Due to a widespread occurrence of multidrug-resistant pathogenic strains of bacteria, there is an urgent need to look for antimicrobial substances, and honey with its antimicrobial properties is a very promising substance. In this study, we examined for the first time antimicrobial properties of novel varietal honeys, i.e., plum, rapeseed, Lime, Phacelia, honeydew, sunflower, willow, and multifloral-P (Prunus spinosa L.), multifloral-AP (Acer negundo L., Prunus spinosa L.), multifloral-Sa (Salix sp.), multifloral-Br (Brassica napus L.). Their antimicrobial activity was tested against bacteria (such as Escherichia coli, Bacillus circulans, Staphylococcus aureus, Pseudomonas aeruginosa), yeasts (such as Saccharomyces cerevisiae and Candida albicans) and mold fungi (such as Aspergillus niger). In tested honeys, phenolic acids constituted one of the most important groups of compounds with antimicrobial properties. Our study found phenolic acids to occur in greatest amount in honeydew honey (808.05 µg GAE/g), with the highest antifungal activity aiming at A. niger. It was caffeic acid that was discovered in the greatest amount (in comparison with all phenolic acids tested). It was found in the highest amount in such honeys as phacelia—356.72 µg/g, multifloral (MSa) and multifloral (MBr)—318.9 µg/g. The highest bactericidal activity against S. aureus was found in multifloral honeys MSa and MBr. Additionally, the highest amount of syringic acid and cinnamic acid was identified in rapeseed honey. Multifloral honey (MAP) showed the highest bactericidal activity against E. coli, and multifloral honey (MSa) against S. aureus. Additionally, multifloral honey (MBr) was effective against E. coli and S. aureus. Compounds in honeys, such as lysozyme-like and phenolic acids, i.e., coumaric, caffeic, cinnamic and syringic acids, played key roles in the health-benefit properties of honeys tested in our study.

The cellular immune response of the greater wax moth Galleria mellonella to Pseudomonas aeruginosa exotoxin A was investigated for the first time. The insects were challenged with a sublethal dose of exoA, and then hemocyte parameters were assessed. The analysis showed a statistically significant decrease in the total hemocyte count (THC), which was associated with significant decreases in the number of granulocytes and plasmatocytes. In turn, no statistically significant changes were observed in the number of spherulocytes and oenocytoides. Fluorescent staining indicated that cells collected from the exoA-challenged larvae exhibited features charac-teristic for apoptotic and autophagic cell death, e.g. cytoplasm vacuolization and chromatin condensation. The flow cytometry analysis revealed a significant increase in the number of phosphatidylserine-and active caspase 3-positive hemocytes challenged with exoA, which proved apoptosis induction. Our results will help in under-standing the role of exotoxin A during P. aeruginosa infections not only in insects but also in mammals, including humans.

In the present study, we have demonstrated a correlation in time between changes in the amount of apolipophorin III (apoLp-III) in the fat body and hemocytes of Galleria mellonella larvae challenged with Pseudomonas aeruginosa exotoxin A (exoA). An increase in the amount of apoLp-III was detected 1-8 h after the challenge; then, a temporary decrease was observed after 15 h followed by an increase in the level of apoLp-III, however to a different extent. The profile of apoLp-III forms in the hemolymph, hemocytes, and fat body of the exoA-challenged larvae was analyzed using two-dimensional electrophoresis (IEF/SDS-PAGE) and immunoblotting with anti-apoLp-III antibodies. Two apoLp-III forms differing in isoelectric point values estimated at ∼ 6.5 and ∼ 6.1 in the hemolymph and ∼ 6.5 and ∼ 5.9 in the hemocytes as well as one isoform with pI ∼ 6.5 in the fat body with an additional apoLp-III-derived polypeptide with estimated pI ∼ 6.9 were detected in the control insects. The injection of exoA caused a significant decrease in the abundance of both apoLp-III isoforms in the insect hemolymph. In the hemocytes, a decrease in the amount of the pI ∼ 5.9 isoform was detected, while the major apoLp-III isoform (pI ∼ 6.5) remained unchanged. In addition, appearance of an additional apoLp-III-derived polypeptide with an estimated pI ∼ 5.2 was observed. Interestingly, there were no statistically significant differences in the amount of the main isoform in the fat body between the control and exoA-challenged insects, but the polypeptide with pI ∼ 6.9 disappeared completely. It should be noted that the decrease in the amount of apoLp-III and other proteins was especially noticeable at the time points when exoA was detected in the studied tissues.

Barciak większy (Galleria mellonella) jest coraz częściej stosowanym owadzim organizmem modelowym. Do zalet gatunku można zaliczyć powszechność występowania, krótki cykl życiowy, oraz stosunkowo duży rozmiar gąsienic, znacznie ułatwiający manipulacje laboratoryjne. Istotne jest to, że owady mogą być inkubowane w temperaturze 37oC, koniecznej w badaniach dotyczących ludzkich patogenów. Warto wspomnieć, że wykazano pozytywną korelację między odpowiedzią gospodarza a wirulencją wielu patogenów w modelach ssaków i owadów. Gąsienice G. mellonella z powodzeniem są wykorzystywane w badaniach interakcji patogen-gospodarz, mechanizmów patogenezy i czynników wirulencji drobnoustrojów chorobotwórczych dla człowieka, m.in. Staphylococcus aureus, Pseudomonas aeruginosa i Candida albicans. Owady okazały się również odpowiednie do testowania skuteczności substancji biologicznie czynnych czy też w terapiach alternatywnych mających na celu zastąpienie antybiotyków.

The role of Pseudomonas aeruginosa exotoxin A in the modulation of humoral immune response parameters in the hemolymph of Galleria mellonella larvae was investigated. Our results indicate that exoA can play a role of a virulence factor by inhibiting insect PO, lysozyme, and antibacterial activity and decreasing the apoLp-III protein level significantly. No peptide bands with molecular mass below 6.5 kDa were detected in the hemolymph of exoA-treated larvae. We provided evidence for involvement of exoA in the pathogenicity of P. aeruginosa against G. mellonella and the usefulness of the insect as a model for analysis of P. aeruginosa toxins.

The work presents identification of antimicrobial peptides and proteins (AMPs) in the hemolymph of Galleria mellonella larvae infected with two Pseudomonas aeruginosa strains (ATCC 27,853 and PA18), differing in the profile of secreted proteases. The insects were immunized with bacteria cultivated in rich (LB) and minimal (M9) media, which resulted in appearance of a similar broad set of AMPs in the hemolymph. Among them, 13 peptides and proteins were identified, i.e. proline-rich peptides 1 and 2, lebocin-like anionic peptide 1 and anionic peptide 2, defensin/galiomicin, cecropin, cecropin D-like peptide, apolipophoricin, gallerimycin, moricin-like peptide B, lysozyme, apolipophorin III, and superoxide dismutase. Bacterial strain- and/or medium-dependent changes in the level of proline-rich peptide 1, anionic peptide 1 and 2, moricin-like peptide B, cecropin D-like and gallerimycin were observed. The analysis of the expression of genes encoding cecropin, gallerimycin, and galiomicin indicated that they were differently affected by the bacterial strain but mainly by the medium used for bacterial culture. The highest expression was found for the LB medium. In addition to the antibacterial and antifungal activity, proteolytic activity was detected in the hemolymph of the P. aeruginosa-infected insects. Based on these results and those presented in our previous reports, it can be postulated that the appearance of AMPs in G. mellonella hemolymph can be triggered not only by P. aeruginosa pathogen associated molecular patterns (PAMPs) but also by bacterial extracellular proteases secreted during infection. However, although there were no qualitative differences in the set of AMPs depending on the P. aeruginosa strain and medium, differences in the level of particular AMPs synthesized in response to the bacteria used were observed.

The role of Pseudomonas aeruginosa alkaline protease, one of the important virulence factors of these bacteria, in the prophenoloxidase (proPO) system activation in Galleria mellonella haemolymph was investigated. Immunization of G. mellonella larvae with alkaline protease led to an increase in phenoloxidase (PO) activity in haemolymph 2-8 h after the injection. However, 15 h after the challenge, almost no PO activity was detected, in contrast to insects immunized with heat-killed P. aeruginosa, where an elevated PO activity in haemolymph persisted at all the time points of the experiment (2-24 h). To test the effect of alkaline protease on already activated PO in vitro, non-immune G. mellonella haemolymph with proPO system pre-activated in vitro by heat-killed P. aeruginosa was used. Subsequently, direct incubation of the protease with non-immune haemolymph was carried out to test the effect of the alkaline protease on the proPO system activation. It can be postulated that P. aeruginosa alkaline protease affected not only the active PO, but also significantly inhibited proPO cascade activation in the haemolymph of G. mellonella.

Thermally induced unfolding and renaturation capability of alkaline proteases (AprA) of three Pseudomonas aeruginosa strains, i.e. ATCC 27853 and two clinical isolates, was examined. Sequence analyses demonstrated a high level of aprA genes identity (99.24-99.8%) in these bacterial strains. The proteases retained 45-60% and 15% of their activity after pre-treatment at 60oC and 80oC, respectively, whereas pre-incubation at 90-95oC resulted in a higher level of activity than at 80oC. Zymography analyses and immunoblotting with AprA antiserum suggested a high thermostability and renaturation capability of the studied enzymes in comparison to another P. aeruginosa protease, elastase B. An intrinsic capability of renaturation of P. aeruginosa AprA was confirmed by fluorescence spectra of the native, thermally denatured, and renatured enzyme. The value of the fluorescence intensity of the denatured and subsequently cooled enzyme recovered to about 80% of the value of the native protein fluorescence intensity. Moreover, pre-incubation of the enzyme at 60oC and 90oC exerted only a slight effect on the intensity of absorbance and the shape of the amide I band, as demonstrated by Fourier transform infrared (FTIR) spectroscopy performed after subsequent cooling of the pre-treated enzyme. The results indicated a high renaturation capability of the P. aeruginosa AprA proteins.

Phenoloxidases are oxidoreducting enzymes whose main function is the oxidation of phenols. The term phenoloxidase is often used interchangeably to describe three different enzymes: tyrosinase (EC 1.14.18.1), catechol oxidase, and laccase. Of these, only tyrosinase has two activities: (1) oxygenase activity to hydroxylate monophenols to ortho-diphenols and (2) oxidase activity responsible for further oxidation of ortho-diphenols to ortho-quinones. Tyrosinase is a key enzyme involved in the melanogenesis process, resulting in the formation of black-brown eumelanin and yellow-red feomelanin. In addition to the pigmentary role, human melanin protects against harmful ultraviolet radiation, while in invertebrate animals melanin is involved in the process of cuticle hardening, wound healing, clot formation, maintenance of intestinal homeostasis and defense reactions. In invertebrates, the tyrosinase is synthesized as a proenzyme that is activated by a serine proteases' cascade known as the phenoloxidase system. This system is considered as one of the innate immunity mechanisms.

Proteolytic enzymes and their inhibitors are crucial in host-pathogen interaction. Metalloproteases secreted by pathogenic microbes play an important role in destroying not only host tissues but also their immune proteins. Metalloproteinase inhibitors, in contrast, may serve as effective therapeutic agents, which is especially important because of the increasing number of microorganisms resistant to known antibiotics. The role of metalloproteases produced by the bacterium Pseudomonas aeruginosa in the colonization of the host organism is described. Attention has also been paid to the role of inhibitors of these enzymes in defense responses and underlined their potential role in inhibiting the development of infection.

The G-protein-coupled receptors (GPCRs) form the largest and most diverse group of membrane receptors engaged in extracellular signals transduction. GPCRs are involved in almost all aspects of vertebrates and invertebrates' life, including regulation of the immune response mechanisms. The paper describes the general structure and classification of GPCRs. Moreover, it presents the mechanisms of GPCR activation and signal transduction as well as the regulation of GPCR activity. Furthermore, basic information about the mechanisms of pathogen recognition by invertebrates is included. The main part of this review shows the most recent data about the involvement of GPCRs in defense mechanisms of invertebrates such as the horseshoe crab (Limulus polyphemus), fruit fly (Drosophila melanogaster), and nematode (Caenorhabditis elegans).

The role of Pseudomonas aeruginosa metalloprotease - alkaline protease in activation of the antimicrobial activity in Galleria mellonella larvae was investigated. The results of our in vivo study showed that injection of alkaline protease at a sublethal dose enhanced the antimicrobial activity in the hemolymph of G. mellonella larvae as a result of induction of defense peptides synthesis. We observed that the antibacterial activity against E. coli appeared in the hemolymph 4 h after the injection of both metalloprotease or heat-killed P. aeruginosa, reached the maximum level 24 h post injection, and next decreased slightly. Antifungal activity against A. niger was detected in the hemolymph 15 h and 24 h after the challenge in the case of the alkaline protease and P. aeruginosa cell treatment, respectively. We also noted that the antimicrobial activity level induced by the presence of the metalloprotease in the hemolymph was higher than the activity measured after the injection of the insects with P. aeruginosa. The results of our in vitro studies indicated that inducible antimicrobial peptides present in the hemolymph of protease- or P. aeruginosa-challenged larvae were digested by alkaline protease.

The greater wax moth G. mellonella is a useful model organism for investigations of pathogenicity and identification of P. aeruginosa virulence factors. Strains differing in the protease profiles elicit different responses of the G. mellonella immune system to infection. The immune response depends on the bacterial strain used and the composition of the bacterial culture medium. Proteases in the initial phase of infection activate the immune system (involving increased lysozyme synthesis, induction of synthesis of immune peptides and metalloproteinase inhibitors, and altered apolipophorin III levels), thereby increasing the antibacterial activity of insect hemolymph. As bacteraemia progresses, they overcome the humoral immune response of the host by inhibiting the phenoloxidase activity, degradation of immune peptides and apolipophorin III. Different proteases exhibit different involvement in the degradation of key elements of insect immune response and/or proteins, while immune peptides display varied sensitivity to proteolytic activity. Elastase B has a double role during infection: it is an inducer of immune response, (involving induction of antimicrobial peptide synthesis and metalloproteinase inhibitors, and increasing expression and activity of lysozyme and the level of apolipophorin III) and a virulence factor involved in the degradation of antimicrobial peptides. Increased quantities and activity of lysozyme in the hemolymph of larvae infected with the P aeruginosa indicates that lysozyme is not very sensitive to the activity of bacterial proteases. Induction of synthesis of humoral immune response factors in G. mellonella larvae in the presence of metalloproteinases (elastase B) implies the "danger" resistance model in this organism.

In numerous studies, the greater wax moth Galleria mellonella has been exploited as an alternative model host for investigating virulence factors of different pathogenic bacteria. In the present paper, we provide evidence that G. mellonella constitutes a useful and convenient model for analysis of the pathogenicity of Pseudomonas aeruginosa clinical strains. In this in vivo study on the G. mellonella–P. aeruginosa interaction, a bidirectional analysis comprising evaluation of humoral immune response of the bacteria-infected larvae and determination of P. aeruginosa proteinases synthesized during the infection was performed. The effects of G. mellonella infection by two clinical strains (PA C124/9 and PA 02/18) and one entomopathogenic strain (ATCC 27853) cultured in a rich LB and minimal M9 medium, known to induce synthesis of different sets of extracellular proteinases, were evaluated. Both clinical isolates were able to establish infection in G. mellonella caterpillars after intrahemocelic injection. However, although the final effect of the larvae infection by each P. aeruginosa strain was their death within ca. 48 h, considerable strain- and medium-dependent differences in the immune response of the insects were detected. The results indicated that G. mellonella larvae distinguished between the three P. aeruginosa strains, which was well reflected by the diverse humoral immune response. The significant differences concerned, among others, the level of phenoloxidase, lysozyme, and antibacterial activity in the hemolymph of the infected insects. An analysis of proteinases performed using specific activity tests, zymography and immunoblotting, revealed that elastase B and alkaline protease were synthesized by each P. aeruginosa strain during the infection. In contrast, a high level of elastase A activity was detected only in the larvae infected by the P. aeruginosa ATCC 27853 strain. It can be postulated that the three P. aeruginosa strains exploit different strategies to avoid and overcome insect immunity. Our results provided further evidence on G. mellonella usefulness as a model for analysis of P. aeruginosa virulence factors and their involvement in pathogenicity.

The proteolytic activity of three Pseudomonas aeruginosa strains, ATCC 27853 - a reference strain, and two clinical isolates was tested. The activity was examined after culturing the bacteria in two different growth media: the minimal M9 medium and rich Luria-Bertani broth (LB). Based on zymograms and protease activity specific assays, it was concluded that the reference strain produced three proteolytic enzymes in the LB medium: protease IV, elastase B and elastase A, while alkaline protease was only produced in the M9 medium. The clinical isolates of P. aeruginosa produced elastase B and alkaline protease when grown in the LB medium and the minimal M9 medium, respectively. PCR analysis confirmed the presence of both the lasB gene encoding elastase B and aprA coding for alkaline protease in the genomes of the three P. aeruginosa strains analyzed. The expression of these genes coding for two important P. aeruginosa virulence factors was dependent on the growth conditions in all the strains studied. The contribution of the extracellular proteinases to the virulence of P. aeruginosa strains used in this study was investigated using an insect model, the greater wax moth Galleria mellonella.