Immunosuppressive effect of cyclosporin A on insect humoral immune response

Cyclosporin A suppressed humoral immune response of Galleria mellonella larvae. Insects were immunized with LPS Pseudomonas aeruginosa and then injected with cyclosporin A. Immunosuppressive effects were expressed both, in larvae treated with cyclosporin A at the initial phase of immune response and at the effector phase of antibacterial immunity. Cyclosporin A moderately decreased lysozyme activity and significantly decreased antibacterial activity peptides against Escherichia coli. Immunosuppressive effects of cyclosporin A were observed after immunoblotting with antibodies anti-G. mellonella lysozyme. Tricine SDS/PAGE shown that synthesis of antibacterial peptides of larvae treated with cyclosporin A was considerably inhibited. Insects of impaired immune response by cyclosporin A action lost protective immunity to insect bacterial pathogen P. aeruginosa.     

Elastase B of Pseudomonas aeruginosa stimulates the humoral immune response in the greater wax moth, Galleria mellonella

The role of Pseudomonas aeruginosa elastase B in activation of the humoral immune response in Galleria mellonella larvae was investigated. The results of our study showed that elastase B injected at a sublethal concentration was responsible for eliciting the humoral immune response in G. mellonella larvae. The insects exhibited increased antibacterial activity, namely, we observed appearance of antimicrobial peptides and a higher level of lysozyme in cell-free hemolymph. Elastase B seems to be a more potent elicitor than thermolysin because similar maximal antibacterial activity levels were observed at a 5-fold lower concentration. We also demonstrated that there were differences in the kinetics of induction of antimicrobial activity between thermolysin and elastase B. The maximum level was observed 18 h post challenge of thermolysin and 38 h after injection of elastase B. It was also shown that, 24 h after elastase injection, the relative levels of apoLp-III in the hemolymph significantly increased in comparison with control G. mellonella larvae. The activation of immune responses in metalloproteinase-challenged larvae involved synthesis of metalloproteinase inhibitors which increased the survival rates of insects both against the lethal dose of thermolysin as well as against viable pathogenic bacterial cells of P. aeruginosa.     

The effect of Habrobracon hebetor venom on the activity of the prophenoloxidase system, the generation of reactive oxygen species and encapsulation in the haemolymph of Galleria mellonella larvae

The cellular and humoral immune reactions in haemolymph of the wax moth Galleria mellonella larvae naturally injected by venom of ectoparasitic wasp Habrobracon hebetor were analyzed. A strong decline of phenoloxidase (PO) activity in the haemolymph and the number of haemocytes with PO activity of envenomated wax moth was observed. In addition, it has been shown that the rate of capsule melanization in the envenomated larvae was half that of the control. Also production of reactive oxygen species (ROS) in the haemolymph of envenomated larvae decreased. The obtained data casts light on the suppression of the main immune reactions in G. mellonella larvae during natural envenomation by H. hebetor.     

Insect immunity: Induction of cecropin and attacin-like antibacterial factors in the haemolymph of Glossina morsitans morsitans

Injections of live Escherichia coli into adult tsetse flies, Glossina morsitans morsitans induced an antibacterial activity in the haemolymph after a lag period of 6–18 hr. Peak activity occurred after 24–72 hr with a dose of 10⁴ bacteria/fly. Acidic electrophoresis of immune haemolymph from G. m. morsitans followed by an antibacterial assay on the gel revealed the presence of cecropin- and attacin-like factors. The induction of antibacterial activity in tsetse was completely blocked by injection of cycloheximide, a known inhibitor of protein synthesis in eukaryotic organisms. Purified InA from Bacillus thuringiensis, a proteolytic enzyme with specificity for cecropins and attacins in haemolymph, inactivated the antibacterial activity in tesetse immune haemolymph. When tested against 10 different bacterial species, the spectrum was the same for the antibacterial activity in immune haemolymph from tsetse and Cecropia.     

Immune reactions of Chironomus larvae (Insecta: Diptera) against bacteria

Humoral encapsulation (“melanization”) represents the predominant defence reaction of Chironomus larvae against injected bacteria. Only low levels of phagocytic activity were observed; cellular encapsulation and nodule formation were completely missing due to low numbers of haemocytes. No other humoral antibacterial activity was detected in normal Chironomus haemolymph and even haemolymph of preinjected (“immunized”) Chironomus larvae showed little inhibition of bacterial growth on agar test plates.Low cellular and lytic activity of Chironomus haemolymph against bacteria is well compensated for by its fast and efficient capacity of humoral encapsulation. Within 5–10 min, even high numbers of injected bacteria (up to 10⁵ per larva) were surrounded by capsular material. Within this range of injection dose, the fates of pathogenic and non-pathogenic strains were identical, and bacteria which are highly pathogenic for many other insects, e.g. larvae of Galleria mellonella, proved to be harmless to Chironomus larvae. The rapidity of humoral encapsulation may prevent the release of toxins or enzymes by which pathogenic bacteria normally damage its host and weaken its immune system.     

Effect of Pseudomonas aeruginosa crude proteolytic fraction on antibacterial activity of Galleria mellonella haemolymph

The antibacterial activity of immune haemolymph Galleria mellonella directed against Escherichia coli D31 was destroyed by Pseudomonas aeruginosa crude proteolytic fraction. This was demonstrated by diffusion well assay and acid gel electrophoresis and subsequent bioautography. On the contrary, lysozyme activity appeared to be insensitive to extracellular proteases of P. aeruginosa when activity was determined using the bioautography method. In addition, no change in lysozyme protein level was observed by immunoblotting with specific antibodies directed against G. mellonella lysozyme, which confirmed that lysozyme was not degraded by the crude proteolytic fraction of P. aeruginosa. However, a significant decrease of lysozyme activity in naive and immune haemolymph exposed to the action of P. aeruginosa proteins determined by using diffusion well assay was observed. Mechanisms of the observed inhibition require further studies.     

Induction kinetics of immune antibacterial proteins in pupae of Galleria mellonella and Pieris brassicae

• 1.1. Pupae of Galleria mellonella and Pieris brassicae given an injection with live, non-pathogenic Enterobacter cloacae or abiotic foreign molecules induce an acquired immunity that corresponds with the synthesis of haemolymph proteins of antibacterial activity.
• 2.2. This humoral defensive response which persists for several days, differs quantitatively between insect species and between the inducers used, although very different foreign bodies induced the same immune proteins in both lepidopteran insects.
• 3.3. A stronger and longer lasting response was consistently noticed in pupae immunized with non-pathogenic bacterium than after sterile nutrient broth injections.
• 4.4. A demonstrably elevated activity of haemolymph lysozyme and trace activity of cecropins found in pupae of Galleria treated with saline W, a salt solution physiological to moths, disappear soon after 36 hr from injection.
• 5.5. In P. brassicae, however, sterile insect Ringer can give a varying, if present at all, immune response.
• 6.6. A mechanical injury (sterile wounding of insect body) can occasionally induce a similar but much weaker response.
• 7.7. The antibacterial activity was drastically reduced in Pieris or completely depressed in most pupae of Galleria when actinomycin D or cycloheximide was given at an early time post-immunization with E. cloacae.
• 8.8. It is concluded that the de novo synthesis of ribonucleic acid and immune proteins is required for expression of antibacterial activity in pupal haemolymphs.
• 9.9. The synthesis of an immune mRNA was completed about 7 hr after the injection of the immunizing bacteria.

     

The virulence of protease and cell surface mutants of Pseudomonas aeruginosa for the larvae of Galleria mellonella

Pseudomonas aeruginosa mucoid strains as well as mutants defective in pili, flagella, lipopolysaccharide, and proteases were isolated and tested for their virulence for the larvae of Galleria mellonella. Of all of the mutants, only the lipopolysaccharide-deficient (rough) strain showed a major decrease in virulence when compared to the wild type. The LD₅₀ of the rough strain was about 30,000 bacteria/larva or roughly 10,000-fold higher than the wild type, suggesting an important role for the lipopolysaccharide in P. aeruginosa infections of G. mellonella larvae.     

Differential synthesis of bacteria-induced proteins of Manduca sexta larvae and pupae

The range of inducible antibacterial and other associated haemolymph proteins in Manduca sexta larvae and pupae was examined by high resolution two-dimensional (2D) isoelectric focusing-polyacrylamide gel electrophoresis. Twenty-two major inducible proteins were consistently resolved on gels of haemolymph from bacteria-injected larvae. Haemolymph from bacteria-injected pupae showed a different pattern of induced proteins. The proteins of the two stages include those which (i) are induced in both stages, (ii) those which are exclusively induced in either larvae or pupae, (iii) those which are inducible in larvae, but consititutively present in pupae, and, (iv) those which are induced in larvae, and which are present at intermediate levels but may be induced to higher levels in pupae.The antibacterial activity of the haemolymph from larvae and pupae was compared on acidpolyacrylamide gels, and the apparent M_r and pI of the inducible proteins determined. Certain of the inducible proteins appear to resemble the cecropin and attacin proteins of Hylophora cecropia.     

Effects of beauverolide L and cyclosporin A on humoral and cellular immune response of the greater wax moth,Galleria mellonella

The effects of beauverolide L and cyclosporin A, cyclic peptidic metabolites, produced by several genera of entomopathogenic fungi on immune responses of last instar larvae of the greater wax moth Galleria mellonella have been examined. Intrahemocoelic injection of either metabolite-coated silica particles or dissolved metabolites in a concentrations ranging between 10 and 30 μg per larva caused no mortality but activated humoral responses in G. mellonella larvae. The challenge induced a significant release of lysozyme and cecropin-like activity into the hemolymph, suggesting stimulatory activity on humoral immune responses. Injected metabolite-coated particles were rapidly surrounded by hemocytes which subsequently accomplished formation of melanized nodules, which increased in size and number compared with controls. In vitro assays with dissolved metabolites indicated no adverse effects of beauverolide L or cyclosporin A on attachment or spreading of isolated plasmatocytes but dose-dependent inhibition of their phagocytic activity. Isolated plasmatocytes incubated with cyclosporin A or beauverolide L exhibited cytoskeleton alterations that differed from those observed in plasmatocytes from infected G. mellonella larvae or reported from other fungal secondary metabolites. The experiments provided further data to elucidate the role of fungal secondary metabolites in development of mycoses in insects.     

Recognition and regulation of metalloproteinase activity in the haemolymph of Galleria mellonella: a new pathway mediating induction of humoral immune responses

Proteolytic activity released within an organism by wounded tissues or invading pathogens can strongly impair the physiological homeostasis when it remains non-regulated. Thus, an efficient mechanism that enables recognition and inactivation of non-regulated proteolytic activity is essential to limit toxic effects. In larvae of the Greater wax moth Galleria mellonella we discovered that injection of bacterial thermolysin at a sublethal concentration mediates both acquired resistance against a subsequently injected lethal concentration of this metalloproteinase and stimulation of humoral immune response accompanied by the synthesis of an inducible metalloproteinase inhibitor (IMPI) which is released within the haemolymph. In search of a putative mechanism mediating recognition and regulation of released microbial metalloproteinases we determined that thermolysin-mediated hydrolysis of G. mellonella haemolymph proteins in vitro yields small (<3 kDa), heat-stable molecules which were discovered to represent potent elicitors of humoral immune responses when injected into untreated larvae. Obtained results allowed to design a model explaining for the first time regulation of released metalloproteinases within the haemolymph of insects. The determined coherence between regulation of released metalloproteinases by IMPI and the simultaneous induction of antimicrobial proteins provides a new insight into the mechanisms leading to expression of genes in course of humoral immune responses.     

Development of Quantitative Proteomics Using iTRAQ Based on the Immunological Response of Galleria mellonella Larvae Challenged with Fusarium oxysporum Microconidia

Galleria mellonella has emerged as a potential invertebrate model for scrutinizing innate immunity. Larvae are easy to handle in host-pathogen assays. We undertook proteomics research in order to understand immune response in a heterologous host when challenged with microconidia of Fusarium oxysporum. The aim of this study was to investigate hemolymph proteins that were differentially expressed between control and immunized larvae sets, tested with F. oxysporum at two temperatures. The iTRAQ approach allowed us to observe the effects of immune challenges in a lucid and robust manner, identifying more than 50 proteins, 17 of them probably involved in the immune response. Changes in protein expression were statistically significant, especially when temperature was increased because this was notoriously affected by F. oxysporum 10⁴ or 10⁶ microconidia/mL. Some proteins were up-regulated upon immune fungal microconidia challenge when temperature changed from 25 to 37°C. After analysis of identified proteins by bioinformatics and meta-analysis, results revealed that they were involved in transport, immune response, storage, oxide-reduction and catabolism: 20 from G. mellonella, 20 from the Lepidoptera species and 19 spread across bacteria, protista, fungi and animal species. Among these, 13 proteins and 2 peptides were examined for their immune expression, and the hypothetical 3D structures of 2 well-known proteins, unannotated for G. mellonella, i.e., actin and CREBP, were resolved using peptides matched with Bombyx mori and Danaus plexippus, respectively. The main conclusion in this study was that iTRAQ tool constitutes a consistent method to detect proteins associated with the innate immune system of G. mellonella in response to infection caused by F. oxysporum. In addition, iTRAQ was a reliable quantitative proteomic approach to detect and quantify the expression levels of immune system proteins and peptides, in particular, it was found that 10⁴ microconidia/mL at 37°C over expressed many more proteins than other treatments.     

Immunity of the greater wax moth Galleria mellonella

Investigation of insect immune mechanisms provides important information concerning innate immunity, which in many aspects is conserved in animals. This is one of the reasons why insects serve as model organisms to study virulence mechanisms of human pathogens. From the evolutionary point of view, we also learn a lot about host–pathogen interaction and adaptation of organisms to conditions of life. Additionally, insect‐derived antibacterial and antifungal peptides and proteins are considered for their potential to be applied as alternatives to antibiotics. While Drosophila melanogaster is used to study the genetic aspect of insect immunity, Galleria mellonella serves as a good model for biochemical research. Given the size of the insect, it is possible to obtain easily hemolymph and other tissues as a source of many immune‐relevant polypeptides. This review article summarizes our knowledge concerning G. mellonella immunity. The best‐characterized immune‐related proteins and peptides are recalled and their short characteristic is given. Some other proteins identified at the mRNA level are also mentioned. The infectious routes used by Galleria natural pathogens such as Bacillus thuringiensis and Beauveria bassiana are also described in the context of host–pathogen interaction. Finally, the plasticity of G. mellonella immune response influenced by abiotic and biotic factors is described.     

5-Adamantan thiadiazole-based thiazolidinones as antimicrobial agents. Design,synthesis, molecular docking and evaluation

In continuation of our efforts to develop new compounds with antimicrobial properties we describe design, synthesis, molecular docking study and evaluation of antimicrobial activity of seventeen novel 2-{[5-(adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-arylidene-1,3-thiazolidin-4-ones. All compounds showed antibacterial activity against eight Gram positive and Gram negative bacterial species. Twelve out of seventeen compounds were more potent than streptomycin and all compounds exhibited higher potency than ampicillin. Compounds were also tested against three resistant bacterial strains: MRSA, P. aeruginosa and E. coli. The best antibacterial potential against ATCC and resistant strains was observed for compound 8 (2-{[5-(adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-(4-nitrobenzylidene)-1,3thiazolidin-4-one). The most sensitive bacterium appeared to be S. typhimirium, followed by B. cereus while L. monocitogenes and M. flavus were the most resistant. Compounds were also tested for their antifungal activity against eight fungal species. All compounds exhibited antifungal activity better than the reference drugs bifonazole and ketokonazole (3-115 times). It was found that compound 8 appeared again to be the most potent. Molecular docking studies on E. coli MurB, MurA as well as C. albicans CYP 51 and dihydrofolate reductase were used for the prediction of mechanism of antibacterial and antifungal activities confirming the experimental results.     

Inflammation in insects: The release of a plasmatocyte depletion factor following interaction between bacteria and haemocytes

Injection of a suspension of the Gram-positive bacterium Bacillus cereus into the haemocoel of Galleria mellonella larvae caused a rapid decrease in the number of circulating plasmatocytes. A similar phenomenon was observed following the injection of cell-free haemolymph collected from larvae previously challenged with bacteria, indicating that plasmatocyte depletion was mediated by a humoral factor released into the haemolymph. The release of plasmatocyte depletion factor could be effected in vitro by incubating adherent haemocytes with a suspension of B. cereus. The in vitro production of the factor did not require the presence of exogenous humoral components, was dose-dependent, was inhibited at low temperatures, and showed the same specificity with respect to bacterial species as observed in vivo. Activity of the plasmatocyte depletion factor destroyed by a 15-min incubation at 56°C. The release of the factor appears to be an early biochemical signal in the complex cellular response of this insect to bacterial infection.     

Studies on the in vivo cellular reactions of insects: Fate of pathogenic and non-pathogenic bacteria in Galleria mellonella nodules

The fate of radioactively-labelled bacteria within nodules of Galleria mellonella was examined over time and the percentage relative viabilities of a pathogen, B. cereus, and a non-pathogen, E. coli K12, were compared. Although large numbers of both species were killed within the nodules during the first six hours, B. cereus eventually overcame the insect's defence mechanisms and multiplied some 200-fold in the haemolymph, inducing a secondary bacteraemia and larval death. In contrast, E. coli remained entrapped in the cell aggregates and failed to kill their hosts. The nature and implication of an active killing mechanism within the nodules is discussed.     

Characterisation of immune responses in the pea aphid, Acyrthosiphon pisum

The innate immune system of insects provides effective defence against a range of parasites and pathogens. The pea aphid, Acyrthosiphon pisum, is a novel study system for investigating host-parasite interactions due to its complex associations with both well-characterised bacterial symbionts and a diversity of pathogens and parasites, including several important biological control agents. However, little is known about the cellular and humoral immune responses of aphids. Here we identify three morphologically distinct types of haemocytes in circulation that we name prohemocytes, granulocytes and oenocytoids. Granulocytes avidly phagocytose Gram negative Escherechia coli and Gram positive Micrococcus luteus while oenocytoids exhibit melanotic activity. Prohaemocytes increase in abundance immediately following an immune challenge, irrespective of the source of stimulus. Pea aphids form melanotic capsules around Sephadex beads but do not form cellular capsules. We also did not detect any antimicrobial peptide activity in the haemolymph using zone of inhibition assays. We discuss these results in relation to recent findings from the pea aphid genome annotation project that suggest that aphids have a reduced immune gene repertoire compared to other insects.     

A peptidomics study reveals the impressive antimicrobial peptide arsenal of the wax moth Galleria mellonella

The complete antimicrobial peptide repertoire of Galleria mellonella was investigated for the first time by LC/MS. Combining data from separate trypsin, Glu-C and Asp-N digests of immune hemolymph allowed detection of 18 known or putative G. mellonella antimicrobial peptides or proteins, namely lysozyme, moricin-like peptides (5), cecropins (2), gloverin, Gm proline-rich peptide 1, Gm proline-rich peptide 2, Gm anionic peptide 1 (P1-like), Gm anionic peptide 2, galiomicin, gallerimycin, inducible serine protease inhibitor 2, 6tox and heliocin-like peptide. Six of these were previously known only as nucleotide sequences, so this study provides the first evidence for expression of these genes. LC/MS data also provided insight into the expression and processing of the antimicrobial Gm proline-rich peptide 1. The gene for this peptide was isolated and shown to be unique to moths and to have an unusually long precursor region (495 bp). The precursor region contained other proline-rich peptides and LC/MS data suggested that these were being specifically processed and were present in hemolymph at very high levels. This study shows that G. mellonella can concurrently release an impressive array of at least 18 known or putative antimicrobial peptides from 10 families to defend itself against invading microbes.