Paweł Mak

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.

Molecular cloning and biochemical characterisation of proteases from Staphylococcus epidermidis.

Abstract We report the complete coding sequence and the partial amino acid sequence (determined by chemical sequencing) of Staphylococcus epidermidis extracellular cysteine (Ecp) and serine (Esp) proteases. The first enzyme shows an extended sequence similarity to Staphylococcus aureus cysteine protease (staphopain) and the second one resembles the serine protease produced by that species. The region directly upstream of the sequence coding for the mature protein in both enzymes displays significant homology to the profragments encoded by sspB and sspA, respectively, thus suggesting that the characterised enzymes may also be produced as proproteins. Furthermore, we report some biological properties of the cysteine protease, contributing to a better understanding of its role as a possible virulence factor. The proteolytic activity of this enzyme was rapidly and efficiently inhibited by human α-2-macroglobulin; however, human kininogen as well as cystatins (A, C and D) were not inhibitory. Moreover, the protease was capable of inactivating, by limited proteolysis, both α-1-antitrypsin and HMWkininogen, but neither α-1-antichymotrypsin nor antithrombin III.

Antibacterial hemoglobin peptides in human menstrual blood

This work documents that normal menstrual vaginal blood of healthy females is exceptionally rich in hemocidins--hemoglobin (Hb) fragments having bactericidal properties. The peptide fractions were isolated from the plasma of vaginal discharge of three healthy nulliparous women and subjected to identification by automatic sequencing as well as by mass spectrometry. All 44 identified peptides originate from Hb (mainly from the N-terminal part of alpha-globin) and all demonstrated differential killing activity toward Escherichia coli. The screening of antimicrobial activity was performed using two synthetic peptides identical to those found in menstrual blood. These peptides were active mainly toward Gram-negative bacteria and to a less degree toward Gram-positive bacteria. Our results confirm recent observations that Hb-derived fragments manifest pronounced antibacterial activity and suggest that these peptides help in maintaining human vaginal homeostasis during physiologic menstrual bleeding.

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.

The increased bactericidal activity of a fatty acid-modified synthetic antimicrobial peptide of human cathepsin G correlates with its enhanced capacity to interact with model membranes

The bactericidal potency of a synthetic peptide (CG 117-136) of human lysosomal cathepsin G (cat G) can be substantially increased by covalent attachment to its N- or C-termini, of saturated, linear fatty acids (FAs), namely those with C-8, C-10 and C-12 hydrocarbon chains. In order to understand better the mechanism by which FA moieties increase the bactericidal activity of CG 117-136, the interaction of N-terminally FA-modified peptides with artificial membranes was studied. First, the content of secondary structure motifs in the modified and unmodified peptides was determined by circular dichroism (CD). A marked increase in the propensity of FA-modified CG 117-136 to form an alpha-helix structure was observed for the C-8, C-10 and C-12 derivatives compared with unmodified/short-chain and long-chain (C-14, C-16, C-18) derivatives. These effects were observed both in the presence of large unilamellar liposomes or in trifuluoroethanol, a membrane-stimulating agent. Second, the capacity of peptides to insert into large unilamellar liposomes as a function of FA length was determined by their ability to release a trapped fluorescent dye. FA derivatives with the highest alpha-helical content were found to be the most effective in releasing a fluorescent dye, compared with an unmodified peptide and/or derivatives having a low alpha-helical content. The ability of the peptides to attain alpha-helical structure in the membrane-like environment and the ability to disrupt the liposomal membrane, therefore correlate remarkably well with their increased ability to kill bacteria. A plausible explanation for improved bactericidal action of the modified peptide is that the FA moiety facilitates formation of the peptide with an alpha-helical structure formation in membranes, which is essential for disrupting the integrity of the bacterial cytoplasmic membrane.

Identification of ADAM10 as a major TNF sheddase in ADAM17-deficient fibroblasts

ADAM17 (a disintegrin and metalloprotease)-deficient murine fibroblasts stably transfected with proTNF cDNA release significant amounts of biologically active soluble TNF. The enzyme responsible for this activity is a membrane protein that hydrolyzes the peptide bond Ala(76):Val(77) within proTNF. Its activity is inhibited by 1,10-phenantroline and GM6001, insusceptible to TIMP-2 (tissue inhibitor of metalloproteinases-2), and stimulated by ionomycin. These characteristics match ADAM10. The moderate silencing of ADAM10 by shRNA resulted in a significant inhibition of TNF shedding. There was no correlation between the level of ADAM10 expression and the presence of active ADAM17. Our results indicate that ADAM10 may function as the TNF sheddase in cells which lack ADAM17 activity.

Menstrual Hemocidin HbB115–146 Is an Acidophilic Antibacterial Peptide Potentiating the Activity of Human Defensins, Cathelicidin and Lysozyme

Our recent studies proved that menstrual discharge is exceptionally rich in bactericidal hemoglobin peptides (hemocidins). Of special interest is the behavior of hemocidins in low pH of the vagina, in different ionic strengths, and in the presence of other specialized antibacterial molecules acting in this organ.

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.

The Role of RCAS1 and Oxytocinase in Immune Tolerance during Pregnancy

Objectives: To determine and compare the level of RCAS1 (receptor-binding cancer antigen expressed in SiSo cells) in placentas at term as well as oxytocinase/cystine amino peptidase (CAP) serum level a few days before labor in order to evaluate their possible role in the regulation of maternal immune response during pregnancy and in initiation of labor. Methods: We estimated the RCAS1 content in 44 placental tissue samples, using Western blot method. We also assessed CAP serum level by its enzymatic activity, using L-cystine-di-β-naphthylamide as a synthetic substrate. The statistical analysis was performed using Shapiro-Wilk procedure. Student’s t test was applied to compare the differences between parametric data. A value of p < 0.05 was considered significant. Results: RCAS1 was found in all placental tissue samples examined. The differences in the RCAS1 relative amount depended on the onset of labor, with the highest level in induced labor and the lowest in spontaneous labor. The differences were also observed in the CAP serum level with the highest level in pregnant women whose labor was induced. Conclusions: We have observed a link between the expression of the two proteins examined and the onset of the labor. Therefore, we posit that RCAS1 and CAP may play a role in the downregulation of the maternal immune response during pregnancy and may participate in the initiation of the labor.