Agnieszka Zdybicka-Barabas

Purification and characterization of eight peptides from Galleria mellonella immune hemolymph

Defense peptides play a crucial role in insect innate immunity against invading pathogens. From the hemolymph of immune-challenged greater wax moth, Galleria mellonella (Gm) larvae, eight peptides were isolated and characterized. Purified Gm peptides differ considerably in amino acid sequences, isoelectric point values and antimicrobial activity spectrum. Five of them, Gm proline-rich peptide 2, Gm defensin-like peptide, Gm anionic peptides 1 and 2 and Gm apolipophoricin, were not described earlier in G. mellonella. Three others, Gm proline-rich peptide 1, Gm cecropin D-like peptide and Galleria defensin, were identical with known G. mellonella peptides. Gm proline-rich peptides 1 and 2 and Gm anionic peptide 2, had unique amino acid sequences and no homologs have been found for these peptides. Antimicrobial activity of purified peptides was tested against gram-negative and gram-positive bacteria, yeast and filamentous fungi. The most effective was Gm defensin-like peptide which inhibited fungal and sensitive bacteria growth in a concentration of 2.9 and 1.9 microM, respectively. This is the first report describing at least a part of defense peptide repertoire of G. mellonella immune hemolymph.

A different repertoire of Galleria mellonella antimicrobial peptides in larvae challenged with bacteria and fungi.

To date, functioning of insect humoral immune response is especially well described in Diptera. The mechanisms of pathogen recognition, activation of signaling pathways and regulation of antimicrobial defense peptide expression are relatively well known. The present paper demonstrates evidence that the immune system of the Lepidoptera moth, Galleria mellonella, is also able to distinguish between different classes of microorganisms and responds to the invading pathogen accordingly. G. mellonella larvae were challenged with Gram-negative and Gram-positive bacteria as well as with yeast and filamentous fungus cells. Subsequently, 24, 48 and 72 h after immunization, the concentrations of lysozyme and six defense peptides were determined in the hemolymph by the HPLC technique. The compounds studied demonstrated variability both in the kinetics of the increase as well as in the concentrations reached. The Gram-negative bacterium and filamentous fungus were particularly effective immunogens, especially affecting the levels of lysozyme, Galleria defensin, proline-rich peptide 2 and cecropin D-like peptide.

Insect antimicrobial peptides show potentiating functional interactions against Gram-negative bacteria

Antimicrobial peptides (AMPs) and proteins are important components of innate immunity against pathogens in insects. The production of AMPs is costly owing to resource-based trade-offs, and strategies maximizing the efficacy of AMPs at low concentrations are therefore likely to be advantageous. Here, we show the potentiating functional interaction of co-occurring insect AMPs (the bumblebee linear peptides hymenoptaecin and abaecin) resulting in more potent antimicrobial effects at low concentrations. Abaecin displayed no detectable activity against Escherichia coli when tested alone at concentrations of up to 200 μM, whereas hymenoptaecin affected bacterial cell growth and viability but only at concentrations greater than 2 μM. In combination, as little as 1.25 μM abaecin enhanced the bactericidal effects of hymenoptaecin. To understand these potentiating functional interactions, we investigated their mechanisms of action using atomic force microscopy and fluorescence resonance energy transfer-based quenching assays. Abaecin was found to reduce the minimal inhibitory concentration of hymenoptaecin and to interact with the bacterial chaperone DnaK (an evolutionarily conserved central organizer of the bacterial chaperone network) when the membrane was compromised by hymenoptaecin. These naturally occurring potentiating interactions suggest that combinations of AMPs could be used therapeutically against Gram-negative bacterial pathogens that have acquired resistance to common antibiotics.

Involvement of apolipophorin III in antibacterial defense of Galleria mellonella larvae.

Apolipophorin III (apoLp-III) is an abundant hemolymph protein involved in lipid transport and immune response in insects. We investigated involvement of apoLp-III in the antibacterial response in Galleria mellonella larvae. Immune challenge with Gram-negative (Escherichia coli, Klebsiella pneumoniae) and Gram-positive (Micrococcus luteus) bacteria led to an increase in the level of apoLp-III in G. mellonella hemolymph, 0.5-2h and 8h after treatment, respectively. ApoLp-III purified from larval hemolymph as well as that present in hemolymph extracts adsorbed on the surface of different bacteria. The adsorption capacity of apoLp-III on bacterial cells prompted us to investigate the effect of this phenomenon on bacterial growth. Our results demonstrate antibacterial activity of apoLp-III against selected Gram-positive and Gram-negative bacteria in vitro. Among bacteria tested, Salmonella typhimurium and K. pneumoniae were the most sensitive to apoLp-III. LIVE/DEAD staining of bacteria incubated with purified apoLp-III revealed their growth inhibition; however, neither morphological changes in the cell shape nor formation of cell aggregates was noticed. The results suggest that apoLp-III is a multifunctional protein in G. mellonella hemolymph.

Synergistic action of Galleria mellonella apolipophorin III and lysozyme against Gram-negative bacteria.

Insect immune response relies on the humoral and cellular mechanisms of innate immunity. The key factors are the antimicrobial polypeptides that act in concert against invading pathogens. Several such components, e.g. apolipophorin III (apoLp-III), lysozyme, and anionic peptide 2, are present constitutively in the hemolymph of non-challenged Galleria mellonella larvae. In the present study, we demonstrate an evidence for a synergistic action of G. mellonella lysozyme and apoLp-III against Gram-negative bacteria, providing novel insights into the mode of action of these proteins in insect antimicrobial defense. It was found that the muramidase activity of G. mellonella lysozyme considerably increased in the presence of apoLp-III. Moreover, apoLp-III enhanced the permeabilizing activity of lysozyme toward Escherichia coli cells. As shown using non-denaturing PAGE, the proteins did not form intermolecular complexes in vivo and in vitro, indicating that the effect observed was not connected with the intermolecular interactions between the proteins. Analysis of AFM images of E. coli cells exposed to G. mellonella lysozyme and/or apoLp-III revealed evident alterations in the bacterial surface structure accompanied by the changes in their biophysical properties. The bacterial cells demonstrated significant differences in elasticity, reflected by Youngs modulus, as well as in adhesive forces and roughness values in comparison to the control ones. The constitutive presence of these two defense molecules in G. mellonella hemolymph and the fact that apoLp-III enhances lysozyme muramidase and perforating activities indicate that they can be regarded as important antibacterial factors acting at the early stage of infection against Gram-negative as well as Gram-positive bacteria.

An atomic force microscopy study of Galleria mellonella apolipophorin III effect on bacteria.

Apolipophorin III (apoLp-III) is an abundant hemolymph protein involved in lipid transport and immune response in insects. As revealed by LIVE/DEAD staining, incubation of Gram-negative and Gram-positive bacteria in the presence of Galleria mellonella apoLp-III led to growth inhibition of selected bacteria. An atomic force microscopy (AFM) study of bacterial cells after apoLp-III treatment showed considerable alterations in the cell surface of Bacillus circulans, Klebsiella pneumoniae and Salmonella typhimurium. Our results clearly demonstrate that apoLp-III disturbed the proper structure of the bacterial cell surface. The alterations were dissimilar to those caused by cationic antimicrobial peptide, cecropin B, suggesting a different mode of action against bacteria. The present results indicate that AFM provides a powerful tool for studying the interactions of apoLp-III with microbial cells.

Synergistic action of Galleria mellonella anionic peptide 2 and lysozyme against Gram-negative bacteria.

Lysozyme and antimicrobial peptides are key factors of the humoral immune response in insects. In the present work lysozyme and anionic defense peptide (GMAP2) were isolated from the hemolymph of the greater wax moth Galleria mellonella and their antibacterial activity was investigated. Adsorption of G. mellonella lysozyme on the cell surface of Gram-positive and Gram-negative bacteria was demonstrated using immunoblotting with anti-G. mellonella lysozyme antibodies. Lysozyme effectively inhibited the growth of selected Gram-positive bacteria, which was accompanied by serious alterations of the cell surface, as revealed by atomic force microscopy (AFM) imaging. G. mellonella lysozyme used in concentrations found in the hemolymph of naive and immunized larvae, perforated also the Escherichia coli cell membrane and the level of such perforation was considerably increased by GMAP2. GMAP2 used alone did not perforate E. coli cells nor influence lysozyme muramidase activity. However, the peptide induced a decrease in the turgor pressure of the bacterial cell. Moreover, in the samples of bacteria treated with a mixture of lysozyme and GMAP2 the sodium chloride crystals were found, suggesting disturbance of ion transport across the membrane leading to cell disruption. These results clearly indicated the synergistic action of G. mellonella lysozyme and anionic peptide 2 against Gram-negative bacteria. The reported results suggested that, thanks to immune factors constitutively present in hemolymph, G. mellonella larvae are to some extent protected against infection caused by Gram-negative bacteria.

Anti-Legionella dumoffii activity of Galleria mellonella defensin and apolipophorin III.

The gram-negative bacterium Legionella dumoffii is, beside Legionella pneumophila, an etiological agent of Legionnaires’ disease, an atypical form of pneumonia. The aim of this study was to determine the antimicrobial activity of Galleria mellonella defense polypeptides against L. dumoffii. The extract of immune hemolymph, containing a mixture of defense peptides and proteins, exhibited a dose-dependent bactericidal effect on L. dumoffii. The bacterium appeared sensitive to a main component of the hemolymph extract, apolipophorin III, as well as to a defense peptide, Galleria defensin, used at the concentrations 0.4 mg/mL and 40 μg/mL, respectively. L. dumoffii cells cultured in the presence of choline were more susceptible to both defense factors analyzed. A transmission electron microscopy study of bacterial cells demonstrated that Galleria defensin and apolipophorin III induced irreversible cell wall damage and strong intracellular alterations, i.e., increased vacuolization, cytoplasm condensation and the appearance of electron-white spaces in electron micrographs. Our findings suggest that insects, such as G. mellonella, with their great diversity of antimicrobial factors, can serve as a rich source of compounds for the testing of Legionella susceptibility to defense-related peptides and proteins.

Studies on the role of insect hemolymph polypeptides: Galleria mellonella anionic peptide 2 and lysozyme.

The lysozymes are well known antimicrobial polypeptides exhibiting antibacterial and antifungal activities. Their antibacterial potential is related to muramidase activity and non-enzymatic activity resembling the mode of action of cationic defense peptides. However, the mechanisms responsible for fungistatic and/or fungicidal activity of lysozyme are still not clear. In the present study, the anti-Candida albicans activity of Galleria mellonella lysozyme and anionic peptide 2 (AP2), defense factors constitutively present in the hemolymph, was examined. The lysozyme inhibited C. albicans growth in a dose-dependent manner. The decrease in the C. albicans survival rate caused by the lysozyme was accompanied by a considerable reduction of the fungus metabolic activity, as revealed by LIVE/DEAD staining. In contrast, although AP2 reduced C. albicans metabolic activity, it did not influence its survival rate. Our results suggest fungicidal action of G. mellonella lysozyme and fungistatic activity of AP2 toward C. albicans cells. In the presence of AP2, the anti-C. albicans activity of G. mellonella lysozyme increased. Moreover, when the fungus was incubated with both defense factors, true hyphae were observed besides pseudohyphae and yeast-like C. albicans cells. Atomic force microscopy analysis of the cells exposed to the lysozyme and/or AP2 revealed alterations in the cell surface topography and properties in comparison with the control cells. The results indicate synergistic action of G. mellonella AP2 and lysozyme toward C. albicans. The presence of both factors in the hemolymph of naive larvae suggests their important role in the early stages of immune response against fungi in G. mellonella.

The effect of Galleria mellonella apolipophorin III on yeasts and filamentous fungi.

Galleria mellonella apolipophorin III (apoLp-III) has been implicated in the innate immune response against bacterial infections. The protein binds components of bacterial cell wall and inhibits growth of selected Gram-positive and Gram-negative bacteria. Interaction of apoLp-III with fungal β-1,3-glucan suggests antifungal properties of the protein. In the present study, the effect of apoLp-III on the growth, metabolic activity and cell surface characteristics of selected yeasts and filamentous fungi was investigated using light, confocal and atomic force microscopy. ApoLp-III bound to the cell surface of different yeasts and filamentous fungi as confirmed by immunoblotting with anti-apoLp-III antibodies. Incubation of the fungi in the presence of apoLp-III induced alterations in growth morphology. Candida albicans underwent transition from yeast-like to hyphal growth with formation of true hyphae, whereas Fusarium oxysporum hyphae exhibited decreased metabolic activity, increased vacuolization and appearance of numerous monophialids with microconidia. Atomic force microscopy imaging demonstrated evident alterations in the fungal cell surface after incubation with apoLp-III, suggesting that the protein affected the cell wall components.