Immune priming modulates Galleria mellonella and Pseudomonas entomophila interaction. Antimicrobial properties of Kazal peptide Pr13a.

Galleria mellonella larvae repeatedly infected with Pseudomonas entomophila bacteria re-induced their immune response. Its parameters, i.e. the defence activities of cell-free hemolymph, the presence and activity of antimicrobial peptides, and the expression of immune-relevant genes were modulated after the re-challenge in comparison to non-primed infected larvae, resulting in better protection. No enhanced resistance was observed when the larvae were initially infected with other microorganisms, and larvae pre-infected with P. entomophila were not more resistant to further infection with other pathogens. Then, the peptide profiles of hemolymph from primed- and non-primed larvae infected with P. entomophila were compared by quantitative RP-HPLC (Reverse Phase - High Performance Liquid Chromatography). The level of carbonic anhydrase, anionic peptide-1, proline peptide-2, and finally, unknown so far, putative Kazal peptide Pr13a was higher in the primed infected animals than in the larvae infected with P. entomophila for the first time. The expression of the Pr13a gene increased two-fold after the infection, but only in the primed animals. To check whether the enhanced level of Pr13a could have physiological significance, the peptide was purified to homogeneity and checked for its defence properties. In fact, it had antibacterial activity: at the concentration of 15 µM and 7.5 µM it reduced the number of P. entomophila and Bacillus thuringiensis CFU, respectively, to about 40%. The antibacterial activity of Pr13a was correlated with changes observed on the surface of the peptide-treated bacteria, e.g. surface roughness and adhesion force. The presented results bring us closer to finding hemolymph constituents responsible for the effect of priming on the immune response in re-infected insects.

Defence response of Galleria mellonella larvae to oral and intrahemocelic infection with Pseudomonasentomophila.

We report differences in the course of infection of G. mellonella larvae with P. entomophila via intrahemocelic and oral routes. Survival curves, larval morphology, histology, and induction of defence response were investigated. Larvae injected with 10 and 50 cells of P. entomophila activated a dose-dependent immune response, which was manifested by induction of immune-related genes and dose-dependent defence activity in larval hemolymph. In contrast, after the oral application of the pathogen, antimicrobial activity was detected in whole hemolymph of larvae infected with the 103 but not 105 dose in spite of the induction of immune response manifested as immune-relevant gene expression and defence activity of electrophoretically separated low-molecular hemolymph components. Among known proteins induced after the P. entomophila infection, we identified proline-rich peptide 1 and 2, cecropin D-like peptide, galiomycin, lysozyme, anionic peptide 1, defensin-like peptide, and a 27 kDa hemolymph protein. The expression of the lysozyme gene and the amount of protein in the hemolymph were correlated with inactivity of hemolymph in insects orally infected with a higher dose of P. entomophila, pointing to its role in the host-pathogen interaction.

Cationic protein 8 plays multiple roles in Galleria mellonella immunity.

Galleria mellonella cationic protein 8 (GmCP8) is a hemolymph protein previously identified as an opsonin and an inhibitor of fungal proteases. In this work, we showed its bactericidal activity toward Pseudomonas entomophila, Pseudomonas aeruginosa, Bacillus thuringiensis, Staphylococcus aureus, and Escherichia coli and against yeast-like fungi Candida albicans. The activity against E. coli was correlated with bacterial membrane permeabilization. In turn, in the case of P. entomophila, B. thuringiensis, and C. albicans, the atomic force microscopy analysis of the microbial surface showed changes in the topography of cells and changes in their nanomechanical properties. GmCP8 also showed the inhibitory activity toward the serine protease trypsin and the metalloproteinase thermolysin. The expression of the gene encoding the GmCP8 protein did not increase either in the gut or in the fat body of G. mellonella after oral infection with P. entomophila. Similarly, the amount of GmCP8 in the hemolymph of G. mellonella did not change in immune-challenged insects. However, when GmCP8 was injected into the G. mellonella hemocel, a change in the survival curve was observed in the infected larvae. Our results shed new light on the function of GmCP8 protein in insect immunity, indicating its role in humoral defence mechanisms.

Current understanding of immune priming phenomena in insects.

It may seem that the most important issues related to insect immunity have already been described. However, novel phenomena observed in recent years shed new light on the understanding of the immune response in insects.The adaptive abilities of insects helped them to populate all ecological land niches.One important adaptive ability of insects that facilitates their success is the plasticity of their immune system. Although they only have innate immune mechanisms, insects can increase their resistance after the first encounter with the pathogen. In recent years, this phenomenon,namedimmunepriming, has become a "hot topic" in immunobiology.Priming can occur within or across generations. In the first case, the resistance of a given individual can increase after surviving a previous infection. Transstadial immune priming occurs when infection takes place at one of the initial developmental stages and increased resistance is observed at the pupal or imago stages. Priming across generations (transgenerationalimmune priming, TGIP) relies on the increased resistance of the offspring when one or both parents are infected during their lifetime.Despite the attention that immune priming has received, basic questions remain to be answered, such as regulation of immune priming at the molecular level. Research indicates that pathogen recognition receptors (PRRs) can be involved in the priming phenomenon. Recent studies have highlighted the special role of microRNAs and epigenetics, which can influence expression of genes that can be transmitted through generations although they are not encoded in the nucleotide sequence. Considerable amounts of research are required to fully understand the mechanisms that regulate priming phenomena. The aim of our work is to analyse thoroughly the most important information on immune priming in insects and help raise pertinent questions such that a greater understanding of this phenomenon can be obtained in the future.

General overview on the role of Peptidoglycan Recognition Proteins in insect immunity.

The insect immune system is responsible for maintaining the homeostasis of organisms. If the pathogen is able to breach the defensive barriers of the host, cellular and humoral mechanisms are triggered. Initiation of effective defence response is possible thanks to pathogen-associated molecular patterns, among which peptidoglycan recognition proteins play a prominent role. They recognize pathogen-associated molecular patterns and some of them also have enzymatic activity. The main aim of peptidoglycan recognition proteins is to activate pathways regulating the synthesis of immune peptides. Some of the peptidoglycan recognition proteins are involved in the phagocytosis process, activation of the prophenoloxidase cascade, and regulation of the xenophagy process. The structural diversity and high specificity of peptidoglycan recognition proteins suggests that they can serve many previously unknown functions in insect's systemic response.

The greater wax moth Galleria mellonella: biology and use in immune studies.

The greater wax moth Galleria mellonella is an invertebrate that is increasingly being used in scientific research. Its ease of reproduction, numerous offspring, short development cycle, and finally, its known genome and immune-related transcriptome provide a convenient research model for investigation of insect immunity at biochemical and molecular levels. Galleria immunity, consisting of only innate mechanisms, shows adaptive plasticity, which has recently become the subject of intensive scientific research. This insect serves as a mini host in studies of the pathogenicity of microorganisms and in vivo tests of the effectiveness of single virulence factors as well as new antimicrobial compounds. Certainly, the Galleria mellonella species deserves our attention and appreciation for its contribution to the development of research on innate immune mechanisms. In this review article, we describe the biology of the greater wax moth, summarise the main advantages of using it as a model organism and present some of the main techniques facilitating work with this insect.

Insect Defense Proteins and Peptides.

The composition of insect hemolymph can change depending on many factors, e.g. access to nutrients, stress conditions, and current needs of the insect. In this chapter, insect immune-related polypeptides, which can be permanently or occasionally present in the hemolymph, are described. Their division into peptides or low-molecular weight proteins is not always determined by the length or secondary structure of a given molecule but also depends on the mode of action in insect immunity and, therefore, it is rather arbitrary. Antimicrobial peptides (AMPs) with their role in immunity, modes of action, and classification are presented in the chapter, followed by a short description of some examples: cecropins, moricins, defensins, proline- and glycine-rich peptides. Further, we will describe selected immune-related proteins that may participate in immune recognition, may possess direct antimicrobial properties, or can be involved in the modulation of insect immunity by both abiotic and biotic factors. We briefly cover Fibrinogen-Related Proteins (FREPs), Down Syndrome Cell Adhesion Molecules (Dscam), Hemolin, Lipophorins, Lysozyme, Insect Metalloproteinase Inhibitor (IMPI), and Heat Shock Proteins. The reader will obtain a partial picture presenting molecules participating in one of the most efficient immune strategies found in the animal world, which allow insects to inhabit all ecological land niches in the world.

Host-pathogen interactions upon the first and subsequent infection of Galleria mellonella with Candida albicans.

Insects are able to develop enhanced resistance in response to repeated infection. This phenomenon is called immune priming. In this work, so-called "primed" Galleria mellonella larvae were re-infected with a lethal dose of Candida albicans 48 h after injection of a non-lethal dose, while "non-primed" larvae were infected only with a lethal dose. The increased resistance of the primed larvae correlated with a slower rate of body colonisation by the fungus. Changes in the protein profiles were detected in the whole hemolymph of the primed insects. The analysis of low-molecular weight proteins and peptides obtained with the use of three different organic solvents and comparative quantitative HPLC analysis thereof showed that the primed larvae did not have higher amounts of any infection-inducible polypeptides than the non-primed larvae. Moreover, electrophoresis of low-molecular weight polypeptides revealed an even lower level of immune-induced peptides in the primed larvae than in the non-primed ones. Furthermore, the defence activity of larval hemolymph, i.e. the antifungal, antibacterial, and lysozyme-type activity, was up-regulated in the primed larvae at the time of re-infection and, consequently, at the early time points after the infection with the lethal dose. Twenty four hours after the infection, these parameters were equally high in the non-primed and primed larvae. Accordingly, at the time of the injection of the lethal dose, certain immune-inducible genes were up-regulated. However, 24 h after the infection with the lethal dose, their expression in both groups was incomparably higher than at the time of the infection and, in most cases, it was as high in the primed larvae as in the non-primed ones. We found that only anti yeast-like activity was enhanced 24 h after the re-infection. This correlated with results obtained by testing the priming effect in heterologous systems: the primed animals did not exhibit higher resistance to the other pathogens tested.