new insights into the role of TEPs in insect immunity. It reveals that TEPs participate in both humoral and cellular arms of immune response in Drosophila. In particular, it shows the importance of TEPs in defence against Gram-positive bacteria and entomopathogenic fungi, notably by promoting Toll pathway activation.

Enhanced hepatotoxicity by acetaminophen in Vanin-1 knockout mice is associated with deficient proliferative and immune responses.

BACKGROUND AND AIMS: Pretreatment with clofibrate, a peroxisome proliferator-activated receptor alpha (PPARa) agonist, protects mice from acetaminophen (APAP) injury. Protection is not due to alterations in APAP metabolism and is dependent on PPARa expression. Gene array analysis revealed that mice receiving clofibrate have enhanced hepatic Vanin-1 (Vnn1) gene expression, a response that is also PPARa dependent. METHODS: We examined the role of Vnn1 by comparing the responses of Vnn1 knockout and wild-type mice following APAP hepatotoxicity. APAP metabolism, hepatotoxicity, and compensatory hepatocyte proliferation and immune responses were assessed. RESULTS: Vnn1 knockout mice are more susceptible to APAP hepatotoxicity despite no differences in hepatic glutathione content, gene expression of APAP metabolizing enzymes, or hepatic capacity to bioactivate or detoxify APAP ex vivo. Together, these data strongly suggest that the susceptibility of Vnn1 knockout mice is not due to differences in APAP metabolism. Immunochemistry revealed a lack of proliferating cell nuclear antigen-positive hepatocytes and F4/80-positive macrophages in and around areas of centrilobular necrosis in APAP-treated Vnn1 knockouts. Hepatic gene induction of pro-inflammatory cytokines was either significantly reduced or completely blunted in these mice. This was correlated with a reduction in early recruitment of cells positive for granulocyte differentiation antigen 1 or integrin alpha M. Heightened toxicity was also observed in CCl4 and ConA hepatitis models in the absence of Vnn1. CONCLUSIONS: These results indicate that mice lacking Vnn1 have deficiencies in compensatory repair and immune responses following toxic APAP exposure and that these mechanisms may contribute to the enhanced hepatotoxicity seen.

Serum pantetheinase/vanin levels regulate erythrocyte homeostasis and severity of malaria.

Tissue pantetheinase, encoded by the VNN1 gene, regulates response to stress, and previous studies have shown that VNN genes contribute to the susceptibility to malaria. Herein, we evaluated the role of pantetheinase on erythrocyte homeostasis and on the development of malaria in patients and in a new mouse model of pantetheinase insufficiency. Patients with cerebral malaria have significantly reduced levels of serum pantetheinase activity (PA). In mouse, we show that a reduction in serum PA predisposes to severe malaria, including cerebral malaria and severe anemia. Therefore, scoring pantetheinase in serum may serve as a severity marker in malaria infection. This disease triggers an acute stress in erythrocytes, which enhances cytoadherence and hemolysis. We speculated that serum pantetheinase might contribute to erythrocyte resistance to stress under homeostatic conditions. We show that mutant mice with a reduced serum PA are anemic and prone to phenylhydrazine-induced anemia. A cytofluorometric and spectroscopic analysis documented an increased frequency of erythrocytes with an autofluorescent aging phenotype. This is associated with an enhanced oxidative stress and shear stress-induced hemolysis. Red blood cell transfer and bone marrow chimera experiments show that the aging phenotype is not cell intrinsic but conferred by the environment, leading to a shortening of red blood cell half-life. Therefore, serum pantetheinase level regulates erythrocyte life span and modulates the risk of developing complicated malaria.

Sox17 regulates liver lipid metabolism and adaptation to fasting.

Liver is a major regulator of lipid metabolism and adaptation to fasting, a process involving PPARalpha activation. We recently showed that the Vnn1 gene is a PPARalpha target gene in liver and that release of the Vanin-1 pantetheinase in serum is a biomarker of PPARalpha activation. Here we set up a screen to identify new regulators of adaptation to fasting using the serum Vanin-1 as a marker of PPARalpha activation. Mutagenized mice were screened for low serum Vanin-1 expression. Functional interactions with PPARalpha were investigated by combining transcriptomic, biochemical and metabolic approaches. We characterized a new mutant mouse in which hepatic and serum expression of Vanin-1 is depressed. This mouse carries a mutation in the HMG domain of the Sox17 transcription factor. Mutant mice display a metabolic phenotype featuring lipid abnormalities and inefficient adaptation to fasting. Upon fasting, a fraction of the PPARα-driven transcriptional program is no longer induced and associated with impaired fatty acid oxidation. The transcriptional phenotype is partially observed in heterozygous Sox17+/- mice. In mutant mice, the fasting phenotype but not all transcriptomic signature is rescued by the administration of the PPARalpha agonist fenofibrate. These results identify a novel role for Sox17 in adult liver as a modulator of the metabolic adaptation to fasting.

PPAR-alpha dependent regulation of vanin-1 mediates hepatic lipid metabolism.

BACKGROUND & AIMS: Peroxisome proliferator-activated receptor alpha (PPARα) is a key regulator of hepatic fat oxidation that serves as an energy source during starvation. Vanin-1 has been described as a putative PPARα target gene in liver, but its function in hepatic lipid metabolism is unknown. METHODS: We investigated the regulation of vanin-1, and total vanin activity, by PPARα in mice and humans. Furthermore, the function of vanin-1 in the development of hepatic steatosis in response to starvation was examined in Vnn1 deficient mice, and in rats treated with an inhibitor of vanin activity. RESULTS: Liver microarray analyses reveals that Vnn1 is the most prominently regulated gene after modulation of PPARα activity. In addition, activation of mouse PPARα regulates hepatic- and plasma vanin activity. In humans, consistent with regulation by PPARα, plasma vanin activity increases in all subjects after prolonged fasting, as well as after treatment with the PPARα agonist fenofibrate. In mice, absence of vanin-1 exacerbates the fasting-induced increase in hepatic triglyceride levels. Similarly, inhibition of vanin activity in rats induces accumulation of hepatic triglycerides upon fasting. Microarray analysis reveal that the absence of vanin-1 associates with gene sets involved in liver steatosis, and reduces pathways involved in oxidative stress and inflammation. CONCLUSIONS: We show that hepatic vanin-1 is under extremely sensitive regulation by PPARα and that plasma vanin activity could serve as a readout of changes in PPARα activity in human subjects. In addition, our data propose a role for vanin-1 in regulation of hepatic TG levels during fasting.

PPARalpha regulates the production of serum Vanin-1 by liver.

The membrane-bound Vanin-1 pantetheinase regulates tissue adaptation to stress. We investigated Vnn1 expression and its regulation in liver. Vnn1 is expressed by centrolobular hepatocytes. Using novel tools, we identify a soluble form of Vnn1 in mouse and human serum and show the contribution of a cysteine to its catalytic activity. We show that liver contributes to Vanin-1 secretion in serum and that PPARalpha is a limiting factor in serum Vnn1 production. Functional PPRE sites are identified in the Vnn1 promoter. These results indicate that serum Vnn1 might be a reliable reporter of PPARalpha activity in liver.