Changes in the apolipophorin III in Galleria mellonella larvae treated with Pseudomonas aeruginosa exotoxin A.

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 âˆ¼ 6.5 and âˆ¼ 6.1 in the hemolymph and âˆ¼ 6.5 and âˆ¼ 5.9 in the hemocytes as well as one isoform with pI âˆ¼ 6.5 in the fat body with an additional apoLp-III-derived polypeptide with estimated pI âˆ¼ 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 âˆ¼ 5.9 isoform was detected, while the major apoLp-III isoform (pI âˆ¼ 6.5) remained unchanged. In addition, appearance of an additional apoLp-III-derived polypeptide with an estimated pI âˆ¼ 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 âˆ¼ 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.

Chemical Composition and Antimicrobial Activity of New Honey Varietals.

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.

Pseudomonas aeruginosa exotoxin A induces apoptosis in Galleria mellonella hemocytes.

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 characteristic 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 understanding the role of exotoxin A during P. aeruginosa infections not only in insects but also in mammals, including humans.

Host-pathogen interactions: The role of Pseudomonas aeruginosa exotoxin A in modulation of Galleria mellonella immune response.

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.

A comparison of the production of antimicrobial peptides and proteins by Galleria mellonella larvae in response to infection with two Pseudomonas aeruginosa strains differing in the profile of secreted proteases.

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.

Pseudomonas aeruginosa alkaline protease exhibits a high renaturation capability.

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.

[Metalloproteases and their inhibitors: role in pathogenesis of selected examples]. / Metaloproteinazy i ich inhibitory: rola w patogenezie na wybranych przykladach.

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.

[Different faces of phenoloxidase in animals]. / Rózne oblicza oksydazy fenolowej u zwierzat.

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.

Diverse effects of Galleria mellonella infection with entomopathogenic and clinical strains of Pseudomonas aeruginosa.

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. mellonella­P. 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.

Galleria mellonella hemocytes destruction after infection with Pseudomonas aeruginosa.

The influence of infection with an entomopathogenic strain of Pseudomonas aeruginosa on Galleria mellonella hemocytes was investigated. Extensive bacteriaemia developed 18 h after infection. This was correlated with significant changes in morphology, viability and the spreading ability of immunocompetent hemocytes, namely granulocytes and plasmatocytes. Since bacteriaemia developed, membrane blebbing, cytoplasm vacuolization, cell and organelle swelling, and chromatin condensation were observed among others. These features are typical for apoptotic and autophagal cell death. A gradually increasing level of procaspase and its activation as well as lack of DNA degradation were also detected. Propidium iodide and acridine orange staining indicated that hemocytes become dead ultimately. Infection of G. mellonella larvae with P. aeruginosa also caused significant changes in the arrangement of the actin cytoskeleton in the hemocytes, which might be correlated with their restricted spreading ability.

Diverse susceptibility of Galleria mellonella humoral immune response factors to the exoproteinase activity of entomopathogenic and clinical strains of Pseudomonas aeruginosa.

We investigated the effects of extracellular proteinases of two Pseudomonas aeruginosa clinical isolates on the essential humoral immune response parameters in hemolymph of the insect model organism Galleria mellonella in vitro. Two culture media, rich LB and minimal M9, known to induce synthesis of different sets of proteinases secreted by P. aeruginosa were used. Changes in lysozyme, antibacterial and antifungal activities, as well as protein and peptide patterns in hemolymph treated with proteolytic fractions were evaluated. The effect of the proteolytic fractions on the apoLp-III level in hemolymph was determined by immunoblotting with antibodies against G. mellonella apolipophorin III (apoLp-III). We found that apoLp-III is hardly degraded by the proteinases of the proteolytic fractions of both clinical P. aeruginosa strains, in contrast to the high susceptibility of the protein to the proteinases of the entomopathogenic strain. The detected differences, together with the changes in the hemolymph protein and peptide patterns caused by the studied fractions, reflected the distinct composition of secreted proteinases of the entomopathogenic P. aeruginosa strain and the clinical strains tested. Our results also suggest the involvement of alkaline protease, the main proteinase of proteolytic fractions of P. aeruginosa grown in minimal medium, in the degradation of G. mellonella antimicrobial factors, such as lysozyme, antibacterial polypeptides, and proteins with antifungal activity. The diverse effects of the P. aeruginosa proteolytic fractions studied on the parameters of G. mellonella immune response indicate that this model insect may be useful in the analysis of the virulence factors of different P. aeruginosa strains.

Three Pseudomonas aeruginosa strains with different protease profiles.

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.

Effect of Pseudomonas aeruginosa elastase B on level and activity of immune proteins/peptides of Galleria mellonella hemolymph.

Abstract Susceptibility of proteins and peptides present in immune hemolymph of Galleria mellonella Fabricius (Lepidoptera: Pyralidae) larvae to proteolytic degradation by purified elastase B of Pseudomonas aeruginosa was studied. Results showed that apoLp-III protein was gradually digested by elastase B in vitro. Additionally, polipeptides with molecular mass 6.5 and 4 kDa were degraded after treatment with the studied enzyme. The lack of these peptides and the decrease in anti-Escherichia coli activity could indicate that inducible antimicrobial peptides were digested by elastase B. On the contrary, no change in the lysosome activity level was observed in immune hemolymph incubated with elastase B. Thus, elastase B might contribute to the pathogenesis of 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 18h post challenge of thermolysin and 38h after injection of elastase B. It was also shown that, 24h 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.

Antibacterial activity in vivo and in vitro in the hemolymph of Galleria mellonella infected with Pseudomonas aeruginosa.

The antibacterial activity of hemolymph from Galleria mellonella infected with entomopathogenic strain of Pseudomonas aeruginosa and non-pathogenic bacterium Escherichia coli was studied. In vivo, the antimicrobial activity appeared shortly after P. aeruginosa infection, reached the maximum level 18 h postinjection, while 30 h later only trace activity was noted. The activity induced by E. coli sustained on the high level until 48 h after infection. We also noted that the antimicrobial activity level induced by the non-pathogenic bacterium was higher in comparison to that measured in insects infected with the pathogenic strain of P. aeruginosa. The results of our in vitro studies indicated that inducible antimicrobial peptides of G. mellonella larvae were digested by P. aeruginosa elastase B. After 1 h incubation of cell-free hemolymph of immune-challenged larvae with elastase B, no antibacterial activity was observed. It was also shown that elastase B degraded synthetic cecropin B while in the presence of 6 mM EDTA antibacterial activity of cell-free hemolymph as well as cecropin B, was not changed which confirmed that the activity was abolished by the metalloprotease.

Changes in Galleria mellonella apolipophorin III level during Pseudomonas aeruginosa infection.

The level of apoLp-III in fat body, hemocytes and plasma from Galleria mellonella larvae infected with Pseudomonas aeruginosa was studied. It was found that the amount of 18kDa protein present in fat body and hemocytes decreased progressively with time after infection. In the case of plasma, an increase in apoLp-III content was observed during the first 19h after infection and then decreased significantly after prolonged infection time. The decreased level of apoLp-III in plasma 24h after infection was accompanied by the appearance of smaller than 18kDa immunoreactive polypeptides. Four intermediate forms with molecular mass of, respectively, 15, 13.3, 12 and 9.5kDa were detectable. The size of polypeptides detected in experiments performed in vivo is comparable with the degradation products of apoLp-III produced by serine protease IV in vitro. In addition, the total proteolytic activity of plasma increased progressively during infection time. The results of our studies suggest that a significant part of total proteolytical activity in the plasma of infected G. mellonella larvae can be attributed to proteases produced by P. aeruginosa during pathogenesis. We discuss the possibility that protease IV of P. aeruginosa is responsible for apoLp-III degradation in vivo.

Apolipophorin III is a substrate for protease IV from Pseudomonas aeruginosa.

Our results demonstrated that Pseudomonas aeruginosa serine protease IV degraded apolipophorin III from the haemolymph of Galleria mellonella larvae. ApoLp-III protein was degraded in a stepwise manner. Four intermediate forms of 15, 13.3, 11.9 and 9.5 kDa were detected after 30 min digestion while only one of 5.6 kDa was released after 1-h incubation time. N-terminal amino acid sequence analysis of 5.6 kDa peptide revealed that it was released from apoLp-III after cleavage between lysine 70 and 71. ApoLp-III degradation by protease IV was inhibited by 1 mM TLCK but not 1 mM EDTA, additionally demonstrating that digestion was catalysed by a serine protease. Our data also indicated apoLp-III degradation in vivo during P. aeruginosa infection of G. mellonella larvae.

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.