Different forms of apolipophorin III in Galleria mellonella larvae challenged with bacteria and fungi.

Apolipophorin III (apoLp-III), a lipid-binding protein and an insect homolog of human apolipoprotein E, plays an important role in lipid transport and immune response in insects. In the present study, we have demonstrated a correlation in time between changes in the apoLp-III abundance occurring in the hemolymph, hemocytes, and fat body after immunization of Galleria mellonella larvae with Gram-negative bacteria Escherichia coli, Gram-positive bacteria Micrococcus luteus, yeast Candida albicans, and a filamentous fungus Fusarium oxysporum. Using two-dimensional electrophoresis (IEF/SDS-PAGE) and immunoblotting with anti-apoLp-III antibodies, the profile of apoLp-III forms in G. mellonella larvae challenged with the bacteria and fungi has been analyzed. Besides the major apoLp-III protein (pI=6.5), one and three additional apoLp-III forms differing in the pI value have been detected, respectively, in the hemolymph, hemocytes, and fat body of non-immunized insects. Also, evidence has been provided that particular apoLp-III-derived polypeptides appear after the immune challenge and are present mainly in the hemolymph and hemocytes. The time of their appearance and persistence in the hemolymph was dependent on the pathogen used. At least two of the apoLp-III forms detected in hemolymph bound to the microbial cell surface. The increasing number of hemolymph apoLp-III polypeptides and differences in their profiles observed in time after the challenge with different immunogens confirmed the important role of apoLp-III in discriminating between pathogens by the insect defense system and in antibacterial as well as antifungal immune response.

Lysozyme and defense peptides as suppressors of phenoloxidase activity in Galleria mellonella.

The prophenoloxidase (proPO) cascade supplies quinones and other reactive compounds for melanin formation, protein cross-linking, hemolymph coagulation, and killing of microbial invaders as well as parasites. The high cytotoxicity of the generated compounds requires a strict control of the activation of the proPO system and phenoloxidase (PO) activity to minimize damage to host tissues and cells. The PO activity in hemolymph of Escherichia coli challenged Galleria mellonella larvae increased, with a temporal drop 1 h after the challenge, reaching the highest level 24 h after the challenge. In the present study, a potential role of G. mellonella defense peptides and lysozyme in controlling the proPO system was investigated. The effects of purified defense peptides (anionic peptides 1 and 2, cecropin D-like peptide, Galleria defensin, proline-rich peptides 1 and 2) and lysozyme were analyzed. Four compounds, namely lysozyme, Galleria defensin, proline-rich peptide 1, and anionic peptide 2, decreased the hemolymph PO activity considerably, whereas the others did not affect the enzyme activity level. Our results indicate that these hemolymph factors could play multiple and distinct roles in the insect immune response.

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.

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.

Protein kinase A activity and protein phosphorylation in the haemocytes of immune-challenged Galleria mellonella larvae.

Protein kinase A (PKA) activity was detected in the haemocytes of greater wax moth, Galleria mellonella larvae using a specific peptide substrate--kemptide. The enzyme was activated in vitro by 1 microM concentration of cAMP, 8-Br-cAMP, 8-Chl-cAMP and BzcMP, whereas in the case of cGMP 10 microM concentration was necessary. Immune challenge of G. mellonella larvae with bacteria led to changes in haemocyte PKA activity. Gram-positive M. luteus was a better inducer of PKA activity than Gram-negative E. coli. The kinetics of activity changes was dependent on the bacteria used and considerably differed from that observed in water-treated insects. Inhibition of PKA activity by cell-permeable, specific inhibitor, Rp-8-Br-cAMPS, induced changes in haemocyte morphology resembling those caused by live bacteria. Four potential PKA substrates of 155 kDa, 44 kDa, 40 kDa and 22 kDa were recognized in the haemocytes of naive larvae by phospho-motif antibodies for PKA phosphorylation consensus site. The modification level of 40 kDa protein changed after water treatment and immune challenge of G. mellonella larvae, whereas that of 155 kDa protein changed only after E. coli and LPS injections. Additionally, in the haemocytes of bacteria- and LPS-challenged insects a transient phosphorylation of 36 kDa protein was detected.

The involvement of protein kinase A in the immune response of Galleria mellonella larvae to bacteria.

The role of protein kinase A (PKA) in the humoral immune response of the greater wax moth Galleria mellonella larvae to live gram-positive bacteria Micrococcus lysodeikticus and gram-negative bacteria Escherichia coli was investigated. The immune challenge of larvae with both kinds of bacteria caused an increase in fat body PKA activity depending on the injected bacteria. Gram-positive M. lysodeikticus was a much better inducer of the enzyme activity than gram-negative E. coli. The PKA activity was increased about 2.5-fold and 1.5-fold, after M. lysodeikticus and E. coli injection, respectively. The in vivo inhibition of the enzyme activity by a cell permeable selective PKA inhibitor, Rp-8-Br-cAMPS, was correlated with considerable changes of fat body lysozyme content and hemolymph antimicrobial activity in bacteria-challenged insects. The kinetics of changes were different and dependent on the bacteria used for the immune challenge of G. mellonella larvae.

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.

Studies on the role of protein kinase A in humoral immune response of Galleria mellonella larvae.

Protein kinase A (PKA) activity was detected in the fat body of Galleria mellonella larvae by a non-radioactive method using a specific peptide substrate-kemptide. The enzyme activity was stimulated by cAMP and its analogues: BzcMP, 8-Chl-cAMP and 8-Br-cAMP in concentrations of 1-4muM. Cyclic GMP was not effective in PKA activation. A two-fold increase in PKA activity was detected in the fat body of G. mellonella LPS-challenged larvae. Selective, membrane-permeable PKA inhibitors, H89 and Rp-8-Br-cAMPS, inhibited protein kinase A activity in the fat body of G. mellonella larvae in vitro and in vivo. The inhibition of PKA activity in vivo was correlated with a considerable lowering of haemolymph antibacterial activity and a decrease in lysozyme content in the fat body of immune challenged larvae. The use of phospho-motif antibodies recognising PKA phosphorylation consensus site allowed identification of four potential PKA phosphorylation substrates of 79, 45, 40 and 36kDa in G. mellonella fat body.

The effect of cAMP and cGMP on the activity and substrate specificity of protein kinase A from methylotrophic yeast Pichia pastoris.

Cyclic AMP dependent protein kinase (PKA) from Pichia pastoris yeast cells was found to be activated by either cAMP or cGMP. Analogs of cAMP such as 8-chloro-cAMP and 8-bromo-cAMP were as potent as cAMP in PKA activation while N6,2'-O-dibutyryl-cAMP did not stimulate the enzyme activity. It was shown that protamine sulfate was almost equally phosphorylated in the presence of 1-2 x 10(-6)M cAMP or cGMP while other substrates such as Kemptide, ribosomal protein S6, were phosphorylated to a lower extent in the presence of cGMP. It was demonstrated that pyruvate kinase is a substrate of PKA which co-purified with the P.pastoris enzyme. Moreover, pyruvate kinase was phosphorylated by PKA in the presence of cAMP and cGMP to comparable levels.

The effect of vanadate on Pichia pastoris growth, protein kinase A activity and ribosomal protein phosphorylation.

It was found that wild type yeast Pichia pastoris can tolerate vanadate concentration as high as 25 mM in the growth medium. Moreover, four vanadate-resistant P. pastoris strains designated JC100/1, JC100/3, JC100/9 and JC100/15 exhibiting tolerance up to 150 mM vanadate were selected. Growth of P. pastoris was correlated with vanadate to vanadyl reduction and its accumulation in the growth medium. In two selected strains, JC100/9 and JC100/15, protein kinase A activity was much higher in comparison to the wild type strain even without vanadate addition to the growth medium. Moreover, in the presence of vanadate, protein kinase A activity was significantly increased in the wild type and the vanadate-resistant JC100/1 and JC100/3 strains. It was also found that phosphorylation of a 40 kDa protein associated with ribosomes occured in all vanadate-resistant strains from the logarithmic, while in the wild type strain from the stationary growth phase. From the presented results it can be concluded that a protein kinase A signalling pathway(s) might be involved in the mechanism of P. pastoris vanadate resistance. The results also indicate a possible role of the 40 kDa protein in protection of P. pastoris against vanadate toxicity.