Glucose and trehalose metabolism through the cyclic pentose phosphate pathway shapes pathogen resistance and host protection in Drosophila.

Activation of immune cells requires the remodeling of cell metabolism in order to support immune function. We study these metabolic changes through the infection of Drosophila larvae by parasitoid wasp. The parasitoid egg is neutralized by differentiating lamellocytes, which encapsulate the egg. A melanization cascade is initiated, producing toxic molecules to destroy the egg while the capsule also protects the host from the toxic reaction. We combined transcriptomics and metabolomics, including 13C-labeled glucose and trehalose tracing, as well as genetic manipulation of sugar metabolism to study changes in metabolism, specifically in Drosophila hemocytes. We found that hemocytes increase the expression of several carbohydrate transporters and accordingly uptake more sugar during infection. These carbohydrates are metabolized by increased glycolysis, associated with lactate production, and cyclic pentose phosphate pathway (PPP), in which glucose-6-phosphate is re-oxidized to maximize NADPH yield. Oxidative PPP is required for lamellocyte differentiation and resistance, as is systemic trehalose metabolism. In addition, fully differentiated lamellocytes use a cytoplasmic form of trehalase to cleave trehalose to glucose and fuel cyclic PPP. Intracellular trehalose metabolism is not required for lamellocyte differentiation, but its down-regulation elevates levels of reactive oxygen species, associated with increased resistance and reduced fitness. Our results suggest that sugar metabolism, and specifically cyclic PPP, within immune cells is important not only to fight infection but also to protect the host from its own immune response and for ensuring fitness of the survivor.

The Entomopathogenic Fungus Conidiobolus coronatus Has Similar Effects on the Cuticular Free Fatty Acid Profile of Sensitive and Resistant Insects.

The mechanisms underlying the recognition of a susceptible host by a fungus and the role of cuticular compounds (CCs) in this process remain unclear; however, accumulated data suggest that this is influenced to a great degree by cuticular lipids. Two insect species differing in their sensitivity to fungal infection, viz. the highly sensitive Galleria mellonella Linnaeus (Lepidoptera: Pyralidae) and the resistant Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae), exhibited significant qualitative and quantitative changes in cuticular free fatty acid (FFA) profiles after exposure to Conidiobolus coronatus (Constantin) Batko (Entomopthorales). Despite being systematically distant, leading different lifestyles in different habitats, both insect species demonstrated similar changes in the same FFAs following exposure to the fungus (C12:0, C13:0, C14:0, C15:0, C16:1, C16:0, C18:1, C18:0), suggesting that these are involved in a contact-induced defense response. As it was not possible to distinguish the share of FFAs present in the conidia that were attached to the cuticle from the FFAs of the cuticle itself in the total number of extracted FFAs, further research is necessary.

Octanoic Acid-An Insecticidal Metabolite of Conidiobolus coronatus (Entomopthorales) That Affects Two Majors Antifungal Protection Systems in Galleria mellonella (Lepidoptera): Cuticular Lipids and Hemocytes.

The food flavour additive octanoic acid (C8:0) is also a metabolite of the entomopathogenic fungus Conidiobolus coronatus, which efficiently infects and rapidly kills Galleria mellonella. GC-MS analysis confirmed the presence of C8:0 in insecticidal fraction FR3 extracted from C. coronatus filtrate. Topical administration of C8:0 had a dose-dependent effect on survival rates of larvae but not on pupation or adult eclosion times of the survivors. Topically applied C8:0 was more toxic to adults than larvae (LD100 for adults 18.33 ± 2.49 vs. 33.56 ± 2.57 µg/mg of body mass for larvae). The administration of C8:0 on the cuticle of larvae and adults, in amounts corresponding to their LD50 and LD100 doses, had a considerable impact on the two main defense systems engaged in protecting against pathogens, causing serious changes in the developmental-stage-specific profiles of free fatty acids (FFAs) covering the cuticle of larvae and adults and damaging larval hemocytes. In vitro cultures of G. mellonella hemocytes, either directly treated with C8:0 or taken from C8:0 treated larvae, revealed deformation of hemocytes, disordered networking, late apoptosis, and necrosis, as well as caspase 1-9 activation and elevation of 8-OHdG level. C8:0 was also confirmed to have a cytotoxic effect on the SF-9 insect cell line, as determined by WST-1 and LDH tests.

Dodecanol, metabolite of entomopathogenic fungus Conidiobolus coronatus, affects fatty acid composition and cellular immunity of Galleria mellonella and Calliphora vicina.

One group of promising pest control agents are the entomopathogenic fungi; one such example is Conidiobolus coronatus, which produces a range of metabolites. Our present findings reveal for the first time that C. coronatus also produces dodecanol, a compound widely used to make surfactants and pharmaceuticals, and enhance flavors in food. The main aim of the study was to determine the influence of dodecanol on insect defense systems, i.e. cuticular lipid composition and the condition of insect immunocompetent cells; hence, its effect was examined in detail on two species differing in susceptibility to fungal infection: Galleria mellonella and Calliphora vicina. Dodecanol treatment elicited significant quantitative and qualitative differences in cuticular free fatty acid (FFA) profiles between the species, based on gas chromatography analysis with mass spectrometry (GC/MS), and had a negative effect on G. mellonella and C. vicina hemocytes and a Sf9 cell line in vitro: after 48 h, almost all the cells were completely disintegrated. The metabolite had a negative effect on the insect defense system, suggesting that it could play an important role during C. coronatus infection. Its high insecticidal activity and lack of toxicity towards vertebrates suggest it could be an effective insecticide.

The type of blood used to feed Aedes aegypti females affects their cuticular and internal free fatty acid (FFA) profiles.

Aedes aegypti, the primary vector of various arthropod-borne viral (arboviral) diseases such as dengue and Zika, is a popular laboratory model in vector biology. However, its maintenance in laboratory conditions is difficult, mostly because the females require blood meals to complete oogenesis, which is often provided as sheep blood. The outermost layer of the mosquito cuticle is consists of lipids which protects against numerous entomopathogens, prevents desiccation and plays an essential role in signalling processes. The aim of this work was to determine how the replacement of human blood with sheep blood affects the cuticular and internal FFA profiles of mosquitoes reared in laboratory culture. The individual FFAs present in cuticular and internal extracts from mosquito were identified and quantified by GC-MS method. The normality of their distribution was checked using the Kolmogorov-Smirnov test and the Student's t-test was used to compare them. GC-MS analysis revealed similar numbers of internal and cuticular FFAs in the female mosquitoes fed sheep blood by membrane (MFSB) and naturally fed human blood (NFHB), however MFSB group demonstrated 3.1 times greater FFA concentrations in the cuticular fraction and 1.4 times the internal fraction than the NFHB group. In the MFSB group, FFA concentration was 1.6 times higher in the cuticular than the internal fraction, while for NFHB, FFA concentration was 1.3 times lower in the cuticular than the internal fraction. The concentration of C18:3 acid was 223 times higher in the internal fraction than the cuticle in the MHSB group but was absent in the NFHB group. MFSB mosquito demonstrate different FFA profiles to wild mosquitoes, which might influence their fertility and the results of vital processes studied under laboratory conditions. The membrane method of feeding mosquitoes is popular, but our research indicates significant differences in the FFA profiles of MFSB and NFHB. Such changes in FFA profile might influence female fertility, as well as other vital processes studied in laboratory conditions, such as the response to pesticides. Our work indicates that sheep blood has potential shortcomings as a substitute feed for human blood, as its use in laboratory studies may yield different results to those demonstrated by free-living mosquitoes.

Infection of Galleria mellonella (Lepidoptera) Larvae With the Entomopathogenic Fungus Conidiobolus coronatus (Entomophthorales) Induces Apoptosis of Hemocytes and Affects the Concentration of Eicosanoids in the Hemolymph.

Apoptosis and autophagy, the mechanisms of programmed cell death, play critical roles in physiological and pathological processes in both vertebrates and invertebrates. Apoptosis is also known to play an important role in the immune response, particularly in the context of entomopathogenic infection. Of the factors influencing the apoptotic process during infection, two of the lesser known groups are caspases and eicosanoids. The aim of this study was to determine whether infection by the entomopathogenic soil fungus Conidiobolus coronatus is associated with apoptosis and changes in caspase activity in the hemocytes of Galleria mellonella larvae, and to confirm whether fungal infection may affect eicosanoid levels in the host. Larvae were exposed for 24 h to fully grown and sporulating fungus. Hemolymph was collected either immediately after termination of exposure (F24 group) or 24 h later (F48 group). Apoptosis/necrosis tests were performed in hemocytes using fluorescence microscopy and flow cytometry, while ELISA tests were used to measure eicosanoid levels. Apoptosis and necrosis occurred to the same degree in F24, but necrosis predominated in F48. Fungal infection resulted in caspase activation, increased PGE1, PGE2, PGA1, PGF2α, and 8-iso-PGF2α levels and decreased TXB2 levels, but had no effect on TXA2 or 11-dehydro-TXB2 concentrations. In addition, infected larvae demonstrated significantly increased PLA2 activity, known to be involved in eicosanoid biosynthesis. Our findings indicate that fungal infection simultaneously induces apoptosis in insects and stimulates general caspase activity, and this may be correlated with changes in the concentrations of eicosanoids.

Metamorphosis-related changes in the free fatty acid profiles of Sarcophaga (Liopygia) argyrostoma (Robineau-Desvoidy, 1830).

The flies of the Sarcophagidae, widespread throughout the temperate zone, are of great significance in Medicine, Veterinary science, Forensics and Entomotoxicology. Lipids are important elements of cell and organelle membranes and a source of energy for embryogenesis, metamorphosis and flight. Cuticular lipids protect from desiccation and act as recognition cues for species, nest mates and castes, and are a source of various pheromones. The free fatty acid (FFA) profile of cuticular and internal extracts of Sarcophaga (Liopygia) argyrostoma (Robineau-Desvoidy, 1830) larvae, pupae and adults was determined by gas chromatography-mass spectrometry (GC-MS). The larvae, pupae and adults contained FFAs from C5:0 to C28:0. The extracts differed quantitatively and qualitatively from each other: C18:1 > C16:1 > C16:0 > C18:0 predominated in the cuticular and internal extracts from the larvae and adults, while 18:1 > C16:0 > C16:1 > C18:0 predominated in the pupae. The FFA profile of the cuticle varies considerably between each development stage: C23:0 and C25:0 are only present in larvae, C28:0 in the pupal cuticle, and C12:1 and C18:3 in internal extracts from adults. The mechanisms underlying this diversity are discussed herein.

Antifungal polybrominated proxyphylline derivative induces Candida albicans calcineurin stress response in Galleria mellonella.

Candida albicans CNB1 plays a role in the response in vitro and in vivo to stress generated by PB-WUT-01, namely 1,3-dimethyl-7-(2-((1-(3-(perbromo-2H-benzo[d][1,2,3]triazol-2-yl)propyl)-1H-1,2,3-triazol-4-yl)methoxy)propyl)-1H-purine-2,6(3H,7H)-dione. The antifungal mechanism involved the calcineurin pathway-regulated genes SAP9-10. Galleria mellonella treated with PB-WUT-01 (at 0.64 µg/mg) showed limited candidiasis and remained within the highest survival rates. The molecular mode of action of PB-WUT-01 was rationalized by in silico docking studies toward both human and C. albicans calcineurin A (CNA) and calcineurin B (CNB) complexes, respectively. PB-WUT-01 acting as a calcineurin inhibitor in the C. albicans cells enhances the cells' susceptibility. Therefore it could be a suitable alternative treatment in patients with candidiasis.

The interaction between cuticle free fatty acids (FFAs) of the cockroaches Blattella germanica and Blatta orientalis and hydrolases produced by the entomopathogenic fungus Conidiobolus coronatus.

The interactions between entomopathogenic fungi and insects serve a classic example of a co-evolutionary arms race between pathogens and their target host. The cuticle, site of the first contact between insects and entomopathogenic fungus, is an important defensive barrier against pathogens. It is covered by a layer of lipids that appears to play a key role in these processes and cuticular free fatty acid (FFA) profiles are consider as a determinant of susceptibility, or resistance, to fungal infections. These profiles are species-specific. The cockroaches Blattella germanica (Blattodea: Blattidae) and Blatta orientalis (Blattodea: Ectobiidae) are unsusceptible to the soil fungus Conidiobolus coronatus (Entomophthorales: Ancylistaceae) infection, therefore we studied the profiles of FFAs in order to understand the defensive capabilities of the cockroaches. The fungus was cultivated for three weeks in minimal medium. Cell-free filtrate was obtained, assayed for elastase, N-acetylglucosaminidase, chitobiosidase and lipase activity, and then used for in vitro hydrolysis of the cuticle from wings and thoraces of adults and oothecae. The amounts of amino acids, N-glucosamine and FFAs released from the hydrolysed cuticle samples were measured after eight hours of incubation. The FFA profiles of the cuticle of adults, and the wings, thoraces and oothecae of both species were established using GC-MS and the results were correlated with the effectiveness of fungal proteases, chitinases and lipases in the hydrolyzation of cuticle samples. Positive correlations would suggest the existence of compounds used by the fungus as nutrients, whereas negative correlations may indicate that these compounds could be engaged in insect defence.

Sulfone derivatives enter the cytoplasm of Candida albicans sessile cells.

Since our study showed that sulfone derivatives' action mode creates a lesser risk of inducing widespread resistance among Candida spp., we continued verifying sulfones' antifungal activity using the following newly synthesized derivatives: bromodichloromethy-4-hydrazinyl-3-nitrophenyl sulfone (S1), difluoroiodomethyl-4-hydrazinyl-3-nitrophenyl sulfone (S2), and chlorodifluoromethyl-4-hydrazinyl-3-nitrophenyl sulfone (S3). As the mechanism by which sulfones gain access to the cytoplasm has not been elucidated yet, in order to track S1-3, we coupled their hydrazine group with BODIPY (final S1-3 BODIPY-labelled were named SB1-3). This approach allowed us to follow the vital internalization and endocytic routing of SB1-3, while BODIPY interacts primarily with fungal surfaces, thus confirming that S1-3 and their counterparts SB1-2 behaved as non-typical agents by damaging the cell membrane and wall after being endocytosed (SB1-3 fluorescence visible inside the unlysed sessile cells). Thus greatly decreasing the likelihood of the appearance of strains resistance. Core sulfones S1-3 are a promising alternative not only to treat planktonic C. albicans but also biofilm-embedded cells. In the flow cytometric analysis, the planktonic cell surface was digested by S1-3, which made the externalized PS accessible to AnnexinV binding and PI input (accidental cell death ACD). The occurrence of ACD as well as apoptosis (crescent-shaped nuclei) and anoikis of sessile cells (regulated cell death by 100%-reduction in attachment to epithelium) was assessed through monitoring the AO/PI/HO342 markers. CLSM revealed the invasion of S1-3 and SB1-3 in C. albicans without inducing cell lysis. This was a novel approach in which QCM-D was used for real-time in situ detection of viscoelastic changes in the C. albicans biofilm, and its interaction with S1 as a representative of the sulfones tested. S1 (not toxic in vivo) is a potent fungicidal agent against C. albicans and could be administered to treat invasive candidiasis as a monotherapy or in combination with antifungal agents of reference to treat C. albicans infections.

Conidiobolus coronatus induces oxidative stress and autophagy response in Galleria mellonella larvae.

Cell homeostasis requires the correct levels of reactive oxygen species (ROS) to be maintained as these regulate the proliferation and differentiation of cells, and control the immune response and inflammation. High levels of ROS can cause oxidative stress, leading to protein, lipid and DNA damage, or even cell death. Under physiological conditions, the rate of autophagy remains stable; however, it can be accelerated by a number of exogenous stimuli such as oxidative stress, starvation or hypoxia, leading to cell death. The present paper examines the effect of Conidiobolus coronatus infection on the immune response, oxidative stress processes and autophagy in the greater wax moth, Galleria mellonella. Fungal infection was found to result in the disorganization of the cytoskeleton of the larval immune cells and the enhancement of oxidative defense processes. Lipid peroxidation and autophagy were also induced in the hemocytes. Our findings show that G. mellonella is an ideal model for exploring immune mechanisms.

New antifungal 4-chloro-3-nitrophenyldifluoroiodomethyl sulfone reduces the Candida albicans pathogenicity in the Galleria mellonella model organism.

Candida albicans represents an interesting microorganism to study complex host-pathogen interactions and for the development of effective antifungals. Our goal was to assess the efficacy of 4-chloro-3-nitrophenyldifluoroiodomethyl sulfone (named Sulfone) against the C. albicans infections in the Galleria mellonella host model. We assessed invasiveness of CAI4 parental strain and mutants: kex2Δ/KEX2 and kex2Δ/kex2Δ in G. mellonella treated with Sulfone. We determined that KEX2 expression was altered following Sulfone treatment in G. mellonella-C. albicans infection model. Infection with kex2Δ/kex2Δ induced decreased inflammation and minimal fault in fitness of larvae vs CAI4. Fifty percent of larvae died within 4-5 days (P value < 0.0001) when infected with CAI4 and kex2Δ/KEX2 at 109 CFU/mL; survival reached 100% in those injected with kex2Δ/kex2Δ. Larvae treated with Sulfone at 0.01 mg/kg 30 min before infection with all C. albicans tested survived infection at 90-100% vs C. albicans infected-PBS-treated larvae. Hypersensitive to Sulfone, kex2Δ/kex2Δ reduced virulence in survival. KEX2 was down-regulated when larvae were treated with Sulfone: 30 min before and 2 h post-SC5314-wild-type infection respectively. kex2Δ/kex2Δ was able to infect larvae, but failed to kill host when treated with Sulfone. Sulfone can be used to prevent or treat candidiasis. G. mellonella facilitates studding of host-pathogen interactions, i.e., testing host vs panel of C. albicans mutants when antifungal is dosed.

Drosophila macrophages switch to aerobic glycolysis to mount effective antibacterial defense.

Macrophage-mediated phagocytosis and cytokine production represent the front lines of resistance to bacterial invaders. A key feature of this pro-inflammatory response in mammals is the complex remodeling of cellular metabolism towards aerobic glycolysis. Although the function of bactericidal macrophages is highly conserved, the metabolic remodeling of insect macrophages remains poorly understood. Here, we used adults of the fruit fly Drosophila melanogaster to investigate the metabolic changes that occur in macrophages during the acute and resolution phases of Streptococcus-induced sepsis. Our studies revealed that orthologs of Hypoxia inducible factor 1α (HIF1α) and Lactate dehydrogenase (LDH) are required for macrophage activation, their bactericidal function, and resistance to infection, thus documenting the conservation of this cellular response between insects and mammals. Further, we show that macrophages employing aerobic glycolysis induce changes in systemic metabolism that are necessary to meet the biosynthetic and energetic demands of their function and resistance to bacterial infection.

Diet influences the bacterial and free fatty acid profiles of the cuticle of Galleria mellonella larvae.

The evolutionary success of insects is arguably due to their ability to build up a complex, highly-adaptable and very effective defense system against numerous pathogens, including entomopathogenic fungi. This system relies on the humoral immune system and cellular defense reactions. The first line of defense against biological pathogens is a cuticle formed of several layers. The cuticular lipids may contain hydrocarbons, free fatty acids (FFA), alcohols, waxes, glycerides, aldehydes and sterols. Cuticular fatty acids may also play a role in defending against fungal invasion. Our present findings show that the diet of insects can have a significant effect on their sensitivity and defense response to pathogens; for example, while G. mellonella larvae fed on beeswax had a similar appearance to those reared on a semi-artificial diet, they possessed a different cuticular free fatty acid (FFA) profile to those fed on a semi-artificial diet, and were less sensitive to Conidiobolus coronatus infection. It is possible that the presence of heneicosenoic acid (C21:1) and other long-chain free fatty acids (C22:0, C24:0, C26:0), as well as Brevibacillus laterosporus bacteria, on the cuticle of larvae fed on beeswax, plays a protective role against fungal invasion. Insect pests represent a global problem. An understanding of the basic mechanisms underlying the fungal infection of insects might provide a clearer insight into their defenses, thus allowing the design of more effective, and environmentally-friendly, means of controlling them. The greater wax moth is an excellent model for the study of immunology resistance. Knowledge of the influence of diet on pathogen resistance in insects can be also useful for creating a model of human diseases caused by pathogens, such as Candia albicans.

Harman and norharman, metabolites of entomopathogenic fungus Conidiobolus coronatus (Entomopthorales), disorganize development of Galleria mellonella (Lepidoptera) and affect serotonin-regulating enzymes.

Naturally occurring entomopathogenic fungi such as Conidiobolus coronatus are important regulatory factors of insect populations. GC-MS analysis of fungal cell-free filtrates showed that C. coronatus synthesizes two ß- carboline alkaloids: harman and norharman. Significantly higher levels of both alkaloids are produced by C. coronatus in minimal postincubation medium than in rich medium. The beta-carboline alkaloids may have an effect on the nervous system of insects and their behavior. Harman and norharman were applied to Galleria mellonella larvae (a parasite of honeybees) either topically or mixed with food. Larvae received alkaloids in three concentrations: 750, 1000 or 1250 ppm. The effect on the survival and further development of larvae was examined. Both harman and norharman delayed pupation and adult eclosion, and inhibit total monoamine oxidase activity. In addition, they increased the serotonin concentration and decreased the monoamine oxidase A level in the heads of the moths. It is likely that the alkaloids were metabolized by the insects, as their effect wore off 24 hours after topical application. This is the first study to show that C. coronatus produces alkaloids. Its aim was to identify the actions of ß-carboline alkaloids on insect development and serotonin-regulating enzymes. Knowledge of the potential role of harman and norharman in the process of fungal infection might lead to the development of more effective and environmentally-friendly means of controlling insect pests.

Cuticular fatty acids of Galleria mellonella (Lepidoptera) inhibit fungal enzymatic activities of pathogenic Conidiobolus coronatus.

The entomopathogenic fungus Conidiobolus coronatus produces enzymes that may hydrolyze the cuticle of Galleria mellonella. Of these enzymes, elastase activity was the highest: this figure being 24 times higher than NAGase activity 553 times higher than chitinase activity and 1844 times higher than lipase activity. The present work examines the differences in the hydrolysis of cuticles taken from larvae, pupae and adults (thorax and wings), by C. coronatus enzymes. The cuticles of the larvae and adult thorax were the most susceptible to digestion by proteases and lipases. Moreover, the maximum concentration of free N-glucosamine was in the hydrolysis of G. mellonella thorax. These differences in the digestion of the various types of cuticle may result from differences in their composition. GC-MS analysis of the cuticular fatty acids isolated from pupae of G. mellonella confirmed the presence of C 8:0, C 9:0, C 12:0, C 14:0, C 15:0, C 16:1, C 16:0, C 17:0, C 18:1, C 18:0, with C 16:0 and C 18:0 being present in the highest concentrations. Additional fatty acids were found in extracts from G. mellonella imagines: C 10:0, C 13:0, C 20:0 and C 20:1, with a considerable dominance of C 16:0 and C 18:1. In larvae, C 16:0 and C 18:1 predominated. Statistically significant differences in concentration (p≤0.05) were found between the larvae, pupae and imago for each fatty acid. The qualitative and quantitative differences in the fatty acid composition of G. mellonella cuticle occurring throughout normal development might be responsible for the varied efficiency of fungal enzymes in degrading larval, pupal and adult cuticles.

Effect of serine protease KEX2 on Candida albicans virulence under halogenated methyl sulfones.

AIM: The effect of KEX2 mutations on C. albicans virulence and resistance to halogenated methyl sulfones was assessed. MATERIALS & METHODS: The mechanism of action of sulfones was studied using flow cytometry and microscopy. Expression of KEX2 and SAP5 was assessed using quantitative Real-Time-PCR. 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide and lactate dehydrogenase assays were elaborated to study, respectively, metabolism of Candida treated with sulfones and their cytotoxicity against tissues. Inflammatory response was detected by ELISA. RESULTS: Lysosome permeabilization and dose-dependent programmed cell death under sulfones were noted. KEX2 induction depended on halogenomethylsulfonyl groups, which affected cell wall biosynthesis and adhesion. CONCLUSION: Sulfones treatment reduced Candida pathogenicity in Galleria mellonella. Sulfones are an alternative for antifungal therapies due to their safety profile and antibiofilm activity.